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.     

Convergence beyond flower morphology? Reproductive biology of hummingbird‐pollinated plants in the Brazilian Cerrado

Convergent reproductive traits in non‐related plants may be the result of similar environmental conditions and/or specialised interactions with pollinators. Here, we documented the pollination and reproductive biology of Bionia coriacea (Fabaceae), Esterhazya splendida (Orobanchaceae) and Ananas ananassoides (Bromeliaceae) as case studies in the context of hummingbird pollination in Cerrado, the Neotropical savanna of Central South America. We combined our results with a survey of hummingbird pollination studies in the region to investigate the recently suggested association of hummingbird pollination and self‐compatibility. Plant species studied here differed in their specialisation for ornithophily, from more generalist A. ananassoides to somewhat specialist B. coriacea and E. splendida. This continuum of specialisation in floral traits also translated into floral visitor composition. Amazilia fimbriata was the most frequent pollinator for all species, and the differences in floral display and nectar energy availability among plant species affect hummingbirds' behaviour. Most of the hummingbird‐pollinated Cerrado plants (60.0%, n = 20), including those studied here, were self‐incompatible, in contrast to other biomes in the Neotropics. Association to more generalist, often territorial, hummingbirds, and resulting reduced pollen flow in open savanna areas may explain predominance of self‐incompatibility. But it is possible that mating system is more associated with the predominance of woody hummingbird plants in the Cerrado plant assemblage than to the pollination system itself.     

Pollination ecology and breeding systems of five Gesneria species from Puerto Rico

Background and Aims

The genus Gesneria diversified in the Greater Antilles giving rise to various floral designs corresponding to different pollination syndromes. The goal of this study was to characterize the pollination and breeding systems of five Puerto Rican Gesneria species.

Methods

The study was conducted in Arecibo and El Yunke National Forest, Puerto Rico, between 2003 and 2007. Floral visitors were documented by human observers and video cameras. Floral longevity and nectar production were recorded for the five study species. Tests for self-compatibility and autonomous selfing were conducted through hand-pollination and bagging experiments.

Key Results

Floral phenology and nectar production schedules agree with nocturnal (in bell-shaped flowered G. pedunculosa and G. viridiflora subsp. sintenisii) or diurnal pollination syndromes (in tubular-flowered G. citrina, G. cuneifolia and G. reticulata). Nectar concentration is consistently low (8–13 %) across species. Gesneria citrina and G. cuneifolia are exclusively pollinated by hummingbirds, while Gesneria reticulata relies mostly on autonomous self-pollination, despite having classic ornithophilous flowers. A variety of floral visitors was recorded for the two species with bell-shaped flowers; however, not all visitors have the ability to transfer pollen. Bats are the primary pollinators of G. pedunculosa, with bananaquits probably acting as secondary pollinators. For G. viridiflora subsp. sintenisii, both bats and hummingbirds contact the flower''s reproductive organs, thus, this species is considered to be a generalist despite its nocturnal floral syndrome. All species are self-compatible but only tubular-flowered Gesneria are capable of autonomous self-pollination.

Conclusions

The visitation patterns described in this study fit the predicted hummingbird and bat pollination syndromes and support both specialization and generalization of pollination systems in Puerto Rican Gesneria. Specialization is associated with low pollinator visitation, particularly by hummingbirds, which may explain the occurrence of autonomous selfing mechanisms in tubular-flowered species.Key words: Autonomous selfing, bat pollination, breeding systems, Gesneria, hummingbird pollination, Puerto Rico     

Repeated evolution of vertebrate pollination syndromes in a recently diverged Andean plant clade

Although specialized interactions, including those involving plants and their pollinators, are often invoked to explain high species diversity, they are rarely explored at macroevolutionary scales. We investigate the dynamic evolution of hummingbird and bat pollination syndromes in the centropogonid clade (Lobelioideae: Campanulaceae), an Andean‐centered group of ∼550 angiosperm species. We demonstrate that flowers hypothesized to be adapted to different pollinators based on flower color fall into distinct regions of morphospace, and this is validated by morphology of species with known pollinators. This supports the existence of pollination syndromes in the centropogonids, an idea corroborated by ecological studies. We further demonstrate that hummingbird pollination is ancestral, and that bat pollination has evolved ∼13 times independently, with ∼11 reversals. This convergence is associated with correlated evolution of floral traits within selective regimes corresponding to pollination syndrome. Collectively, our results suggest that floral morphological diversity is extremely labile, likely resulting from selection imposed by pollinators. Finally, even though this clade's rapid diversification is partially attributed to their association with vertebrate pollinators, we detect no difference in diversification rates between hummingbird‐ and bat‐pollinated lineages. Our study demonstrates the utility of pollination syndromes as a proxy for ecological relationships in macroevolutionary studies of certain species‐rich clades.     

Reproductive biology of a hummingbird‐pollinated Billbergia: light influence on pollinator behaviour and specificity in a Brazilian semi‐deciduous forest

• Ornithophily has evolved in parallel several times during evolution of angiosperms. Bird pollination is reported for 65 families, including Bromeliaceae. One of the most diverse bromeliad is Billbergia, which comprises species pollinated mainly by hummingbirds.
• Based on investigations on flowering phenology, morpho‐anatomy, volume and concentration of nectar, pollinators and breeding system, this paper explores the reproductive biology and pollinator specificity of B. distachia in a mesophytic semi‐deciduous forest of southeastern Brazil.
• The results have show that B. distachia is pollinated by a single species of hermit hummingbird, Phaethornis eurynome, which search for nectar produced by a septal nectary, where the secretory tissue is located above the placenta. The species is self‐incompatible. The combination of pollinator specificity, due to long corolla tubes that exclude visitation of short‐billed hummingbirds, complete self‐incompatibility and non‐territorial behaviour of pollinators, it is very important to reduce pollen loss and increase gene flow within population.
• Our results indicate that studies on pollination biology and reproduction are essential to understand the evolutionary history of pollination systems of plants since, at least in Billbergia, variation in the pollinator spectrum has been recorded for different habitats among Brazilian forests. Furthermore, according to our data, foraging of Phaethornis on flowers is independent of air temperature and humidity, while the main factor influencing hummingbird visitation is daylight. Considering current knowledge on climatic parameters influencing hummingbird foraging, pollination and reproductive biology of Neotropical flora and environment of the hermit hummingbird in tropical forests, new insights on plant–pollinator interaction are provided.

     

同域分布的紫堇属三种植物的繁育系统和传粉差异

为研究近缘物种之间繁育系统分化对传粉环境的适应性意义,本文针对湖北地区的3种紫堇属植物（紫堇Corydalis edulis Maxim.、尖距紫堇C.shearer S.Moore.和小花黄堇C.racemosa（Thunb.） Pers.）进行了传粉生态学研究,对比分析了它们在花部特征、分布模式、花期、交配系统、传粉系统等方面的差异。3种紫堇属植物常见伴生现象,花期有一定重叠;花色、距长、花蜜量等方面均有显著差异;尖距紫堇交配系统为自交不亲和、依靠传粉者异花授粉;而紫堇和小花黄堇交配系统为自交亲和,兼有自交和异交的混合交配系统。3个物种均由蜂类传粉,且花部性器官与传粉者的接触部位一致,但访花频率差异较大。在混合群落中,传粉者访花具有较高的忠实性,花部特征差异可能是传粉者选择性访花的原因。对于紫堇属3个物种,花部特征和交配系统的分化有助于其避免传粉过程的相互干扰,交配系统与传粉环境具有一定相关性,混合交配系统可能有利于提高植物对不同生境和气候的适应性。     

POLLINATOR‐MEDIATED REPRODUCTIVE ISOLATION AMONG DIOECIOUS FIG SPECIES (FICUS,MORACEAE)

The extent of isolation among closely related sympatric plant species engaged in obligate pollination mutualisms depends on the fitness consequences of interspecies floral visitation. In figs (Ficus), interspecific gene flow may occur when pollinating wasps (Agaonidae) visit species other than their natal fig species. We studied reproductive isolation in a clade of six sympatric dioecious fig species in New Guinea. Microsatellite genotyping and Bayesian clustering analysis of the fig community indicated strong reproductive barriers among sympatric species. A total of 1–2% of fig populations consisted of hybrid individuals. A new experimental method of manipulating fig wasps investigated the reproductive consequences of conspecific and heterospecific pollinator visitation for both mutualists. Fig wasps introduced to Ficus hispidioides pollinated and oviposited in receptive figs. Seed development and seedling growth were largely comparable between conspecific and heterospecific crosses. Heterospecific pollinator fitness, however, was significantly less than that of conspecific pollinators. Heterospecific pollinators induced gall formation but offspring did not develop to maturity in the new host. Selection on pollinators maintaining host specificity appears to be an important mechanism of contemporary reproductive isolation among these taxa that could potentially influence their diversification.     

Selective pollination by fungus gnats potentially functions as an alternative reproductive isolation among five Arisaema species

Background and AimsInterspecific difference in pollinators (pollinator isolation) is important for reproductive isolation in flowering plants. Species-specific pollination by fungus gnats has been discovered in several plant taxa, suggesting that they can contribute to reproductive isolation. Nevertheless, their contribution has not been studied in detail, partly because they are too small for field observations during flower visitation. To quantify their flower visitation, we used the genus Arisaema (Araceae) because the pitcher-like spathe of Arisaema can trap all floral visitors.MethodsWe evaluated floral visitor assemblage in an altitudinal gradient including five Arisaema species. We also examined interspecific differences in altitudinal distribution (geographic isolation) and flowering phenology (phenological isolation). To exclude the effect of interspecific differences in altitudinal distribution on floral visitor assemblage, we established ten experimental plots including the five Arisaema species in high- and low-altitude areas and collected floral visitors. We also collected floral visitors in three additional sites. Finally, we estimated the strength and contribution of these three reproductive barriers using a unified formula for reproductive isolation.Key ResultsEach Arisaema species selectively attracted different fungus gnats in the altitudinal gradient, experimental plots and additional sites. Altitudinal distribution and flowering phenology differed among the five Arisaema species, whereas the strength of geographic and phenological isolations were distinctly weaker than those in pollinator isolation. Nevertheless, the absolute contribution of pollinator isolation to total reproductive isolation was weaker than geographic and phenological isolations, because pollinator isolation functions after the two early-acting barriers in plant life history.ConclusionsOur results suggest that selective pollination by fungus gnats potentially contributes to reproductive isolation. Since geographic and phenological isolations can be disrupted by habitat disturbance and interannual climate change, the strong and stable pollinator isolation might compensate for the weakened early-acting barriers as an alternative reproductive isolation among the five Arisaema species.     

Floral traits and isolation of three sympatric Aquilegia species in the Qinling Mountains, China

Floral isolation has been considered to be an important reproductive mechanism governing the species diversification in many genera. In a classic example Aquilegia, sympatric species from North America with diverse floral traits are generally associated with specialized pollinators that prohibit interspecific hybridization. It remains unclear whether species diversification in the genus from Eurasia is also maintained by floral isolation. We investigated floral phenology, floral characteristics and pollinators in three sympatric Aquilegia species (A. ecalcarata, A. incurvata and A. yabeana) in the Qinling Mountains, Shanxi Province, China from 2001 to 2005. The spurless A. ecalcarata flowers earlier than the other two species with nectar spurs but their floral phenology overlaps. Major pollinators of A. ecalcarata are syrphid flies while bumblebees are major for A. incurvata and A. yabeana. Therefore our observations confirm that mechanical isolation through differential pollinators could contribute reproductive isolation between spurless and spurred species, as demonstrated by studies from North America. Whether floral isolation plays a major role in the reproductive isolation between two spurred species (A. incurvata and A. yabeana), however, remains to be seen. Further studies are required to quantify the potential role of geographical isolation because they occupy different habitats.     

The invasive <Emphasis Type="Italic">Lespedeza cuneata</Emphasis> attracts more insect pollinators than native congeners in tallgrass prairie with variable impacts

Invasive plant species can potentially exert competitive or facilitative effects on insect pollination services of native species. Factors that influence these effects include the degree of shared pollinator species, synchronous flowering phenology, similar flower morphology and color, relatedness of invasive and natives, and showiness and densities of flowers. We investigated such plant-pollinator dynamics by comparing the invasive Lespedeza cuneata and three native congeners, all sympatric with synchronous flowering, using in situ populations over 2 years during peak floral displays. Insect visitation rates of the invasive were significantly higher per plant in both years than on the native species. The invasive exerted a competitive effect on visitation of the two native species with fewer shared pollinators, and a facilitative effect on visitation of the native species with the highest degree of shared insect visitors. Positive correlations were found between floral density and visitation rate per plant in all the native species. Although no such correlation was found for the invasive, floral density in L. cuneata was at least twenty times higher than in the native species and likely saturated the response of the pollinator community. Analyses of insect visitor taxonomic data indicated the insect communities visiting each of the Lespedeza species were generally similar though with species-specific differences. The main exception was that the common honeybee, Apis mellifera, was a primary visitor to the invasive plant species, yet was never observed on the native Lespedeza species.     

Reproductive Biology of Erythrina falcata (Fabaceae: Papilionoideae)1

Reproductive phenology, floral biology, degree of self‐incompatibility, and floral visitors of Erythrina falcata were studied in an Argentinean population. Flowering occurs during the dry season from late August to late October. Flower lifetime is 5–6 d. Phylogenetic studies indicate that E. falcata, together with E. fusca and E. crista‐galli, are included in a basal clade within Erythrina. Its phylogenetic position, floral morphology, and nectar characteristics suggest a hummingbird–passerine mixed pollination system. The flowers are nontubular, and the vexillum (the upper petal of the corolla) covers the other remaining floral parts until displaced by a visiting passerine (Icterus cayanensis) or a hummingbird (Amazilia chionogaster). Both birds act as pollen vectors. Bees were observed as occasional pollinators. Nectar production begins at anther dehiscence and coincides with maximum stigmatic receptivity. The base of the keel forms a secondary nectar reservoir. Controlled pollinations showed that this species is self‐incompatible, although a few fruits develop from selfing. Pollen:ovule ratio (43,200:7) is as expected for a xenogamous plant. Only 1 percent of the flowers set seeds under natural conditions. Possible explanations for the low reproductive success are discussed.     

Limited phenological and pollinator-mediated isolation among selfing and outcrossing Arabidopsis lyrata populations

Transitions from outcrossing to selfing have been a frequent evolutionary shift in plants and clearly play a role in species divergence. However, many questions remain about the initial mechanistic basis of reproductive isolation during the evolution of selfing. For instance, how important are pre-zygotic pre-pollination mechanisms (e.g. changes in phenology and pollinator visitation) in maintaining reproductive isolation between newly arisen selfing populations and their outcrossing ancestors? To test whether changes in phenology and pollinator visitation isolate selfing populations of Arabidopsis lyrata from outcrossing populations, we conducted a common garden experiment with plants from selfing and outcrossing populations as well as their between-population hybrids. Specifically, we asked whether there was isolation between outcrossing and selfing plants and their between-population hybrids through differences in (1) the timing or intensity of flowering; and/or (2) pollinator visitation. We found that phenology largely overlapped between plants from outcrossing and selfing populations. There were also no differences in pollinator preference related to mating system. Additionally, pollinators preferred to visit flowers on the same plant rather than exploring nearby plants, creating a large opportunity for self-fertilization. Overall, this suggests that pre-zygotic pre-pollination mechanisms do not strongly reproductively isolate plants from selfing and outcrossing populations of Arabidopsis lyrata.     

How to be an attractive male: floral dimorphism and attractiveness to pollinators in a dioecious plant

Background  

Sexual selection theory predicts that males are limited in their reproductive success by access to mates, whereas females are more limited by resources. In animal-pollinated plants, attraction of pollinators and successful pollination is crucial for reproductive success. In dioecious plant species, males should thus be selected to increase their attractiveness to pollinators by investing more than females in floral traits that enhance pollinator visitation. We tested the prediction of higher attractiveness of male flowers in the dioecious, moth-pollinated herb Silene latifolia, by investigating floral signals (floral display and fragrance) and conducting behavioral experiments with the pollinator-moth, Hadena bicruris.     

Pollen dispersal and fruit production in Vaccinium oxycoccos and comparison with its sympatric congener V. uliginosum

Investigating plant–pollinator interactions and pollen dispersal are particularly relevant for understanding processes ensuring long‐term viability of fragmented plant populations. Pollen dispersal patterns may vary strongly, even between similar congeneric species, depending on the mating system, pollinator assemblages and floral traits. We investigated pollen dispersal and fruit production in a population of Vaccinium oxycoccos, an insect‐pollinated shrub, and compared the pollen dispersal pattern with a co‐flowering, sympatric congener, V. uliginosum. We examined whether they share pollinators (through interspecific fluorescent dye transfers) and may differently attract pollinators, by comparing their floral colour as perceived by insects. Fluorescent dyes were mainly dispersed over short distances (80% within 40.4 m (max. 94.5 m) for V. oxycoccos and 3.0 m (max. 141.3 m) for V. uliginosum). Dye dispersal in V. oxycoccos was not significantly affected by plant area, floral display or the proximity to V. uliginosum plants. Interspecific dye transfers were observed, indicating pollinator sharing. The significantly lower dye deposition on V. oxycoccos stigmas suggests lower visitation rates by pollinators, despite higher flower density and local abundance. The spectral reflectance analysis indicates that bees are unlikely to be able to discriminate between the two species based on floral colour alone. Fruit production increased with increasing floral display, but was not affected by proximity to V. uliginosum plants. Our study highlights that fragmented populations of V. oxycoccos, when sympatric with co‐flowering V. uliginosum, might incur increased competition for the shared pollinators in the case of pollination disruption, which might then reduce outcrossed seed set.     

Relative impact of mate versus pollinator availability on pollen limitation and outcrossing rates in a mass‐flowering species

Plant mating systems are driven by several pre‐pollination factors, including pollinator availability, mate availability and reproductive traits. We investigated the relative contributions of these factors to pollination and to realized outcrossing rates in the patchily distributed mass‐flowering shrub Rhododendron ferrugineum. We jointly monitored pollen limitation (comparing seed set from intact and pollen‐supplemented flowers), reproductive traits (herkogamy, flower size and autofertility) and mating patterns (progeny array analysis) in 28 natural patches varying in the level of pollinator availability (flower visitation rates) and of mate availability (patch floral display estimated as the total number of inflorescences per patch). Our results showed that patch floral display was the strongest determinant of pollination and of the realized outcrossing rates in this mass‐flowering species. We found an increase in pollen limitation and in outcrossing rates with increasing patch floral display. Reproductive traits were not significantly related to patch floral display, while autofertility was negatively correlated to outcrossing rates. These findings suggest that mate limitation, arising from high flower visitation rates in small plant patches, resulted in low pollen limitation and high selfing rates, while pollinator limitation, arising from low flower visitation rates in large plant patches, resulted in higher pollen limitation and outcrossing rates. Pollinator‐mediated selfing and geitonogamy likely alleviates pollen limitation in the case of reduced mate availability, while reduced pollinator availability (intraspecific competition for pollinator services) may result in the maintenance of high outcrossing rates despite reduced seed production.     

Identification of major quantitative trait loci underlying floral pollination syndrome divergence in Penstemon

Distinct floral pollination syndromes have emerged multiple times during the diversification of flowering plants. For example, in western North America, a hummingbird pollination syndrome has evolved more than 100 times, generally from within insect-pollinated lineages. The hummingbird syndrome is characterized by a suite of floral traits that attracts and facilitates pollen movement by hummingbirds, while at the same time discourages bee visitation. These floral traits generally include large nectar volume, red flower colour, elongated and narrow corolla tubes and reproductive organs that are exerted from the corolla. A handful of studies have examined the genetic architecture of hummingbird pollination syndrome evolution. These studies find that mutations of relatively large effect often explain increased nectar volume and transition to red flower colour. In addition, they suggest that adaptive suites of floral traits may often exhibit a high degree of genetic linkage, which could facilitate their fixation during pollination syndrome evolution. Here, we explore these emerging generalities by investigating the genetic basis of floral pollination syndrome divergence between two related Penstemon species with different pollination syndromes—bee-pollinated P. neomexicanus and closely related hummingbird-pollinated P. barbatus. In an F₂ mapping population derived from a cross between these two species, we characterized the effect size of genetic loci underlying floral trait divergence associated with the transition to bird pollination, as well as correlation structure of floral trait variation. We find the effect sizes of quantitative trait loci for adaptive floral traits are in line with patterns observed in previous studies, and find strong evidence that suites of floral traits are genetically linked. This linkage may be due to genetic proximity or pleiotropic effects of single causative loci. Interestingly, our data suggest that the evolution of floral traits critical for hummingbird pollination was not constrained by negative pleiotropy at loci that show co-localization for multiple traits.     

Pre-and post-zygotic reproductive isolation between co-occurring Mussaenda pubescens var.alba and M. shikokiana(Rubiaceae)

Reproductive isolation is a fundamental requirement for speciation and includes several sequential stages.Few studies have determined the relative contributions of pre-and post-zygotic reproductive isolation in plants,especially between relative species with clear differentiation in flower form.To investigate the mechanisms responsible for reproductive isolation in sympatric Mussaenda pubescens var.alba and Mussaenda shikokiana(Rubiaceae)in Guangxi Province,China,we made observations of flowering phenology,patterns of insect visitation,and conducted pollination experiments,including artificial hybridization.The two species had overlapping flowering times and were pollinated by overlapping pollinators;however,their relative importance differed significantly with M.pubescens visited more commonly by bees and M.shikokiana more frequently by butterflies.Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals.Hybrids werecharacterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence.Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility.Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.     

Floral divergence, pollinator partitioning and the spatiotemporal pattern of plant–pollinator interactions in three sympatric Adenophora species

Floral divergence among congeners may relate to differential utilization of pollinators and contribute to reducing overlap in pollination niches. To investigate whether and how floral differences are associated with differential utilization of pollinators in three sympatric Adenophora species, we analyzed floral traits and evaluated the contribution of different visitors to pollination. We compared visitation rates of different pollinator categories in different years and sites. A suite of floral traits differed among the three Adenophora species, suggesting adaptation to diurnal versus nocturnal pollination and an intermediate condition. However, many visitor species were shared among the three plant species, suggesting that floral traits did not rigorously filter visitors. Effective pollinators were large bees and moths. The importance of large bees as pollinators decreased whereas that of moths increased along the gradient from typically bee-pollinated to moth-pollinated flowers. The intermediate species (A. khasiana) differed substantially from the other two species in pollinator species but not in pollinator categories. The principal pollinator category of each species was constant across years and sites except in the intermediate species where it differed between two sites. Overall, the three sympatric species of Adenophora partition pollinators by floral divergence and the principal pollinators coincide with the predictions based on floral syndromes.     

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.     

Floral volatile alleles can contribute to pollinator‐mediated reproductive isolation in monkeyflowers (Mimulus)

Pollinator‐mediated reproductive isolation is a major factor in driving the diversification of flowering plants. Studies of floral traits involved in reproductive isolation have focused nearly exclusively on visual signals, such as flower color. The role of less obvious signals, such as floral scent, has been studied only recently. In particular, the genetics of floral volatiles involved in mediating differential pollinator visitation remains unknown. The bumblebee‐pollinated Mimulus lewisii and hummingbird‐pollinated Mimulus cardinalis are a model system for studying reproductive isolation via pollinator preference. We have shown that these two species differ in three floral terpenoid volatiles – d ‐limonene, β‐myrcene, and E‐β‐ocimene – that are attractive to bumblebee pollinators. By genetic mapping and in vitro analysis of enzyme activity we demonstrate that these interspecific differences are consistent with allelic variation at two loci, LIMONENE‐MYRCENE SYNTHASE (LMS) and OCIMENE SYNTHASE (OS). Mimulus lewisii LMS (MlLMS) and OS (MlOS) are expressed most strongly in floral tissue in the last stages of floral development. Mimulus cardinalis LMS (McLMS) is weakly expressed and has a nonsense mutation in exon 3. Mimulus cardinalis OS (McOS) is expressed similarly to MlOS, but the encoded McOS enzyme produces no E‐β‐ocimene. Recapitulating the M. cardinalis phenotype by reducing the expression of MlLMS by RNA interference in transgenic M. lewisii produces no behavioral difference in pollinating bumblebees; however, reducing MlOS expression produces a 6% decrease in visitation. Allelic variation at the OCIMENE SYNTHASE locus is likely to contribute to differential pollinator visitation, and thus promote reproductive isolation between M. lewisii and M. cardinalis. OCIMENE SYNTHASE joins a growing list of ‘speciation genes’ (‘barrier genes’) in flowering plants.