Evidence of introduced honeybees (Apis mellifera) as pollen wasters in orchid pollination.

Biological invasions threaten global biodiversity, altering landscapes, ecosystems, and mutualistic relationships like pollination. Orchids are one of the most threatened plant families, yet the impact of invasive bees on their reproduction remains poorly understood. We conduct a global literature survey on the incidence of invasive honeybees (Apis mellifera) on orchid pollination, followed by a study case on Australian orchids. Our literature survey shows that Apis mellifera is the primary alien bee visiting orchids worldwide. However, in most cases, introduced honeybees do not deposit orchid pollen. We also test the extent to which introduced honeybees affect orchid pollination using Diuris brumalis and D. magnifica. Diuris brumalis shows higher fruit set and pollination in habitats with both native and invasive bees compared to habitats with only introduced bees. Male and female reproductive success in D. magnifica increases with native bee abundance, while conversely pollinator efficiency decreases with honeybee abundance and rises with habitat size. Our results suggest that introduced honeybees are likely involved in pollen removal but do not effectively deposit orchid pollen, acting as pollen wasters. However, Apis mellifera may still contribute to pollination of Diuris where native bees no longer exist. Given the global occurrence of introduced honeybees, we warn that certain orchids may suffer from pollen depletion by these invaders, especially in altered habitats with compromised pollination communities.

Structural Variants and Speciation: Multiple Processes at Play.

Research on the genomic architecture of speciation has increasingly revealed the importance of structural variants (SVs) that affect the presence, abundance, position, and/or direction of a nucleotide sequence. SVs include large chromosomal rearrangements such as fusion/fissions and inversions and translocations, as well as smaller variants such as duplications, insertions, and deletions (CNVs). Although we have ample evidence that SVs play a key role in speciation, the underlying mechanisms differ depending on the type and length of the SV, as well as the ecological, demographic, and historical context. We review predictions and empirical evidence for classic processes such as underdominance due to meiotic aberrations and the coupling effect of recombination suppression before exploring how recent sequencing methodologies illuminate the prevalence and diversity of SVs. We discuss specific properties of SVs and their impact throughout the genome, highlighting that multiple processes are at play, and possibly interacting, in the relationship between SVs and speciation.

Mimicking orchids lure bees from afar with exaggerated ultraviolet signals.

Flowers have many traits to appeal to pollinators, including ultraviolet (UV) absorbing markings, which are well-known for attracting bees at close proximity (e.g., <1 m). While striking UV signals have been thought to attract pollinators also from far away, if these signals impact the plant pollinia removal over distance remains unknown. Here, we report the case of the Australian orchid Diuris brumalis, a nonrewarding species, pollinated by bees via mimicry of the rewarding pea plant Daviesia decurrens. When distant from the pea plant, Diuris was hypothesized to enhance pollinator attraction by exaggeratedly mimicking the floral ultraviolet (UV) reflecting patterns of its model. By experimentally modulating floral UV reflectance with a UV screening solution, we quantified the orchid pollinia removal at a variable distance from the model pea plants. We demonstrate that the deceptive orchid Diuris attracts bee pollinators by emphasizing the visual stimuli, which mimic the floral UV signaling of the rewarding model Daviesia. Moreover, the exaggerated UV reflectance of Diuris flowers impacted pollinators' visitation at an optimal distance from Da. decurrens, and the effect decreased when orchids were too close or too far away from the model. Our findings support the hypothesis that salient UV flower signaling plays a functional role in visual floral mimicry, likely exploiting perceptual gaps in bee neural coding, and mediates the plant pollinia removal at much greater spatial scales than previously expected. The ruse works most effectively at an optimal distance of several meters revealing the importance of salient visual stimuli when mimicry is imperfect.

Hidden genetic variation in plasticity provides the potential for rapid adaptation to novel environments.

Rapid environmental change is forcing populations into environments where plasticity will no longer maintain fitness. When populations are exposed to novel environments, evolutionary theory predicts that genetic variation in fitness will increase and should be associated with genetic differences in plasticity. If true, then genetic variation in plasticity can increase adaptive potential in novel environments, and population persistence via evolutionary rescue is more likely. To test whether genetic variation in fitness increases in novel environments and is associated with plasticity, we transplanted 8149 clones of 314 genotypes of a Sicilian daisy (Senecio chrysanthemifolius) within and outside its native range, and quantified genetic variation in fitness, and plasticity in leaf traits and gene expression. Although mean fitness declined by 87% in the novel environment, genetic variance in fitness increased threefold and was correlated with plasticity in leaf traits. High fitness genotypes showed greater plasticity in gene expression, but lower plasticity in most leaf traits. Interestingly, genotypes with the highest fitness in the novel environment had the lowest fitness at the native site. These results suggest that standing genetic variation in plasticity could help populations to persist and adapt to novel environments, despite remaining hidden in native environments.

Adaptive divergence generates distinct plastic responses in two closely related Senecio species.

The evolution of plastic responses to external cues allows species to maintain fitness in response to the environmental variations they regularly experience. However, it remains unclear how plasticity evolves during adaptation. To test whether distinct patterns of plasticity are associated with adaptive divergence, we quantified plasticity for two closely related but ecologically divergent Sicilian daisy species (Senecio, Asteraceae). We sampled 40 representative genotypes of each species from their native range on Mt. Etna and then reciprocally transplanted multiple clones of each genotype into four field sites along an elevational gradient that included the native elevational range of each species, and two intermediate elevations. At each elevation, we quantified survival and measured leaf traits that included investment (specific leaf area), morphology, chlorophyll fluorescence, pigment content, and gene expression. Traits and differentially expressed genes that changed with elevation in one species often showed little changes in the other species, or changed in the opposite direction. As evidence of adaptive divergence, both species performed better at their native site and better than the species from the other habitat. Adaptive divergence is, therefore, associated with the evolution of distinct plastic responses to environmental variation, despite these two species sharing a recent common ancestor.

Hopping or Jumping on the Cliffs: The Unusual Phylogeographical and Demographic Structure of an Extremely Narrow Endemic Mediterranean Plant.

Several past and recent climatic and geological events have greatly influenced the current distribution of coastal species around the Mediterranean Basin. As a consequence, the reconstruction of the distributional history of these species is challenging. In this study, we used both chloroplast and nuclear SNPs to assess the levels of genetic differentiation, contemporary/historical levels of gene flow, and demographic history for the three only known (one mainland and two insular) populations of Eokochia saxicola, a rare Mediterranean coastal rocky halophyte. Plastid genome analysis revealed very low intraspecific haplotype variation and partial admixture among Capri and Palinuro populations with at least two independent colonization events for the Strombolicchio islet. Nuclear SNPs variation consistently identified three distinct genetic clusters corresponding to our sampling localities. Furthermore, strong genetic isolation was confirmed by both historical and contemporary levels of migration among the three populations. The DIYABC analysis identified two introductions temporally separated from Palinuro to Capri (ca.25 Mya) and subsequently to Strombolicchio (ca.09 Mya) as the most likely hypothesis for the current distribution of E. saxicola. Regardless of their small population sizes, all study sites supported high-genetic diversity maintained by outcrossing and random mating between individuals owing largely to wind pollination, an exclusive trait among Mediterranean narrow endemics. In conclusion, the patterns observed confirm that some Mediterranean endemics are not necessarily "evolutionary dead-ends" but rather represent species that have extensive demographic stability and a strong evolutionary legacy.

Ecological and phylogenetic constraints determine the stage of anthetic ovule development in orchids.

PREMISE: Unlike most flowering plants, orchid flowers have under-developed ovules that complete development only after pollination. Classical studies reported variation in the stage in which ovule development is arrested, but the extent of this variation and its evolutionary and ecological significance are unclear. METHODS: Here, we used light microscopy to observe ovule development at anthesis for 39 species not previously studied and surveyed the literature gaining information on 94 orchid species. Tropical and temperate members of all five orchid subfamilies as well as species with contrasting pollination strategies (rewarding versus deceptive) and life forms (epiphytic versus terrestrial) were represented. We analyzed the data using statistical comparisons and a phylogenetic generalized least square (PGLS) analysis. RESULTS: Apostasioideae, the sister to the rest of the orchids, have mature ovules similar to other Asparagales, while under-differentiated ovules are present in the other subfamilies. Ovule developmental stages showed high variation even among closely related groups. Ovules were more developed in terrestrial than in epiphytic, in temperate than in tropical, and in rewarding than in deceptive pollination orchid species. This latter comparison was also significant in the PGLS analysis. CONCLUSIONS: These results suggest that ovule developmental stage in orchids can be shaped by ecological factors, such as seasonality and pollination strategy, and can be selected for optimizing female reproductive investment.

Do floral and ecogeographic isolation allow the co-occurrence of two ecotypes of Anacamptis papilionacea (Orchidaceae)?

Ecotypes are relatively frequent in flowering plants and considered central in ecological speciation as local adaptation can promote the insurgence of reproductive isolation. Without geographic isolation, gene flow usually homogenizes the allopatrically generated phenotypic and ecological divergences, unless other forms of reproductive isolation keep them separated. Here, we investigated two orchid ecotypes with marked phenotypic floral divergence that coexist in contact zones. We found that the two ecotypes show different ecological habitat preferences with one being more climatically restricted than the other. The ecotypes remain clearly morphologically differentiated both in allopatry and in sympatry and differed in diverse floral traits. Despite only slightly different flowering times, the two ecotypes achieved floral isolation thanks to different pollination strategies. We found that both ecotypes attract a wide range of insects, but the ratio of male/female attracted by the two ecotypes was significantly different, with one ecotype mainly attracts male pollinators, while the other mainly attracts female pollinators. As a potential consequence, the two ecotypes show different pollen transfer efficiency. Experimental plots with pollen staining showed a higher proportion of intra- than interecotype movements confirming floral isolation between ecotypes in sympatry while crossing experiments excluded evident postmating barriers. Even if not completely halting the interecotypes pollen flow in sympatry, such incipient switch in pollination strategy between ecotypes may represent a first step on the path toward evolution of sexual mimicry in Orchidinae.

DNA barcoding of native Caucasus herbal plants: potentials and limitations in complex groups and implications for phylogeographic patterns.

DNA barcoding has rapidly become a useful complementary tool in floristic investigations particularly for identifying specimens that lack diagnostic characters. Here, we assess the capability of three DNA barcode markers (chloroplast rpoB, accD and nuclear ITS) for correct species assignment in a floristic survey on the Caucasus. We focused on two herbal groups with potential for ornamental applications, namely orchids and asterids. On these two plant groups, we tested whether our selection of barcode markers allows identification of the "barcoding gap" in sequence identity and to distinguish between monophyletic species when employing distance-based methods. All markers successfully amplified most specimens, but we found that the rate of species-level resolution amongst selected markers largely varied in the two plant groups. Overall, for both lineages, plastid markers had a species-level assignment success rate lower than the nuclear ITS marker. The latter confirmed, in orchids, both the existence of a barcoding gap and that all accessions of the same species clustered together in monophyletic groups. Further, it also allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the best performing barcode for asterids; however, none of the three tested markers showed high discriminatory ability. Even if ITS were revealed as the most promising plant barcode marker, we argue that the ability of this barcode for species assignment is strongly dependent on the evolutionary history of the investigated plant lineage.

Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes.

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication.

Phenotypic expression of floral traits in hybrid zones provides insights into their genetic architecture.

Information on the genetic architecture of phenotypic traits is helpful for constructing and testing models of the ecoevolutionary dynamics of natural populations. For plant groups with long life cycles there is a lack of line cross experiments that can unravel the genetic architecture of loci underlying quantitative traits. To fill this gap, we propose the use of variation for phenotypic traits expressed in natural hybrid zones as an alternative approach. We used data from orchid hybrid zones and compared expected and observed patterns of phenotypic trait expression in different early-generation hybrid classes identified by molecular genetic markers. We found evidence of additivity, dominance, and epistatic interactions for different phenotypic traits. We discuss the potential of this approach along with its limitations and suggest that it may represent a realistic way to gain an initial insight into the heritability and genomic architecture of traits in organismal groups with complex life history, such as orchids and many others.

The effect of seasonality on developmental stages of anthetic ovule integuments in Mediterranean orchids.

Orchids, differently from most flowering plants, have under-differentiated ovules at anthesis that require pollination to complete differentiation. This ovule developmental stage has been often observed in tropical species in which the absence of an evident seasonality may allow plants to extend their phenology beneficiating of a long time for post-pollination events. Here, we used scanning electron microscopy (SEM) to detect ovule integument developmental stages in 21 species of Mediterranean Orchidoideae and Epidendroideae and in 11 tropical Epidendroideae with the aim of understanding whether species with a seasonal constraint and shorter time for post-pollination ovule maturation are characterized by different stages of ovule development at anthesis. We found that Mediterranean orchids (both Orchidoideae and Epidendroideae) have more developed ovule integuments than tropical orchids. Most species show partially to fully developed ovules at anthesis with the exception of Cephalanthera where ovules are arrested in a very early developmental stage. Despite the phylogenetic signal, anthetic ovule integument development differs between related species (with different pollination strategies or blooming times), suggesting the presence of some ecological constraints. The synchronization between ovule integuments and megagametophyte development, as found in tropical orchids, is also confirmed in Mediterranean orchids. Our data show that Mediterranean and tropical orchids clearly differ in anthetic ovule developmental stages, likely depending on seasonality.

Hybridization and geographic distribution shapes the spatial genetic structure of two co-occurring orchid species.

Multiple ecological and life-history traits shape the fine-scale spatial genetic structure (FSGS) of a given population. The occurrence in core versus peripheral populations, levels of outcrossing, pollen and seed dispersal, and hybridization are important biological properties that influence the kinship of individuals within populations. We examined spatial genetic structure within 15 populations of Epidendrum fulgens and E. puniceoluteum distributed along a linear gradient of Brazilian coastal vegetation, including both allopatric and sympatric populations where the two orchid species hybridize. We analyzed 581 mapped specimens using nine simple sequence repeat loci, aiming to investigate how geographic distribution and hybridization shape within-population FSGS. A significant increase in FSGS was found towards peripheral populations, compared to core populations. Analysis of short-distance and long-distance components of FSGS identified biparental inbreeding and higher levels of FSGS at peripheral populations, when compared to core populations. In contrast, the relatively high density of reproductive adults in core populations potentially leads to highly overlapping seed and pollen movement, decreasing FSGS. Hybridization was an important factor shaping within-population spatial genetic structure at sympatric sites, decreasing the FSGS observed in parental species. Our results indicate that different ecological forces act in concert to create a gradient of FSGS along species distribution ranges, shaped by extensive levels of intraspecific and interspecific gene exchange.

The Complete Plastome Sequences of Eleven Capsicum Genotypes: Insights into DNA Variation and Molecular Evolution.

Members of the genus Capsicum are of great economic importance, including both wild forms and cultivars of peppers and chilies. The high number of potentially informative characteristics that can be identified through next-generation sequencing technologies gave a huge boost to evolutionary and comparative genomic research in higher plants. Here, we determined the complete nucleotide sequences of the plastomes of eight Capsicum species (eleven genotypes), representing the three main taxonomic groups in the genus and estimated molecular diversity. Comparative analyses highlighted a wide spectrum of variation, ranging from point mutations to small/medium size insertions/deletions (InDels), with accD, ndhB, rpl20, ycf1, and ycf2 being the most variable genes. The global pattern of sequence variation is consistent with the phylogenetic signal. Maximum-likelihood tree estimation revealed that Capsicum chacoense is sister to the baccatum complex. Divergence and positive selection analyses unveiled that protein-coding genes were generally well conserved, but we identified 25 positive signatures distributed in six genes involved in different essential plastid functions, suggesting positive selection during evolution of Capsicum plastomes. Finally, the identified sequence variation allowed us to develop simple PCR-based markers useful in future work to discriminate species belonging to different Capsicum complexes.

The complete plastid genomes of Ophrys iricolor and O. sphegodes (Orchidaceae) and comparative analyses with other orchids.

Sexually deceptive orchids of the genus Ophrys may rapidly evolve by adaptation to pollinators. However, understanding of the genetic basis of potential changes and patterns of relationships is hampered by a lack of genomic information. We report the complete plastid genome sequences of Ophrys iricolor and O. sphegodes, representing the two most species-rich lineages of the genus Ophrys. Both plastomes are circular DNA molecules (146754 bp for O. sphegodes and 150177 bp for O. iricolor) with the typical quadripartite structure of plastid genomes and within the average size of photosynthetic orchids. 213 Simple Sequence Repeats (SSRs) (31.5% polymorphic between O. iricolor and O. sphegodes) were identified, with homopolymers and dipolymers as the most common repeat types. SSRs were mainly located in intergenic regions but SSRs located in coding regions were also found, mainly in ycf1 and rpoC2 genes. The Ophrys plastome is predicted to encode 107 distinct genes, 17 of which are completely duplicated in the Inverted Repeat regions. 83 and 87 putative RNA editing sites were detected in 25 plastid genes of the two Ophrys species, all occurring in the first or second codon position. Comparing the rate of nonsynonymous (dN) and synonymous (dS) substitutions, 24 genes (including rbcL and ycf1) display signature consistent with positive selection. When compared with other members of the orchid family, the Ophrys plastome has a complete set of 11 functional ndh plastid genes, with the exception of O. sphegodes that has a truncated ndhF gene. Comparative analysis showed a large co-linearity with other related Orchidinae. However, in contrast to O. iricolor and other Orchidinae, O. sphegodes has a shift of the junction between the Inverted Repeat and Small Single Copy regions associated with the loss of the partial duplicated gene ycf1 and the truncation of the ndhF gene. Data on relative genomic coverage and validation by PCR indicate the presence, with a different ratio, of the two plastome types (i.e. with and without ndhF deletion) in both Ophrys species, with a predominance of the deleted type in O. sphegodes. A search for this deleted plastid region in O. sphegodes nuclear genome shows that the deleted region is inserted in a retrotransposon nuclear sequence. The present study provides useful genomic tools for studying conservation and patterns of relationships of this rapidly radiating orchid genus.

Masquerading as pea plants: behavioural and morphological evidence for mimicry of multiple models in an Australian orchid.

Background and Aims: While there is increasing recognition of Batesian floral mimicry in plants, there are few confirmed cases where mimicry involves more than one model species. Here, we test for pollination by mimicry in Diuris (Orchidaceae), a genus hypothesized to attract pollinators via mimicry of a range of co-occurring pea plants (Faboideae). Methods: Observations of pollinator behaviour were made for Diuris brumalis using arrays of orchid flowers. An analysis of floral traits in the co-flowering community and spectral reflectance measurements were undertaken to test if Di. brumalis and the pea plants showed strong similarity and were likely to be perceived as the same by bees. Pollen removal and fruit-set were recorded at 18 sites over two years to test if fitness of Di. brumalis increased with the abundance of the model species. Key Results: Diuris brumalis shares the pollinator species Trichococolletes capillosus and T. leucogenys (Hymenoptera: Colletidae) with co-flowering Faboideae from the genus Daviesia. On Di. brumalis, Trichocolletes exhibited the same stereotyped food-foraging and mate-patrolling behaviour that they exhibit on Daviesia. Diuris and pea plants showed strong morphological similarity compared to the co-flowering plant community, while the spectral reflectance of Diuris was similar to that of Daviesia spp. Fruit-set and pollen removal of Di. brumalis was highest at sites with a greater number of Daviesia flowers. Conclusions: Diuris brumalis is pollinated by mimicry of co-occurring congeneric Faboideae species. Evidence for mimicry of multiple models, all of which share pollinator species, suggests that this may represent a guild mimicry system. Interestingly, Di. brumalis belongs to a complex of species with similar floral traits, suggesting that this represents a useful system for investigating speciation in lineages that employ mimicry of food plants.

The relative contribution of diurnal and nocturnal pollinators to plant female fitness in a specialized nursery pollination system.

Plants involved in specialized pollinator interactions, such as nursery pollination, may experience trade-offs in their female fitness, as the larvae of their pollinators may also consume seeds produced by the flowers they pollinate. These interactions could potentially shift between mutualism and parasitism, depending on the presence and abundance of both the nursery pollinator and of other pollinators. We investigated the fitness trade-off in a Mediterranean plant (Silene latifolia), which has a specialist nocturnal nursery pollinator moth (Hadena bicruris) and is also visited by several diurnal pollinators. We estimated the pollination rates and fecundity of S. latifolia in both natural and experimental populations in the Mediterranean. We estimated natural pollination rates in different flowering times and with presence/absence of the H. bicruis moth. Then by exposing plants to each pollinator group either during the day or at night, we quantified the contribution of other diurnal pollinators and the specialized nocturnal nursery pollinator to plant female fitness. We found no difference in plant fruit set mediated by diurnal versus nocturnal pollinators, indicating that non-specialist pollinators contribute to plant female fitness. However, in both natural and experimental populations, H. bicruris was the most efficient pollinator in terms of seeds produced per fruit. These results suggest that the female fitness costs generated by nursery pollination can be overcome through higher fertilization rates relative to predation rates, even in the presence of co-pollinators. Quantifying such interactions is important for our understanding of the selective pressures that promote highly specialized mutualisms, such as nursery pollination, in the Mediterranean region, a centre of diversification of the carnation family.

Fluctuating selection across years and phenotypic variation in food-deceptive orchids.

Nectarless flowers that deceive pollinators offer an opportunity to study asymmetric plant-insect interactions. Orchids are a widely used model for studying these interactions because they encompass several thousand species adopting deceptive pollination systems. High levels of intra-specific phenotypic variation have been reported in deceptive orchids, suggesting a reduced consistency of pollinator-mediated selection on their floral traits. Nevertheless, several studies report on widespread directional selection mediated by pollinators even in these deceptive orchids. In this study we test the hypothesis that the observed selection can fluctuate across years in strength and direction thus likely contributing to the phenotypic variability of this orchid group. We performed a three-year study estimating selection differentials and selection gradients for nine phenotypic traits involved in insect attraction in two Mediterranean orchid species, namely Orchis mascula and O. pauciflora, both relying on a well-described food-deceptive pollination strategy. We found weak directional selection and marginally significant selection gradients in the two investigated species with significant intra-specific differences in selection differentials across years. Our data do not link this variation with a specific environmental cause, but our results suggest that pollinator-mediated selection in food-deceptive orchids can change in strength and in direction over time. In perennial plants, such as orchids, different selection differentials in the same populations in different flowering seasons can contribute to the maintenance of phenotypic variation often reported in deceptive orchids.

Floral scent and species divergence in a pair of sexually deceptive orchids.

Speciation is typically accompanied by the formation of isolation barriers between lineages. Commonly, reproductive barriers are separated into pre- and post-zygotic mechanisms that can evolve with different speed. In this study, we measured the strength of different reproductive barriers in two closely related, sympatric orchids of the Ophrys insectifera group, namely Ophrys insectifera and Ophrys aymoninii to infer possible mechanisms of speciation. We quantified pre- and post-pollination barriers through observation of pollen flow, by performing artificial inter- and intraspecific crosses and analyzing scent bouquets. Additionally, we investigated differences in mycorrhizal fungi as a potential extrinsic factor of post-zygotic isolation. Our results show that floral isolation mediated by the attraction of different pollinators acts apparently as the sole reproductive barrier between the two orchid species, with later-acting intrinsic barriers seemingly absent. Also, the two orchids share most of their fungal mycorrhizal partners in sympatry, suggesting little or no importance of mycorrhizal symbiosis in reproductive isolation. Key traits underlying floral isolation were two alkenes and wax ester, present predominantly in the floral scent of O. aymoninii. These compounds, when applied to flowers of O. insectifera, triggered attraction and a copulation attempt of the bee pollinator of O. aymoninii and thus led to the (partial) breakdown of floral isolation. Based on our results, we suggest that adaptation to different pollinators, mediated by floral scent, underlies species isolation in this plant group. Pollinator switches may be promoted by low pollination success of individuals in dense patches of plants, an assumption that we also confirmed in our study.