BACKGROUND
angiosperms and their insect pollinators have flourished with extraordinary diversity through parallel and successive "explosive" radiations over the past  <dig> million years.
it has been suggested that the intimate relationships between flowering plants and their pollinators have fuelled each other's diversification  <cit>  and led to some of today's textbook cases of pollinator-mediated radiation such as in the polemoniaceae family  <cit> .
the examination of plant-pollinator interactions indicates that discrete floral differences among closely-related plant species can induce assortative pollinator attraction and contribute to reproductive isolation  <cit> .
the origin of floral novelties  can therefore be considered as an important driving force in the diversification of flowering plants.
differences in floral phenotype can be generated by allelic variation at sometimes only one or a few loci  <cit> , yet it has been shown that more "emergent" floral novelties  can also originate via other processes such as polyploidy  <cit>  or hybridisation between sympatric taxa  <cit> .
hybridisation can affect several phenotypic traits and niche dimensions which made this phenomenon and its creative potential a particularly important driving force in angiosperm evolution and diversification  <cit> .
the acquisition of novel combinations of floral traits can help recombinant hybrids invade a vacant pollinator "niche", unexploited by its progenitors, which represents one route to adaptive evolutionary divergence, sympatric establishment and in some instances the origin of new species  <cit> .
the orchid family and its ca.
<dig>  species described to date  represent a particularly attractive group of flowering plants for studies addressing the ecological and evolutionary consequences of hybridisation.
indeed, the unusually high degree of specificity in pollinator attraction  <cit>  and the apparent frequent formation of hybrids between orchid species, genera and even subtribes  <cit>  offer a fertile loam for studies on the role of floral traits, including floral scents, in pollinator attraction.
this is particularly true for some of the species-rich genera of so-called sexually deceptive orchids, like the european genus ophrys, where pollinator attraction is brought about by a form of floral mimicry known as sexual deception.
in this plant-pollinator interaction, a range of male insects  pollinate the flowers during an attempted copulation  on the female decoys on the labellum  <cit> .
although floral colours/contrasts might be important in the detection of the flowers, the major floral attractant in this mimicry system is the floral scent, which mimics the female sex pheromone bouquet of a narrow range of targeted insect species  <cit> .
since sex pheromone communication channels are usually species-specific , most ophrys species are de facto reproductively isolated through the species-specific attraction of only one or a few closely related insect taxa  <cit> .
furthermore, when sympatric ophrys taxa share the same pollinator species, cross-pollination is usually prevented by the attachment of the pollen masses  of the orchid on different body parts of the insect  during pseudocopulation  <cit> .
however, despite the apparent strength of pollinator-mediated reproductive isolation in ophrys  <cit> , a considerable proportion of these orchids that clearly belong to different and diagnosable species do hybridise in nature  <cit> .
hybridisation in ophrys therefore provides unique opportunities for the formation of novel combinations of floral traits, particularly the composition of the floral scent, that can potentially drive shifts in pollinator niches and hence the rapid evolution of reproductive isolation between the hybrids and their sympatric parent species .
in this paper, we investigated the potential of ophrys hybridisation to generate novel combinations of floral traits that could induce a pollinator shift.
we used a combination of comparative chemical analyses of floral scents, genetic analyses of orchid taxa with aflp molecular markers, in situ hand pollinations as well as behavioural bioassays with pollinators to uncover the origin and evolutionary consequences of hybridisation.
specifically, we ask the following questions:  how did the hybrids originate?
;  are there differences in floral scent composition between the parental orchid species and the hybrids?
;  can these floral differences induce a "niche shift" in the hybrids towards the attraction of a "new" pollinator species not exploited by any of the parental orchid species?
RESULTS
behavioural experiments with fresh inflorescences
our observations of pollinator behaviour during pseudocopulations with fresh, unpollinated flowers of the orchid taxa illustrate that the pollinators of each parent taxon systematically initiated copulation attempts on the orchid labellum in the expected position .
however, we have observed that the insects' copulatory activity on the flowers regularly led to changes in their position on the labellum and occasionally to the subsequent uptake of pollinaria when in the alternative position .
the supplementary video material  shows a male c. cunicularius pseudocopulating on the labellum of o. lupercalis, the other parent species, and withdrawing pollinaria on both its head and its abdomen tip during a single visit on the flowers.
behavioural experiments with floral odour extracts
results from our bioassays provide evidence for cross-attraction among orchid taxa towards patrolling males of c. cunicularius and a. nigroaenea .
specifically, we found that for each pollinator, floral odour extracts of the most commonly associated ophrys taxa  were more attractive than floral odour extracts of the other parent species.
the results from these bioassays further show that the floral odour extracts of hybrids triggered significantly less inspecting flights and contacts than those of the two parental species , except for the bioassays with a. nigroaenea where no significant difference in attractiveness was found  between the attractiveness of male bees towards floral odour extracts of the hybrids and of o. arachnitiformis .
by performing bioassays with males of a. vaga, a species not initially involved in the pollination of the parent ophrys species, we found that the floral odour of the hybrids triggered significantly more approaching flights to and contacts with the odour source than the floral odour of any of the two parents  .
floral odour differentiation - hybrids and their progenitors
our analyses of floral odour extracts of each orchid taxa in the hybrid zone have detected the presence of  <dig> individual compounds in total, including the biologically active compounds identified for a. nigroaenea, the pollinator of o. lupercalis  <cit> , and for c. cunicularius, the pollinator of o. arachnitiformis  .
a comparison of the number of floral odour compounds found in each taxon is provided in table  <dig>  with a total of  <dig> odour compounds, the hybrids produce more odour compounds  in their floral odour than any of their parent species.
we also found some compounds to be taxon-specific, in particular  <dig> "new" odour compounds produced exclusively by the hybrids whose gc-ms spectra indicate that they are straight-chained alkenes  of  <dig> and  <dig> carbon chain length, respectively.
summary of floral odour compound production in each orchid taxon of the hybrid zone investigated.
a total of  <dig> odour compounds have been identified,  <dig>  of which were produced by all three taxa simultaneously.
a canonical discriminant function  analysis of odour compounds resolved the hybrids and their progenitor species into non-overlapping groups .
this cdf analysis rejects the null hypothesis of homogeneity of covariance matrices .
the high discriminatory ability of the canonical discriminant functions  <dig> and  <dig> plotted in figure  <dig> provide evidence for the importance of the independent variables  to the discriminant analysis.
canonical correlation values close to  <dig>  associated with the two cdf's plotted further account for the significant contribution of the first two canonical discriminant functions to the resolving of all three orchid taxa into non-overlapping groups.
the cdf's  <dig> and  <dig> account for 100%  of the total odour variance among orchid taxa, which further indicates their great discriminatory ability in the model .
overall,  <dig> % of all cross-validated samples were assigned correctly to their taxa by the two cdf's .
the analysis of the partitioning of the floral odour variance yielded a significantly higher proportion of the variance among taxa  over within taxa .
the analysis of overall floral odour similarity  among samples was performed using unweighted pair groups with arithmetic averages .
our results show that, besides the fact that all but one sample  grouped together according to taxa, the floral odour of the hybrids is chemically asymmetric towards o. lupercalis.
the upgma cladogram revealed two discrete clusters, the first one comprising all the samples of o. arachnitiformis  and the second consisting of two subclusters containing o. lupercalis and the hybrids .
molecular hybrid index scores
the eight primer pair combinations used for aflp analysis produced a total of  <dig> polymorphic markers.
using the 100% difference criterion, we identified  <dig> markers as species-specific for ophrys arachnitiformis and o. lupercalis.
of these,  <dig> were present as bands in o. arachnitiformis and  <dig> in o. lupercalis.
the hybrid index analysis to allocated individuals to the specific hybrid class with high confidence .
the hybrid index analysis based on species-specific markers revealed exactly the same pattern of hybrid index analysis calculated with all polymorphic markers .
the mean ml hybrid index ) for hybrid individuals was  <dig>   with diagnostic species-specific markers.
we found no evidence for patterns of genetic variation suggestive of gene introgression into parental classes in the hybrid zone investigated .
crossing experiments and flow cytometry analyses
none of the hand-pollinations performed between the hybrids and the parents resulted in capsule formation.
hand-pollinations between hybrids yielded only one capsule that contained few seeds that were devoid of embryo, which we therefore considered unviable.
we did not check whether the pollen tubes made it all the way to the ovules in the back-crosses and the f1xf <dig> crosses.
the flow cytometry profiles produced consistent results within taxa and revealed differences in ploidy level among the ophrys species investigated.
our analyses of pollinia and leaf fragments showed that o. bilunulata , o. sphegodes  and o. arachnitiformis  have the same ploidy level, namely that they are all diploid .
the samples of o. lupercalis, however, contained twice as much dna as those of the aforementioned species, which suggests that all the o. lupercalis individuals investigated in the hybrid zone are tetraploid .
finally, the analyses of dna contents of both pollinia and leaf fragments of the hybrids revealed a ploidy level intermediate to that of the two parents, which supports the scenario that the hybrids sampled in the hybrid zone are triploid .
discussion
the origin of ophrys hybrids
our study provided evidence for the occasional breakdown of both ethological and mechanical isolation barriers mediated by the pollinators, which provides opportunities for the formation of hybrids between o. arachnitiformis and o. lupercalis when they are found in sympatry.
the cross-attraction of each parent orchid species to each pollinator was allowed by the emission of  overlapping patterns of biologically-active compounds for each pollinator species .
it is generally assumed that sexually deceptive orchids are strongly isolated via pre-pollination barriers, and more weakly through post-zygotic  mechanisms, which contrasts to the situation observed in food deceptive orchids that have weak pre-pollination  <cit>  but strong post-zygotic isolation barriers  <cit> .
yet in this study, we have been able to show that pre-pollination  isolation barriers, which are generally thought to represent strong isolation mechanisms in flowering plants, especially when they act in concert , could be more permeable than previously thought in the genus ophrys .
many species of solitary bees that act as pollinators in ophrys use specific female sex pheromone that are often based on a "variation on a theme", i.e.
identical compounds in different ratios  <cit> .
this phenomenon enhances the probability for an ophrys species to be cross-attractive to different pollinators, even though the flowers might receive significantly more visits by their most commonly associated pollinator .
hence, reproductive isolation is usually maintained but does not prevent opportunities for gene flow between sympatric species, at least theoretically.
besides, the attractiveness of orchids to alternative pollinators observed in o. arachnitiformis and o. lupercalis  might enhance the reproductive output of these orchids at a local scale, a mechanism that should be favoured by selection since orchids in general and deceptive ones in particular are often pollinator-limited in their reproductive success  <cit> .
hybrid floral novelty and pollinator shift
a prerequisite for adaptive evolutionary divergence of hybrids is the invasion of an alternative "niche" in which the hybrids are subjected to different selection pressures, and the parallel evolution of reproductive isolation between the parents and the hybrids.
the results from our analyses and bioassays support these two requirements, first because we have shown that the f <dig> hybrids emit "emergent" combinations of floral scent compouds, i.e.
they are not intermediate between their parents or have the sum of their parents' traits, but instead they have developed an "emergent" floral scent novelty with completely new compounds that are not predictable from the parents' phenotypes.
second, this floral scent novelty leads to the acquisition of a novel pollinator "niche" by hybrids  that can drive the rapid evolution of reproductive isolation between the hybrids and their parents .
these data are consistent with previous studies reporting that even small changes in floral scent chemistry in ophrys have the potential to mediate assortative pollinator attraction  <cit>  and hence promote rapid reproductive isolation between diverging sympatric orchid taxa if maintained over generations  <cit> .
furthermore, our results challenge the view that hybrids often experience reduced fitness compared to their parents  <cit>  by showing that they can also outcompete their parents under certain ecological conditions .
finally, the last major step towards fully autonomous establishment requires the hybrids to be fertile  <cit> .
here, none of our bidirectional and controlled hand-pollinations resulted in capsule formation, and hand-pollinations within the group of hybrids yielded only one capsule that contained seeds devoid of embryo, which suggests that the hybrids tested here between o. arachnitiformis and o. lupercalis are both maternally and paternally sterile, a likely consequence of their triploidy.
although the triploid hybrids investigated have been shown to be sterile, their evolutionary potential should not be dismissed a priori.
indeed, studies on allotriploids obtained via diploid and tetraploid parent species indicate that many of the gametes produced by triploids are not functional, because they possess aneuploid, unbalanced chromosome numbers.
however, triploids may occasionally generate small numbers of euploid  gametes and they can also produce 3x gametes via non-reduction .
this may enable allotriploids to produce fertile allotetraploids by selfing or backcrossing with diploid parents  <cit>  without otherwise significant changes in the genomic architecture of the polyploid individuals  <cit> .
several studies have shown that massive expression changes often accompany polyploid formation but autopolyploidy might produce less dramatic expression changes than allopolyploidy.
hybridisation is likely to cause more dramatic phenotypic changes than genome doubling per se because it results in transcriptional effects following the combination of differentiated genomes, with their divergent regulatory machinery, into a common nucleus .
if filled, these requirements could potentially open up a new route to the emergence of a hybrid neospecies through the attraction of a novel pollinator, provided potential pollinators are present locally.
this scenario could also help explain the occurrence of several endemic species of ophrys frequently characterised by a tetraploid genome  <cit> .
the parental species pair investigated in this study turned out to have different ploidy levels, yet reports indicate that o. lupercalis might be diploid in other regions of the mediterranean basin  <cit> .
these contact sites between o. arachnitiformis and o. lupercalis should be investigated by using the present study as a conceptual touchstone to ultimately determine whether or not the local hybrids are at least partially fertile.
other sympatric species of ophrys that have the same ploidy level are known to produce hybrids that set seed when they are experimentally pollinated with pollinia from other sympatric hybrids  <cit> .
among these hybrids, several have been reported to have a significantly different floral odour bouquet compared to their parents  <cit> .
our results should therefore encourage investigations in these hybrid zones to test the extent to which hybrids can escape the ecological niche of their parents through the attraction of pollinators that are not involved in the pollination of their parents  <cit> .
CONCLUSIONS
in this study, we have found evidence that hybridisation in sexually deceptive orchids can produce novel combinations of floral traits, particularly their floral odour , that can in turn lead to a pollinator shift .
however, the development of the hybrids under study into novel "ecological" species is hindered by their sterility, which is also expected to impede the formation of later-generation progeny via selfing, sib-crossing with neighbouring hybrid individuals, or back-crossing with any of the sympatric parental species.
yet, although several assumptions are not met for making plausible ecological speciation by hybridisation in the hybrids between o. arachnitiformis and o. lupercalis, our study provides a unique window into the stepwise process by which apparent reproductive barriers can be broken down and how new combinations of floral traits can be generated, leading to the evolution of novel, highly specific plant-pollinator interactions.

 angiosperms and their insect pollinators have flourished with extraordinary diversity through parallel and successive "explosive" radiations over the past million years. it has been suggested that the intimate relationships between flowering plants and their pollinators have fuelled each other's diversification and led to some of today's textbook cases of pollinator-mediated radiation such as in the polemoniaceae family. the examination of plant-pollinator interactions indicates that discrete floral differences among closely-related plant species can induce assortative pollinator attraction and contribute to reproductive isolation. the origin of floral novelties can therefore be considered as an important driving force in the diversification of flowering plants. differences in floral phenotype can be generated by allelic variation at sometimes only one or a few loci, yet it has been shown that more "emergent" floral novelties can also originate via other processes such as polyploidy or hybridisation between sympatric taxa. hybridisation can affect several phenotypic traits and niche dimensions which made this phenomenon and its creative potential a particularly important driving force in angiosperm evolution and diversification. the acquisition of novel combinations of floral traits can help recombinant hybrids invade a vacant pollinator "niche", unexploited by its progenitors, which represents one route to adaptive evolutionary divergence, sympatric establishment and in some instances the origin of new species. the orchid family and its ca. species described to date represent a particularly attractive group of flowering plants for studies addressing the ecological and evolutionary consequences of hybridisation. indeed, the unusually high degree of specificity in pollinator attraction and the apparent frequent formation of hybrids between orchid species, genera and even subtribes offer a fertile loam for studies on the role of floral traits, including floral scents, in pollinator attraction. this is particularly true for some of the species-rich genera of so-called sexually deceptive orchids, like the european genus ophrys, where pollinator attraction is brought about by a form of floral mimicry known as sexual deception. in this plant-pollinator interaction, a range of male insects pollinate the flowers during an attempted copulation on the female decoys on the labellum. although floral colours/contrasts might be important in the detection of the flowers, the major floral attractant in this mimicry system is the floral scent, which mimics the female sex pheromone bouquet of a narrow range of targeted insect species. since sex pheromone communication channels are usually species-specific, most ophrys species are de facto reproductively isolated through the species-specific attraction of only one or a few closely related insect taxa. furthermore, when sympatric ophrys taxa share the same pollinator species, cross-pollination is usually prevented by the attachment of the pollen masses of the orchid on different body parts of the insect during pseudocopulation. however, despite the apparent strength of pollinator-mediated reproductive isolation in ophrys, a considerable proportion of these orchids that clearly belong to different and diagnosable species do hybridise in nature. hybridisation in ophrys therefore provides unique opportunities for the formation of novel combinations of floral traits, particularly the composition of the floral scent, that can potentially drive shifts in pollinator niches and hence the rapid evolution of reproductive isolation between the hybrids and their sympatric parent species. in this paper, we investigated the potential of ophrys hybridisation to generate novel combinations of floral traits that could induce a pollinator shift. we used a combination of comparative chemical analyses of floral scents, genetic analyses of orchid taxa with aflp molecular markers, in situ hand pollinations as well as behavioural bioassays with pollinators to uncover the origin and evolutionary consequences of hybridisation. specifically, we ask the following questions: how did the hybrids originate?; are there differences in floral scent composition between the parental orchid species and the hybrids?; can these floral differences induce a "niche shift" in the hybrids towards the attraction of a "new" pollinator species not exploited by any of the parental orchid species?  behavioural experiments with fresh inflorescences our observations of pollinator behaviour during pseudocopulations with fresh, unpollinated flowers of the orchid taxa illustrate that the pollinators of each parent taxon systematically initiated copulation attempts on the orchid labellum in the expected position. however, we have observed that the insects' copulatory activity on the flowers regularly led to changes in their position on the labellum and occasionally to the subsequent uptake of pollinaria when in the alternative position. the supplementary video material shows a male c. cunicularius pseudocopulating on the labellum of o. lupercalis, the other parent species, and withdrawing pollinaria on both its head and its abdomen tip during a single visit on the flowers. behavioural experiments with floral odour extracts results from our bioassays provide evidence for cross-attraction among orchid taxa towards patrolling males of c. cunicularius and a. nigroaenea. specifically, we found that for each pollinator, floral odour extracts of the most commonly associated ophrys taxa were more attractive than floral odour extracts of the other parent species. the results from these bioassays further show that the floral odour extracts of hybrids triggered significantly less inspecting flights and contacts than those of the two parental species, except for the bioassays with a. nigroaenea where no significant difference in attractiveness was found between the attractiveness of male bees towards floral odour extracts of the hybrids and of o. arachnitiformis. by performing bioassays with males of a. vaga, a species not initially involved in the pollination of the parent ophrys species, we found that the floral odour of the hybrids triggered significantly more approaching flights to and contacts with the odour source than the floral odour of any of the two parents. floral odour differentiation - hybrids and their progenitors our analyses of floral odour extracts of each orchid taxa in the hybrid zone have detected the presence of individual compounds in total, including the biologically active compounds identified for a. nigroaenea, the pollinator of o. lupercalis, and for c. cunicularius, the pollinator of o. arachnitiformis. a comparison of the number of floral odour compounds found in each taxon is provided in table with a total of odour compounds, the hybrids produce more odour compounds in their floral odour than any of their parent species. we also found some compounds to be taxon-specific, in particular "new" odour compounds produced exclusively by the hybrids whose gc-ms spectra indicate that they are straight-chained alkenes of and carbon chain length, respectively. summary of floral odour compound production in each orchid taxon of the hybrid zone investigated. a total of odour compounds have been identified, of which were produced by all three taxa simultaneously. a canonical discriminant function analysis of odour compounds resolved the hybrids and their progenitor species into non-overlapping groups. this cdf analysis rejects the null hypothesis of homogeneity of covariance matrices. the high discriminatory ability of the canonical discriminant functions and plotted in figure provide evidence for the importance of the independent variables to the discriminant analysis. canonical correlation values close to associated with the two cdf's plotted further account for the significant contribution of the first two canonical discriminant functions to the resolving of all three orchid taxa into non-overlapping groups. the cdf's and account for 100% of the total odour variance among orchid taxa, which further indicates their great discriminatory ability in the model. overall, % of all cross-validated samples were assigned correctly to their taxa by the two cdf's. the analysis of the partitioning of the floral odour variance yielded a significantly higher proportion of the variance among taxa over within taxa. the analysis of overall floral odour similarity among samples was performed using unweighted pair groups with arithmetic averages. our results show that, besides the fact that all but one sample grouped together according to taxa, the floral odour of the hybrids is chemically asymmetric towards o. lupercalis. the upgma cladogram revealed two discrete clusters, the first one comprising all the samples of o. arachnitiformis and the second consisting of two subclusters containing o. lupercalis and the hybrids. molecular hybrid index scores the eight primer pair combinations used for aflp analysis produced a total of polymorphic markers. using the 100% difference criterion, we identified markers as species-specific for ophrys arachnitiformis and o. lupercalis. of these, were present as bands in o. arachnitiformis and in o. lupercalis. the hybrid index analysis to allocated individuals to the specific hybrid class with high confidence. the hybrid index analysis based on species-specific markers revealed exactly the same pattern of hybrid index analysis calculated with all polymorphic markers. the mean ml hybrid index ) for hybrid individuals was with diagnostic species-specific markers. we found no evidence for patterns of genetic variation suggestive of gene introgression into parental classes in the hybrid zone investigated. crossing experiments and flow cytometry analyses none of the hand-pollinations performed between the hybrids and the parents resulted in capsule formation. hand-pollinations between hybrids yielded only one capsule that contained few seeds that were devoid of embryo, which we therefore considered unviable. we did not check whether the pollen tubes made it all the way to the ovules in the back-crosses and the f1xf crosses. the flow cytometry profiles produced consistent results within taxa and revealed differences in ploidy level among the ophrys species investigated. our analyses of pollinia and leaf fragments showed that o. bilunulata, o. sphegodes and o. arachnitiformis have the same ploidy level, namely that they are all diploid. the samples of o. lupercalis, however, contained twice as much dna as those of the aforementioned species, which suggests that all the o. lupercalis individuals investigated in the hybrid zone are tetraploid. finally, the analyses of dna contents of both pollinia and leaf fragments of the hybrids revealed a ploidy level intermediate to that of the two parents, which supports the scenario that the hybrids sampled in the hybrid zone are triploid. discussion the origin of ophrys hybrids our study provided evidence for the occasional breakdown of both ethological and mechanical isolation barriers mediated by the pollinators, which provides opportunities for the formation of hybrids between o. arachnitiformis and o. lupercalis when they are found in sympatry. the cross-attraction of each parent orchid species to each pollinator was allowed by the emission of overlapping patterns of biologically-active compounds for each pollinator species. it is generally assumed that sexually deceptive orchids are strongly isolated via pre-pollination barriers, and more weakly through post-zygotic mechanisms, which contrasts to the situation observed in food deceptive orchids that have weak pre-pollination but strong post-zygotic isolation barriers. yet in this study, we have been able to show that pre-pollination isolation barriers, which are generally thought to represent strong isolation mechanisms in flowering plants, especially when they act in concert, could be more permeable than previously thought in the genus ophrys. many species of solitary bees that act as pollinators in ophrys use specific female sex pheromone that are often based on a "variation on a theme", i.e. identical compounds in different ratios. this phenomenon enhances the probability for an ophrys species to be cross-attractive to different pollinators, even though the flowers might receive significantly more visits by their most commonly associated pollinator. hence, reproductive isolation is usually maintained but does not prevent opportunities for gene flow between sympatric species, at least theoretically. besides, the attractiveness of orchids to alternative pollinators observed in o. arachnitiformis and o. lupercalis might enhance the reproductive output of these orchids at a local scale, a mechanism that should be favoured by selection since orchids in general and deceptive ones in particular are often pollinator-limited in their reproductive success. hybrid floral novelty and pollinator shift a prerequisite for adaptive evolutionary divergence of hybrids is the invasion of an alternative "niche" in which the hybrids are subjected to different selection pressures, and the parallel evolution of reproductive isolation between the parents and the hybrids. the results from our analyses and bioassays support these two requirements, first because we have shown that the f hybrids emit "emergent" combinations of floral scent compouds, i.e. they are not intermediate between their parents or have the sum of their parents' traits, but instead they have developed an "emergent" floral scent novelty with completely new compounds that are not predictable from the parents' phenotypes. second, this floral scent novelty leads to the acquisition of a novel pollinator "niche" by hybrids that can drive the rapid evolution of reproductive isolation between the hybrids and their parents. these data are consistent with previous studies reporting that even small changes in floral scent chemistry in ophrys have the potential to mediate assortative pollinator attraction and hence promote rapid reproductive isolation between diverging sympatric orchid taxa if maintained over generations. furthermore, our results challenge the view that hybrids often experience reduced fitness compared to their parents by showing that they can also outcompete their parents under certain ecological conditions. finally, the last major step towards fully autonomous establishment requires the hybrids to be fertile. here, none of our bidirectional and controlled hand-pollinations resulted in capsule formation, and hand-pollinations within the group of hybrids yielded only one capsule that contained seeds devoid of embryo, which suggests that the hybrids tested here between o. arachnitiformis and o. lupercalis are both maternally and paternally sterile, a likely consequence of their triploidy. although the triploid hybrids investigated have been shown to be sterile, their evolutionary potential should not be dismissed a priori. indeed, studies on allotriploids obtained via diploid and tetraploid parent species indicate that many of the gametes produced by triploids are not functional, because they possess aneuploid, unbalanced chromosome numbers. however, triploids may occasionally generate small numbers of euploid gametes and they can also produce 3x gametes via non-reduction. this may enable allotriploids to produce fertile allotetraploids by selfing or backcrossing with diploid parents without otherwise significant changes in the genomic architecture of the polyploid individuals. several studies have shown that massive expression changes often accompany polyploid formation but autopolyploidy might produce less dramatic expression changes than allopolyploidy. hybridisation is likely to cause more dramatic phenotypic changes than genome doubling per se because it results in transcriptional effects following the combination of differentiated genomes, with their divergent regulatory machinery, into a common nucleus. if filled, these requirements could potentially open up a new route to the emergence of a hybrid neospecies through the attraction of a novel pollinator, provided potential pollinators are present locally. this scenario could also help explain the occurrence of several endemic species of ophrys frequently characterised by a tetraploid genome. the parental species pair investigated in this study turned out to have different ploidy levels, yet reports indicate that o. lupercalis might be diploid in other regions of the mediterranean basin. these contact sites between o. arachnitiformis and o. lupercalis should be investigated by using the present study as a conceptual touchstone to ultimately determine whether or not the local hybrids are at least partially fertile. other sympatric species of ophrys that have the same ploidy level are known to produce hybrids that set seed when they are experimentally pollinated with pollinia from other sympatric hybrids. among these hybrids, several have been reported to have a significantly different floral odour bouquet compared to their parents. our results should therefore encourage investigations in these hybrid zones to test the extent to which hybrids can escape the ecological niche of their parents through the attraction of pollinators that are not involved in the pollination of their parents.  in this study, we have found evidence that hybridisation in sexually deceptive orchids can produce novel combinations of floral traits, particularly their floral odour, that can in turn lead to a pollinator shift. however, the development of the hybrids under study into novel "ecological" species is hindered by their sterility, which is also expected to impede the formation of later-generation progeny via selfing, sib-crossing with neighbouring hybrid individuals, or back-crossing with any of the sympatric parental species. yet, although several assumptions are not met for making plausible ecological speciation by hybridisation in the hybrids between o. arachnitiformis and o. lupercalis, our study provides a unique window into the stepwise process by which apparent reproductive barriers can be broken down and how new combinations of floral traits can be generated, leading to the evolution of novel, highly specific plant-pollinator interactions. 
collectively, our results illustrate that the process of hybridisation can lead to the generation of evolutionary novelties, and that novel combinations of floral traits can drive pollinator shifts and rapid reproductive isolation in highly specific plant-pollinator interactions. our chemical analyses of floral scents show that the ophrys f hybrids investigated here produce more compounds, significantly different ratios, as well as new compounds in their floral odour compared to their progenitors. in particular, we have investigated the extent to which these ophrys hybrids generate "emergent" combinations of floral traits, and how these phenotypic novelties, particularly the odour blends emitted by the flower, could facilitate the invasion of a novel pollinator "niche" and induce the rapid formation of reproductive isolation, a prerequisite for adaptive evolutionary divergence. when tested for their attractiveness to the pollinator of each parent orchid species, we found that floral scent extracts of the hybrids triggered less inspecting flights and contacts by the male bees with the scented dummy than those of the parental orchid species. sexually deceptive orchids of the genus ophrys attract their pollinators, male insects, on a highly specific basis through the emission of odour blends that mimic the female sex pheromone of the targeted species. however, a series of additional behavioural bioassays revealed that the novel floral scent of the hybrids was significantly more attractive than either of the two parents to a pollinator species not initially involved in the pollination of any of the parent ophrys species. in this study, we have investigated a contact site between ophrys arachnitiformis and o. lupercalis, two sympatric orchid species that are usually reproductively isolated via the exploitation of different pollinator "niches", but occasionally hybridise despite their apparent combination of ethological and mechanical isolation barriers.
 sexually deceptive orchids of the genus ophrys attract their pollinators, male insects, on a highly specific basis through the emission of odour blends that mimic the female sex pheromone of the targeted species. in this study, we have investigated a contact site between ophrys arachnitiformis and o. lupercalis, two sympatric orchid species that are usually reproductively isolated via the exploitation of different pollinator "niches", but occasionally hybridise despite their apparent combination of ethological and mechanical isolation barriers. in particular, we have investigated the extent to which these ophrys hybrids generate "emergent" combinations of floral traits, and how these phenotypic novelties, particularly the odour blends emitted by the flower, could facilitate the invasion of a novel pollinator "niche" and induce the rapid formation of reproductive isolation, a prerequisite for adaptive evolutionary divergence.  our chemical analyses of floral scents show that the ophrys f hybrids investigated here produce more compounds, significantly different ratios, as well as new compounds in their floral odour compared to their progenitors. when tested for their attractiveness to the pollinator of each parent orchid species, we found that floral scent extracts of the hybrids triggered less inspecting flights and contacts by the male bees with the scented dummy than those of the parental orchid species. however, a series of additional behavioural bioassays revealed that the novel floral scent of the hybrids was significantly more attractive than either of the two parents to a pollinator species not initially involved in the pollination of any of the parent ophrys species.  collectively, our results illustrate that the process of hybridisation can lead to the generation of evolutionary novelties, and that novel combinations of floral traits can drive pollinator shifts and rapid reproductive isolation in highly specific plant-pollinator interactions. 