Phenotype-fitness relationships and pollen-transfer efficiency of five orchid species with different pollination strategies.

PREMISE: Deceptive pollination, a fascinating mechanism that independently originated in several plant families for benefiting from pollinators without providing any reward, is particularly widespread among orchids. Pollination efficiency is crucial in orchids due to the aggregated pollen in a pollinarium, which facilitates pollen transfer and promotes cross-pollination as pollinators leave after being deceived. METHODS: In this study, we compiled data on reproductive ecology from five orchid species with different pollination strategies: three deceptive-strategy species (shelter imitation, food deception, sexual deception), one nectar-rewarding species, and one shelter-imitation but spontaneously selfing species. We aimed to compare the reproductive success (female fitness: fruit set; male fitness: pollinarium removal) and pollination efficiency of species representing these strategies. We also investigated pollen limitation and inbreeding depression among the pollination strategies. RESULTS: Male and female fitness were strongly correlated in all species but the spontaneously selfing species, which had high fruit set and low pollinarium removal. As expected, pollination efficiency was highest for the rewarding species and the sexually deceptive species. Rewarding species had no pollen limitation but did have high cumulative inbreeding depression; deceptive species had high pollen limitation and moderate inbreeding depression; and spontaneously selfing species did not have pollen limitation or inbreeding depression. CONCLUSIONS: Pollinator response to deception is critical to maintain reproductive success and avoid inbreeding in orchid species with non-rewarding pollination strategies. Our findings contribute to a better understanding of the trade-offs associated with different pollination strategies in orchids and highlight the importance of pollination efficiency in orchids due to the pollinarium.

Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus.

BACKGROUND: Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus. RESULTS: The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log2 FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and ß-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination. CONCLUSIONS: This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo.

Mechanisms of Male-Male Interference during Dispersal of Orchid Pollen.

AbstractSiring success of flowering plants depends on the fates of male gametophytes, which compete for access to stigmas, stylar resources, and ovules. Although rarely considered, pollen may often compete during dispersal, affecting the processes required for export to stigmas: pollen pickup, transport, and deposition. We quantified dispersal interference by tracking bee-mediated dispersal of stained Anacamptis morio (Orchidaceae) pollen from individual donor flowers and inferred the affected dispersal mechanisms on the basis of the fit of a process-based model. During individual trials, all recipient flowers were either emasculated, precluding interference with donor pollen, or intact, adding potentially interfering pollen to the pollinator. The presence of competing pollinaria on bees reduced pickup of additional pollinaria, doubled the overall proportion of lost donor pollen, and reduced total pollen export by 27%. Interference specifically increased loss of donor pollen between successive flower visits and variation in deposition among trials, and it likely also reduced pollen contact with stigmas and pollen deposition when contact occurred. Thus, by altering pollen removal, transport, and deposition, male-male interference during pollen dispersal can significantly-and perhaps commonly-limit plant-siring success.

So small and so rich: diversity of floral resources in miniature Oncidiinae (Orchidaceae) and their relation to pollinators.

Oncidiinae is one of the most important subtribes among the Neotropical orchids, with an enormous diversity of floral morphology and secretory structures. This subtribe attracts a diverse array of pollinators which explore a variety of floral resources of its flowers. In this paper we provide a detailed investigation of the floral anatomy of 32 species of micro Oncidiinae. We applied histochemical tests in order to determine the diversity of the glands and rewards. The diversity of secretory flower structures and rewards was related to the group of pollinators known for this subtribe. We verified that half of the species (16 species, 50%) secrete oil as a resource, being pollinated by female of solitary bees. Species of some distinct nectar-secreting genera (four species, 12.5%) are pollinated by a range of nectar-searching animals. Species of the genus Notylia (four species, 12.5%) release floral perfumes that reward male Euglossini bees. Most of the investigated species (six species, 18.75%) possess osmophores that are involved in pollinator attraction. Two species of Capanemia (6.25%) do not offer any floral reward, suggesting that pollination by food deception is involved. There are strong variations in the anatomy of reward-producing structures and resources in Oncidiinae. The diversity of floral rewards affects the range of pollinators, which are related to the diversification of this subtribe throughout the Neotropics. The understanding of relationships between Oncidiinae species and their pollinators is crucial to our knowledge of the evolution of pollination systems in this huge plant family represented by the orchids.

Food Reward Chemistry Explains a Novel Pollinator Shift and Vestigialization of Long Floral Spurs in an Orchid.

During the evolutionary history of flowering plants, transitions between pollinator groups (pollinator shifts) have been frequent,1 and contributed to the spectacular radiation of angiosperms.2 Although the evolution of floral traits during pollinator shifts has been studied in real time under controlled laboratory conditions,3 it is challenging to study in nature and therefore poorly understood.4-7 Using a comparative, multidisciplinary approach, we dissect the evolution of floral traits during a pollinator shift in the long-spurred African orchid Satyrium longicauda. Phylogenetic analysis and ecological experiments revealed a shift from moth- to oil-collecting bee pollination. Remarkably, flowers of the bee-pollinated form are similar in morphology, color, and overall volatile chemistry to those of moth-pollinated forms, but differ in having spurs that are mostly devoid of nectar, and have an elevated presence of the oil-derived compound diacetin, which oil-collecting bees use as a cue for oil presence.8 Experiments demonstrated that long spurs are critical for pollination of a moth-pollinated form, but are not needed for pollination of the bee-pollinated form. We conclude that the pollinator shift in Satyrium was mediated by a switch in chemistry of the pollinator reward. The ancestral presence of diacetin might have served as a pre-adaptation for bee pollination, whereas the current mismatch between flower morphology and bees is due to the retention of vestigial floral spurs. These results elucidate the sequence of floral evolution in the early stages of pollinator shifts and help to explain the assembly of suites of co-varying traits through pre-adaptation and vestigialization.9-12.

Pollen viability of Euro-Mediterranean orchids under different storage conditions: The possible effects of climate change.

The future impact of climate change and a warmer world is a matter of great concern. We therefore aimed to evaluate the effects of temperature on pollen viability and fruit set of Mediterranean orchids. The in vitro and controlled pollination experiments were performed to evaluate the ability of pollinia stored at lower and higher temperatures to germinate and produce fruits and seeds containing viable embryos. In all of the examined orchids, pollen stored at -20 °C remained fully viable for up to 3 years, reducing its percentage germination from year 4 onwards. Pollinia stored at higher temperatures had a drastic reduction in vitality after 2 days at 41-44 °C, while pollinia stored at 47-50 °C did not show any pollen tube growth. The different levels of pollen viability duration among the examined orchids can be related to their peculiar reproductive biology and pollination ecology. The germinability of pollinia stored at lower temperatures for long periods suggests that orchid pollinia can be conserved ex situ. In contrast, higher temperatures can have harmful effects on the vitality of pollen and consequently on reproductive success of the plants. To our knowledge, this is the first report demonstrating the effects of global change on orchid pollen, and on pollen ability to tolerate, or not, higher air temperatures. Although vegetative reproduction allows orchids to survive a few consecutive warm years, higher temperatures for several consecutive years can have dramatic effects on reproductive success of orchids.

A cognitive analysis of deceptive pollination: associative mechanisms underlying pollinators' choices in non-rewarding colour polymorphic scenarios.

Intraspecific floral colour polymorphism is a common trait of food deceptive orchids, which lure pollinators with variable, attractive signals, without providing food resources. The variable signals are thought to hinder avoidance learning of deceptive flowers by pollinators. Here, we analysed the cognitive mechanisms underlying the choice of free-flying stingless bees Scaptotrigona aff. depilis trained to visit a patch of artificial flowers that displayed the colours of Ionopsis utricularioides, a food deceptive orchid. Bees were trained in the presence of a non-rewarding colour and later tested with that colour vs. alternative colours. We simulated a discrete-polymorphism scenario with two distinct non-rewarding test colours, and a continuous-polymorphism scenario with three non-rewarding test colours aligned along a chromatic continuum. Bees learned to avoid the non-rewarding colour experienced during training. They thus preferred the novel non-rewarding colour in the discrete-polymorphic situation, and generalized their avoidance to the adjacent colour of the continuum in the continuous-polymorphism situation, favouring thereby the most distant colour. Bees also visited less flowers and abandoned faster a non-rewarding monomorphic patch than a non-rewarding polymorphic patch. Our cognitive analyses thus reveal that variable deceptive orchids disrupt avoidance learning by pollinators and exploit their generalization abilities, which make them favour distinct morphs.

Noctuid moths as pollinators of Habenaria sagittifera (Orchidaceae): floral adaptations for the transfer of pollinaria on the thoraxes of moths.

Orchids attach their pollinaria (cohesive masses of pollen) to specific body parts of flower visitors, but usually not to the hairy and scaly body parts of flower-visiting moths, because hairs and scales are easily detached. We demonstrate that pollinaria of Habenaria sagittifera (Orchidaceae) are transferred among flowers on the hairy thoraxes of moths in Japan. Diurnal and nocturnal insects visited the orchid flowers. However, pollinaria were attached only to the hairy thoraxes of plusiine moths (Lepidoptera: Noctuidae). All pollinaria were directly attached to the ventral thorax surfaces at the bases of hairs. Orchid spur lengths matched plusiine proboscis lengths so that nectar-feeding moths contacted the viscidia (sticky pads of pollinaria) and stigma. Other flower visitors did not contact the viscidia or stigmas while feeding on nectar. Habenaria sagittifera appears to have a floral morphology that is adaptive for the transfer of pollinaria on the thoraxes of plusiine moths.

Conflicting selection on floral scent emission in the orchid Gymnadenia conopsea.

Floral scent is a crucial trait for pollinator attraction. Yet only a handful of studies have estimated selection on scent in natural populations and no study has quantified the relative importance of pollinators and other agents of selection. In the fragrant orchid Gymnadenia conopsea, we used electroantennographic data to identify floral scent compounds detected by local pollinators and quantified pollinator-mediated selection on emission rates of 10 target compounds as well as on flowering start, visual display and spur length. Nocturnal pollinators contributed more to reproductive success than diurnal pollinators, but there was significant pollinator-mediated selection on both diurnal and nocturnal scent emission. Pollinators selected for increased emission of two compounds and reduced emission of two other compounds, none of which were major constituents of the total bouquet. In three cases, pollinator-mediated selection was opposed by nonpollinator-mediated selection, leading to weaker or no detectable net selection. Our study demonstrates that minor scent compounds can be targets of selection, that pollinators do not necessarily favour stronger scent signalling, and that some scent compounds are subject to conflicting selection from pollinators and other agents of selection. Hence, including floral scent traits into selection analysis is important for understanding the mechanisms behind floral evolution.

Cryopreservation of Orchids - A Review.

BACKGROUND: The orchids are one of the beautiful creations of nature which stand apart from any other assemblage of flowering plants. They are highly evolutionary and ecologically significant group of plants that have effectively occupied almost every habitat on the earth. Indiscriminate collections and extermination of their natural habitats have threatened many species of orchids with extinction, resulting in a severe reduction of their genetic resources in nature according to recent patents. It is necessary to adopt sound scientific protocols for the preservation of orchid species. METHOD: This cost-effective technique provides large storage time for the conservation of germplasm. Presently, efforts have been made to explore various cryopreservation techniques utilized so far and factors affecting the longevity of the propagules (in vivo and in vitro) while cryopreserving them. The sample to be cryopreserved is freeze-preserved in two ways, a) stepwise at two different subzero temperatures and b) in the rapid method, the samples are placed directly in the liquid nitrogen. RESULTS: The orchid seeds and pollen are the most suitable propagules for cryopreservation of orchids due to their minute size and less space requirement. CONCLUSION: Among the tissues (such as seeds, pollen, protocorms etc.) seeds are the most reliable. The present article reviews the cryopreservation techniques and factors effecting the cryopreservation, for in vitro conservation of orchid gene pool.

An experimental evaluation of traits that influence the sexual behaviour of pollinators in sexually deceptive orchids.

Pollination by sexual deception of male insects is perhaps one of the most remarkable cases of mimicry in the plant kingdom. However, understanding the influence of floral traits on pollinator behaviour in sexually deceptive plants is challenging, due to the risk of confounding changes in floral odour when manipulating morphology. Here, we investigated the floral traits influencing the sexual response of male Zaspilothynnus nigripes (Tiphiidae) wasps, a pollinator of two distantly related sexually deceptive orchids with contrasting floral architecture, Caladenia pectinata and Drakaea livida. In D. livida, the chemical sexual attractant is emitted from the labellum, whereas in C. pectinata, it is produced from the distal sepal tips, allowing manipulative experiments. When controlling for visual cues, there was no difference in long-distance attraction, although the floral odour of D. livida induced copulation more frequently than that of C. pectinata. The role of colour in pollinator sexual attraction was equivocal, indicating that colour may not be a strong constraint on the initial evolution of sexual deception. The frequency of wasp visitors landing on C. pectinata decreased when the amount of floral odour was reduced, but attempted copulation rates were enhanced when the source of floral odour was associated with the labellum. These latter variables may represent axes of selection that operate across many sexually deceptive species. Nonetheless, the observed variation in floral traits suggests flexibility among species in how sexual deception can be achieved.

Novel computed tomography-based tools reliably quantify plant reproductive investment.

The flower is a bisexual reproductive unit where both genders compete for resources. Counting pollen and ovules in flowers is essential to understand how much is invested in each gender. Classical methods to count very numerous pollen grains and ovules are inefficient when pollen grains are tightly aggregated, and when fertilization rates of ovules are unknown. In this study we have therefore developed novel counting techniques based on computed tomography. In order to demonstrate the potential of our methods in very difficult cases, we counted pollen and ovules across inflorescences of deceptive and rewarding species of European orchids, which possess both very large numbers of pollen grains (tightly aggregated) and ovules. Pollen counts did not significantly vary across inflorescences and pollination strategies, whereas deceptive flowers had significantly more ovules than rewarding flowers. The within-inflorescence variance of pollen-to-ovule ratios in rewarding flowers was four times higher than in deceptive flowers, possibly demonstrating differences in the constraints acting on both pollination strategies. We demonstrate the inaccuracies and limitations of previously established methods, and the broad applicability of our new techniques: they allow measurement of reproductive investment without restriction on object number or aggregation, and without specimen destruction.

Comparative reproductive biology and pollinator specificity among sympatric Gomesa (Orchidaceae: Oncidiinae).

The incredible pollination mechanisms displayed by orchid flowers has inspired biologists over the centuries. Based on the intriguing flower structures, the relationship among orchid species and their pollinators has been frequently regarded as very specialised. Given that visits on flowers pollinated by oil-collecting bees are regularly rare, and in Oncidiinae the flowers frequently attractexclusively species that act as effective pollinators, the comparative reproductive biology and pollinator specificity of two sympatric Gomesa (G. varicosa and G. montana; Oncidiinae) were analysedbased on records of floral morphology, production of floral rewards, pollinators and pollination mechanisms. Furthermore, experimental pollinations were carried out in order to examine the breeding systems. The results have show that in the studied population, both Gomesa are visited by several bee species, but these orchids present a specific pollination system.Pollinaria are deposited on the head of Centridini (G. varicosa and G. montana) and Epicharitini (G. varicosa) bees when landed on the central callus of the labellumto collect lipoidal substances produced by glandular elaiophores on lateral lobes of the labellum. Both species are dependent on a biotic pollen vector to set fruits. Gomesamontana is completely self-incompatible, while G. varicosa is partially self-compatible. Our results indicate that although the occurrence of self-sterile species seems to be common in Oncidiinae, in partially self-incompatible species, as is the case of G. varicosa, self-compatibility has been considered as an important factor favouring reproductive assurance in populations with low visitation frequencies, despite occurrence of inbreeding depression.

Experimental reduction in interaction intensity strongly affects biotic selection.

The link between biotic interaction intensity and strength of selection is of fundamental interest for understanding biotically driven diversification and predicting the consequences of environmental change. The strength of selection resulting from biotic interactions is determined by the strength of the interaction and by the covariance between fitness and the trait under selection. When the relationship between trait and absolute fitness is constant, selection strength should be a direct function of mean population interaction intensity. To test this prediction, we excluded pollinators for intervals of different length to induce five levels of pollination intensity within a single plant population. Pollen limitation (PL) increased from 0 to 0.77 across treatments, accompanied by a fivefold increase in the opportunity for selection. Trait-fitness covariance declined with PL for number of flowers, but varied little for other traits. Pollinator-mediated selection on plant height, corolla size, and spur length increased by 91%, 34%, and 330%, respectively, in the most severely pollen-limited treatment compared to open-pollinated plants. The results indicate that realized biotic selection can be predicted from mean population interaction intensity when variation in trait-fitness covariance is limited, and that declines in pollination intensity will strongly increase selection on traits involved in the interaction.

Telipogon peruvianus (Orchidaceae) Flowers Elicit Pre-Mating Behaviour in Eudejeania (Tachinidae) Males for Pollination.

Several neotropical orchid genera have been proposed as being sexually deceptive; however, this has been carefully tested in only a few cases. The genus Telipogon has long been assumed to be pollinated by male tachinid flies during pseudocopulatory events but no detailed confirmatory reports are available. Here, we have used an array of methods to elucidate the pollination mechanism in Telipogon peruvianus. The species presents flowers that have a mean floral longevity of 33 days and that are self-compatible, although spontaneous self-pollination does not occur. The flowers attract males of four tachinid species but only the males of an undescribed Eudejeania (Eudejeania aff. browni; Tachinidae) species are specific pollinators. Males visit the flowers during the first few hours of the day and the pollination success is very high (42% in one patch) compared with other sexually deceptive species. Female-seeking males are attracted to the flowers but do not attempt copulation with the flowers, as is usually described in sexually deceptive species. Nevertheless, morphological analysis and behavioural tests have shown an imperfect mimicry between flowers and females suggesting that the attractant stimulus is not based only on visual cues, as long thought. Challenging previous conclusions, our chemical analysis has confirmed that flowers of Telipogon release volatile compounds; however, the role of these volatiles in pollinator behaviour remains to be established. Pollinator behaviour and histological analyses indicate that Telipogon flowers possess scent-producing structures throughout the corolla. Our study provides the first confirmed case of (i) a sexually deceptive species in the Onciidinae, (ii) pollination by pre-copulatory behaviour and (iii) pollination by sexual deception involving tachinid flies.

Different pollinator assemblages ensure reproductive success of Cleisostoma linearilobatum (Orchidaceae) in fragmented holy hill forest and traditional tea garden.

Orchids are generally recognized to have specialist pollination systems and low fruit set is often thought to be characteristic of the family. In this study, we investigated the reproductive ecology of Cleisostoma linearilobatum, an epiphytic tropical orchid, in a holy hill forest fragment and a traditional tea garden in SW China using comparable methods. C. linearilobatum is self-compatible and dependent on insects for pollination. Fruit production in natural conditions was both pollinator- and resource-limited. However, the natural fruit set remained stable over multiple years at both sites. Pollination observations showed that C. linearilobatum has a generalized pollination system and seven insect species were observed as legitimate pollinators. Although the visit frequencies of different pollinators were different in the two sites, the pollinator assemblages ensured reproductive success of C. linearilobatum in both study sites over multiple years. The results partly explain why C. linearilobatum is so successful in the area, and also suggest that holy hill forest fragments and traditional tea gardens in Xishuangbanna are important in preserving orchids, especially those with generalist pollination.

Strong pollinator-mediated selection for increased flower brightness and contrast in a deceptive orchid.

Contrasting flower color patterns that putatively attract or direct pollinators toward a reward are common among angiosperms. In the deceptive orchid Anacamptis morio, the lower petal, which makes up most of the floral display, has a light central patch with dark markings. Within populations, there is pronounced variation in petal brightness, patch size, amount of dark markings, and contrast between patch and petal margin. We tested whether pollinators mediate selection on these color traits and on morphology (plant height, number of flowers, corolla size, spur length), and whether selection is consistent with facilitated or negative frequency-dependent pollination. Pollinators mediated strong selection for increased petal brightness (Δßpoll = 0.42) and contrast (Δßpoll = 0.51). Pollinators also tended to mediate stabilizing selection on brightness (Δγpoll = -0.27, n.s.) favoring the most common phenotype in the population. Selection for reduced petal brightness among hand-pollinated plants indicated a fitness cost associated with brightness. The results demonstrate that flower color traits influence pollination success and seed production in A. morio, indicating that they affect attractiveness to pollinators, efficiency of pollen transfer, or both. The documented selection is consistent with facilitated pollination and selection for color convergence toward cooccurring rewarding species.

Pollination ecology of two species of Elleanthus (Orchidaceae): novel mechanisms and underlying adaptations to hummingbird pollination.

Relationships among floral biology, floral micromorphology and pollinator behaviour in bird-pollinated orchids are important issues to understand the evolution of the huge flower diversity within Orchidaceae. We aimed to investigate floral mechanisms underlying the interaction with pollinators in two hummingbird-pollinated orchids occurring in the Atlantic forest. We assessed floral biology, nectar traits, nectary and column micromorphologies, breeding systems and pollinators. In both species, nectar is secreted by lip calli through spaces between the medial lamellar surfaces of epidermal cells. Such a form of floral nectar secretion has not been previously described. Both species present functional protandry and are self-compatible yet pollinator-dependent. Fruit set in hand-pollination experiments was more than twice that under natural conditions, evidencing pollen limitation. The absence of fruit set in interspecific crosses suggests the existence of post-pollination barriers between these sympatric co-flowering species. In Elleanthus brasiliensis, fruits resulting from cross-pollination and natural conditions were heavier than those resulting from self-pollination, suggesting advantages to cross-pollination. Hummingbirds pollinated both species, which share at least one pollinator species. Species differences in floral morphologies led to distinct pollination mechanisms. In E. brasiliensis, attachment of pollinarium to the hummingbird bill occurs through a lever apparatus formed by an appendage in the column, another novelty to our knowledge of orchid pollination. In E. crinipes, pollinarium attachment occurs by simple contact with the bill during insertion into the flower tube, which fits tightly around it. The novelties described here illustrate the overlooked richness in ecology and morphophysiology in Orchidaceae.

[Pollen viability, stigma receptivity and fruiting characteristics of botanical origin of Jinxianlian].

The viability and life span of pollen were evaluated by TTC (2,3,5-triphenyl tetrazlium chloride) and the peroxidase solution, the stigma receptivity were estimated by benzidine-H2O2 method and the fruiting characteristics were investigated. The results showed that (1) Anoectochilus roxburghii and A. formosanus appeared the same up-and-down trend of the pollen viability, increased and then decreased. The storage temperature and storage time had significant impact on the pollen viability. With the extension of storage time, the pollen activity decreased. 4 degrees C refrigerator storage may be extended the pollen vitality. (2) The stigma had receptivity in 1st day and reached the highest level in the 4th day after blooming. A. roxburghii lost receptivity in the 8th day while A. formosanus lost receptivity in the 10th day after blooming. (3) The different pollination had significant impact on seed setting rate. The seed setting rate of artificial cross-pollination was higher than that of the artificial self-pollination. Collecting pollen in the 3rd day and carrying out artificial cross-pollination in the 4th day after blooming can significantly improve seed setting rate. The results provided technical assurance for A. roxburghii and A. formosanus breeding of new varieties and seed breeding.

Experimental fertilization increases amino acid content in floral nectar, fruit set and degree of selfing in the orchid Gymnadenia conopsea.

Floral traits have evolved to maximize reproductive success by attracting pollinators and facilitating pollination. Highly attractive floral traits may, however, also increase the degree of self-pollination, which could become detrimental for plant fitness through inbreeding depression. Floral nectar is a trait that is known to strongly mediate pollinator attraction and plant reproductive success, but the particular role of the nectar amino acid (AA) composition is poorly understood. Therefore, we experimentally manipulated the nectar AA composition and abundance of the Lepidoptera-pollinated orchid Gymnadenia conopsea through soil fertilization, and we quantified AA content and AA composition through high performance anion exchange chromatography with pulsed amperometric detection. Mixed models were then used to evaluate differences in pollinia removal, fruit set, seed set and degree of selfing between fertilized and control individuals. Selfing rates were estimated using microsatellite markers. We found that fertilized individuals had a significantly higher nectar AA content and an altered AA composition, whereas plant height, number of flowers, nectar volume and sugar concentration remained unchanged. Fertilized individuals also had significantly more pollinia removed and a higher fruit set, whereas control plants that did not receive the fertilization treatment had significantly fewer selfed seeds, and more viable seeds. Although we cannot exclude a role of changes in floral scent following the fertilization treatment, our results strongly suggest a relation among nectar AA composition, fruiting success and selfing rates. Our results also indicate potential consequences of nutrient pollution for plant reproductive success, through the induced changes in nectar AA composition.