Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids

Floral isolation is an important component of pollinator-driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators' mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F(1) hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.     

Associations between floral specialization and species diversity: cause, effect, or correlation?

It has been proposed frequently, from Darwin’s time onwards, that specialized pollination increases speciation rates and thus the diversity of plant species (i.e. clade species richness). We suggest here that the correlation between clade species richness and floral specialization is real, but that clade species richness is frequently the cause, not the result of floral specialization. We urge a broader, variance-partitioning perspective for assessing the causes of this correlation by suggesting four models of how the diversity-specialization correlation might come about: (1) floral specialization promotes initial reproductive isolation (“Initial-RI” model), (2) floral specialization promotes reinforcement of reproductive isolation upon secondary contact (“Reinforcement” model), (3) floral specialization reduces the extinction rate by promoting tighter species packing (“Extinction” model), (4) floral specialization is the result of high clade species richness, which increases the number of related species in communities, and thus selects for floral character displacement (“Character-Displacement” model). These hypotheses are evaluated by comparing the relationships between species richness, speciation mechanisms, and pollination precision, accuracy, and specialization in the broader literature and, more specifically, in four study systems: Dalechampia (Euphorbiaceae), Collinsia (Plantaginaceae), Burmeistera (Campanulaceae), and Stylidium (Stylidiaceae). These systems provide stronger support for the character-displacement hypothesis, wherein local species diversity drives the evolution of specialized pollination. Although the two reproductive-isolation hypotheses may hold for plants like orchids, with extremely precise pollination systems, the reproductive character-displacement hypothesis seems likely to be more important for plant groups with less precise pollination systems.     

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.     

Orchid diversity: an evolutionary consequence of deception?

The Orchidaceae are one of the most species-rich plant families and their floral diversity and pollination biology have long intrigued evolutionary biologists. About one-third of the estimated 18,500 species are thought to be pollinated by deceit. To date, the focus has been on how such pollination evolved, how the different types of deception work, and how it is maintained, but little progress has been made in understanding its evolutionary consequences. To address this issue, we discuss here how deception affects orchid mating systems, the evolution of reproductive isolation, speciation processes and neutral genetic divergence among species. We argue that pollination by deceit is one of the keys to orchid floral and species diversity. A better understanding of its evolutionary consequences could help evolutionary biologists to unravel the reasons for the evolutionary success of orchids.     

Is floral divergence sufficient to maintain species boundaries upon secondary contact in Mediterranean food-deceptive orchids?

Analyzing the processes that determine whether species boundaries are maintained on secondary contact may shed light on the early phase of speciation. In Anacamptis morio and Anacamptis longicornu, two Mediterranean orchid sister-species, we used molecular and morphological analyses, together with estimates of pollination success and experimental crosses, to assess whether floral isolation can shelter the species' genomes from genetic admixture on secondary contact. We found substantial genetic and morphological homogenization in sympatric populations in combination with an apparent lack of postmating isolation. We further detected asymmetric introgression in the sympatric populations and an imbalance in cytotype representation, which may be due either to a difference in flowering phenology or else be a consequence of cytonuclear incompatibilities. Estimates of genetic clines for markers across sympatric zones revealed markers that significantly deviated from neutral expectations. We observed a significant correlation between spur length and reproductive success in sympatric populations, which may suggest that directional selection is the main cause of morphological differentiation in this species pair. Our results suggest that allopatric divergence has not led to the evolution of sufficient reproductive isolation to prevent genomic admixture on secondary contact in this orchid species pair.     

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.     

Speciation in the Orchidaceae: confronting the challenges

The Orchidaceae is renowned for its large number of species (19,500) and its many diverse, even bizarre, specialized pollination systems. One unusual feature of orchids is the high frequency of food deception whereby animal pollination is achieved without providing nectar, pollen or other food rewards. Food-deceptive pollination is estimated to occur in approximately one-third of all orchids. Equally intriguing is pollination by sexual deception whereby pollination is achieved by the sexual attraction of male insects to the orchid flower. Sexual deception is found in several hundred species representing multiple lineages. Given their rich species diversity and extraordinary plant-animal interactions, orchids clearly offer exciting research opportunities in pollination biology, reproductive isolation and speciation, yet surprisingly they remain under-represented in scientific investigations both in these fields and more generally. In this special issue of Molecular Ecology, Moccia et al. provide an exemplar study that combine multiple lines of evidence to illuminate the mechanism of reproductive isolation between two closely related food-deceptive orchids. Their study demonstrates that many of the challenges that confront orchid researchers and impede progress in our understanding of speciation in the Orchidaceae can be overcome by the creative application and integration of both old and new tools in ecology and genetics.     

An informational diversity framework,illustrated with sexually deceptive orchids in early stages of speciation

Reconstructing evolutionary history for emerging species complexes is notoriously difficult, with newly isolated taxa often morphologically cryptic and the signature of reproductive isolation often restricted to a few genes. Evidence from multiple loci and genomes is highly desirable, but multiple inputs require ‘common currency’ translation. Here we deploy a Shannon information framework, converting into diversity analogue, which provides a common currency analysis for maternally inherited haploid and bi‐parentally inherited diploid nuclear markers, and then extend that analysis to construction of minimum‐spanning networks for both genomes. The new approach is illustrated with a quartet of cryptic congeners from the sexually deceptive Australian orchid genus Chiloglottis, still in the early stages of speciation. Divergence is more rapid for haploid plastids than for nuclear markers, consistent with the effective population size differential (N_ep < N_en), but divergence patterns are broadly correlated for the two genomes. There are nevertheless intriguing discrepancies between the emerging plastid and nuclear signals of early phylogenetic radiation of these taxa, and neither pattern is entirely consistent with the available information on the sexual cues used by the orchids to lure the pollinators enforcing reproductive isolation. We describe possible extensions of this methodology to multiple ploidy levels and other types of markers, which should increase the range of application to any taxonomic assemblage in the very early stages of reproductive isolation and speciation.     

COMPARATIVE APPROACHES TO THE EVOLUTION OF REPRODUCTIVE ISOLATION: A COMMENT ON SCOPECE ET AL. 2007

Speciation can be driven by the evolution of many forms of reproductive isolation. Comparative study is a powerful approach for elucidating the relative importance of individual isolating barriers in the speciation process. A recent contribution by Scopece and colleagues provides comparative data for two groups of deceptive pollination orchids and aims to test hypotheses about which forms of isolation are most important in the two clades. The authors compare pollinator isolation and postmating isolation between the two orchid groups, and conclude that food-deceptive orchid species have less isolation by pollinator specificity than sexually deceptive species, and that postmating isolation is more important in the food-deceptive clade. Although we find this approach to be novel and potentially powerful, these conclusions are called into question by the methods used to define and select species and quantify pollinator isolation. Definition and selection of taxa were performed in a biased manner that undermines the ability to infer general patterns of speciation. Furthermore, pollinator isolation was calculated inconsistently for the two groups under study, effectively nullifying the comparison.     

Effects of distance from models on the fitness of floral mimics

• Rewardless plants can attract pollinators by mimicking floral traits of rewarding heterospecific plants. This should result in the pollination success of floral mimics being dependent on the relative abundance of their models, as pollinator abundance and conditioning on model signals should be higher in the vicinity of the models. However, the attraction of pollinators to signals of the models may be partially innate, such that spatial isolation of mimics from model species may not strongly affect pollination success of mimics.
• We tested whether pollination rates and fruit set of the rewardless orchid Disa pulchra were influenced by proximity and abundance of its rewarding model species, Watsonia lepida.
• Pollination success of the orchid increased with proximity to the model species, while fruit set of the orchid increased with local abundance of the model species. Orchids that were experimentally translocated outside the model population experienced reduced pollinaria removal and increased pollinator‐mediated self‐pollination.
• These results confirm predictions that the pollination success of floral mimics should be dependent on the proximity and abundance of model taxa, and thus highlight the importance of ecological facilitation among species involved in mimicry systems.

     

Divergent selection on flowering phenology but not on floral morphology between two closely related orchids

Closely related species often differ in traits that influence reproductive success, suggesting that divergent selection on such traits contribute to the maintenance of species boundaries. Gymnadenia conopsea ss. and Gymnadenia densiflora are two closely related, perennial orchid species that differ in (a) floral traits important for pollination, including flowering phenology, floral display, and spur length, and (b) dominant pollinators. If plant–pollinator interactions contribute to the maintenance of trait differences between these two taxa, we expect current divergent selection on flowering phenology and floral morphology between the two species. We quantified phenotypic selection via female fitness in one year on flowering start, three floral display traits (plant height, number of flowers, and corolla size) and spur length, in six populations of G. conopsea s.s. and in four populations of G. densiflora. There was indication of divergent selection on flowering start in the expected direction, with selection for earlier flowering in two populations of the early‐flowering G. conopsea s.s. and for later flowering in one population of the late‐flowering G. densiflora. No divergent selection on floral morphology was detected, and there was no significant stabilizing selection on any trait in the two species. The results suggest ongoing adaptive differentiation of flowering phenology, strengthening this premating reproductive barrier between the two species. Synthesis: This study is among the first to test whether divergent selection on floral traits contribute to the maintenance of species differences between closely related plants. Phenological isolation confers a substantial potential for reproductive isolation, and divergent selection on flowering time can thus greatly influence reproductive isolation and adaptive differentiation.     

Modeling the two‐locus architecture of divergent pollinator adaptation: how variation in SAD paralogs affects fitness and evolutionary divergence in sexually deceptive orchids

Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n‐alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species‐specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator‐mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two‐locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator‐mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator‐driven ecological speciation.     

Effects of conspecific and heterospecific floral density on the pollination of two related rewarding orchids

Variation in within-population floral density can affect interactions between plants and pollinators, resulting in variable pollen export for plants. We investigated the effects of conspecific and heterospecific floral densities on pollination success both of two related, self-compatible, nectar-rewarding orchid species in Ireland, Spiranthes romanzoffiana (rare and listed as endangered) and its congener, S. spiralis (more abundant and not of conservation concern). Floral densities, insect visitation rates, and orchid pollen transport were recorded in multiple quadrats in four populations of both orchid species over their flowering season. We found that conspecific and heterospecific co-flowering plant density affected pollination in both orchid species. For S. romanzoffiana, higher heterospecific density increased pollen removal. For S. spiralis, higher conspecific visitation increased pollen removal and increased heterospecific density decreased pollen deposition. In addition, increased conspecific density increased pollen deposition in both species. This study shows that plants may interact to facilitate or compete for different components of the pollination process, namely; pollinator attraction, pollen removal and deposition. Such interactions have immediate consequences for endangered plant species, as increases in both conspecific and heterospecific coflowering density may ameliorate the negative effects of rarity on pollination, hence overall reproductive success.     

Patterns of reproductive isolation in Mediterranean deceptive orchids

The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.     

Chemical ecology and pollinator-driven speciation in sexually deceptive orchids

Sexually deceptive orchids mimic females of their pollinator species to attract male insects for pollination. Pollination by sexual deception has independently evolved in European, Australian, South African, and South American orchid taxa. Reproductive isolation is mainly based on pre-mating isolation barriers, the specific attraction of males of a single pollinator species, mostly bees, by mimicking the female species-specific sex-pheromone. However, in rare cases post-mating barriers have been found. Sexually deceptive orchids are ideal candidates for studies of sympatric speciation, because key adaptive traits such as the pollinator-attracting scent are associated with their reproductive success and with pre-mating isolation.During the last two decades several investigations studied processes of ecological speciation in sexually deceptive orchids of Europe and Australia. Using various methods like behavioural experiments, chemical, electrophysiological, and population-genetic analyses it was shown that minor changes in floral odour bouquets might be the driving force for pollinator shifts and speciation events. New pollinators act as an isolation barrier towards other sympatrically occurring species. Hybridization occurs because of similar odour bouquets of species and the overlap of flowering periods. Hybrid speciation can also lead to the displacement of species by the hybrid population, if its reproductive success is higher than that in the parental species.     

Reproductive isolation is mediated by pollen incompatibility in sympatric populations of two Arnebia species

To explore uncertain aspects of the processes that maintain species boundaries, we evaluated contributions of pre‐ and postpollination reproductive isolation mechanisms in sympatric populations of Arnebia guttata and A. szechenyi. For this, we investigated their phylogenetic relationships, traits, microenvironments, pollinator visits, action of natural selection on floral traits, and the outcome of hand pollination between the two species. Phylogenetic analysis indicates that A. szechenyi is a derived species that could be closely related to A. guttata, and both could be diploid species. Arnebia guttata flowers have larger parts than A. szechenyi flowers, but smaller nectar guides. Soil supporting A. szechenyi had higher water contents than soil supporting neighboring populations of A. guttata (in accordance with their geographical distributions). The pollinators shared by the two species preferred A. szechenyi flowers, but interspecific visitations were frequent. We found evidence of conflicting selection pressures on floral tube length, flower diameter and nectar guide size mediated via male fitness, and on flower diameter and floral tube diameter via female fitness. Hand‐pollination experiments indicate complete pollen incompatibility between the two species. Our results suggest that postpollination prezygotic mechanisms are largely responsible for reproductive isolation of sympatric populations of the two Arnebia species.     

Floral scent evolution in the section Pseudophrys: pollinator‐mediated selection or phylogenetic constraints?

• Sexually deceptive orchid species from the Mediterranean genus Ophrys usually interact with one or a few pollinator species by means of specific floral scents. In this study, we investigated the respective role of pollinator‐mediated selection and phylogenetic constraints in the evolution of floral scents in the section Pseudophrys.
• We built a phylogenetic tree of 19 Pseudophrys species based on three nuclear loci; we gathered a dataset on their pollination interactions from the literature and from our own field data; and we extracted and analysed their floral scents using solid phase microextraction and gas chromatography‐mass spectrometry. We then quantified the phylogenetic signal carried by floral scents and investigated the link between plant–pollinator interactions and floral scent composition using phylogenetic comparative methods.
• We confirmed the monophyly of the section Pseudophrys and demonstrated the existence of three main clades within this section. We found that floral scent composition is affected by both phylogenetic relationships among Ophrys species and pollination interactions, with some compounds (especially fatty acid esters) carrying a significant phylogenetic signal and some (especially alkenes and alkadienes) generating dissimilarities between closely related Pseudophrys pollinated by different insects.
• Our results show that in the section Pseudophrys, floral scents are shaped both by pollinator‐mediated selection and by phylogenetic constraints, but that the relative importance of these two evolutionary forces differ among compound classes, probably reflecting distinct selective pressures imposed upon behaviourally active and non‐active compounds.

     

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA

Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen–stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.     

Divergence on floral traits and vertebrate pollinators of two endemic Encholirium bromeliads

Shifts in pollen vectors favour diversification of floral traits, and differences in pollination strategies between congeneric sympatric species can contribute to reproductive isolation. Divergence in flowering phenology and selfing could also reduce interspecific crossing between self‐compatible species. We investigated floral traits and visitation rates of pollinators of two sympatric Encholirium species on rocky outcrops to evaluate whether prior knowledge of floral characters could indicate actual pollinators. Data on flowering phenology, visitation rates and breeding system were used to evaluate reproductive isolation. Flowering phenology overlapped between species, but there were differences in floral characters, nectar volume and concentration. Several hummingbird species visited flowers of both Encholirium spp., but the endemic bat Lonchophylla bokermanni and an unidentified sphingid only visited E. vogelii. Pollination treatments demonstrated that E. heloisae and E. vogelii were partially self‐compatible, with weak pollen limitation to seed set. Herbivores feeding on inflorescences decreased reproductive output of both species, but for E. vogelii the damage was higher. Our results indicate that actual pollinators can be known beforehand through floral traits, in agreement with pollination syndromes stating that a set of floral traits can be associated with the attraction of specific groups of pollinators. Divergence on floral traits and pollinator assemblage indicate that shifts in pollination strategies contribute to reproductive isolation between these Encholirium species, not divergence on flowering phenology or selfing. We suggest that hummingbird pollination might be the ancestral condition in Encholirium and that evolution of bat pollination made a substantial contribution to the diversification of this clade.