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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.

Results

As found in previous studies, male plants produced more but smaller flowers. Male flowers, however, emitted significantly larger amounts of scent than female flowers, especially of the pollinator-attracting compounds. In behavioral tests we showed that naïve pollinator-moths preferred male over female flowers, but this preference was only significant for male moths.

Conclusion

Our data suggest the evolution of dimorphic floral signals is shaped by sexual selection and pollinator preferences, causing sexual conflict in both plants and pollinators.

An important mechanism in stabilizing tightly linked host-parasitoid and prey-predator interactions is the presence of refuges that protect organisms from their natural enemies. However, the presence and quality of refuges can be strongly affected by the environment. We show that infection of the host plant Silene latifolia by its specialist fungal plant pathogen Microbotryum violaceum dramatically alters the enemy-free space of a herbivore, the specialist noctuid seed predator Hadena bicruris, on their shared host plant. The pathogen arrests the development of seed capsules that serve as refuges for the herbivore's offspring against the specialist parasitoid Microplitis tristis, a major source of mortality of H. bicruris in the field. Pathogen infection resulted both in lower host-plant food quality, causing reduced adult emergence, and in twofold higher rates of parasitism of the herbivore. We interpret the strong oviposition preference of H. bicruris for uninfected plants in the field as an adaptive response, positioning offspring on refuge-rich, high-quality hosts. To our knowledge, this is the first demonstration that plant-inhabiting micro-organisms can affect higher trophic interactions through alteration of host refuge quality. We speculate that such interference can potentially destabilize tightly linked multitrophic interactions.

Background

Expressed sequence tag (EST) databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. One class of markers derived from EST sequences are simple sequence repeat (SSR) markers, also known as EST-SSRs. These are useful in plant genetic and evolutionary studies because they are located in transcribed genes and a putative function can often be inferred from homology searches. Another important feature of EST-SSR markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. In the present study we constructed a normalized EST library from floral tissue of Silene latifolia with the aim to identify expressed genes and to develop polymorphic molecular markers.

Results

We obtained a total of 3662 high quality sequences from a normalized Silene cDNA library. These represent 3105 unigenes, with 73% of unigenes matching genes in other species. We found 255 sequences containing one or more SSR motifs. More than 60% of these SSRs were trinucleotides. A total of 30 microsatellite loci were identified from 106 ESTs having sufficient flanking sequences for primer design. The inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. Tests for crossamplification of the EST-SSR loci in other Silene species established their applicability to related species.

Conclusion

The newly characterized genes and gene-derived markers from our Silene EST library represent a valuable genetic resource for future studies on Silene latifolia and related species. The polymorphism and transferability of EST-SSR markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus Silene.

The actinomycete Streptomyces setonii 75Vi2 demethylates vanillic acid and guaiacol to protocatechuic acid and catechol, respectively, and then metabolizes the products by the β-ketoadipate pathway. UV spectroscopy showed that this strain could also metabolize veratrole (1,2-dimethoxybenzene). When grown in veratrole-containing media supplemented with 2,2′-dipyridyl to inhibit cleavage of the aromatic ring, S. setonii accumulated catechol, which was detected by both liquid chromatography and gas chromatography. Reduced cell extracts from veratrole-grown cultures, but not sodium succinate-grown cultures, produced a carbon monoxide difference spectrum with a peak at 450 nm that indicated the presence of soluble cytochrome P-450. Addition of veratrole or guaiacol to oxidized cell extracts from veratrole-grown cultures produced difference spectra that indicated that these compounds were substrates for cytochrome P-450. My results suggest that S. setonii produces a cytochrome P-450 that is involved in the demethylation of veratrole and guaiacol to catechol, which is then catabolized by the β-ketoadipate pathway.

Background and Aims

Studies of the effects of pollination on floral scent and bee visitation remain rare, particularly in agricultural crops. To fill this gap, the hypothesis that bee visitation to flowers decreases after pollination through reduced floral volatile emissions in highbush blueberries, Vaccinium corymbosum, was tested. Other sources of variation in floral emissions and the role of floral volatiles in bee attraction were also examined.

Methods

Pollinator visitation to blueberry flowers was manipulated by bagging all flowers within a bush (pollinator excluded) or leaving them unbagged (open pollinated), and then the effect on floral volatile emissions and future bee visitation were measured. Floral volatiles were also measured from different blueberry cultivars, times of the day and flower parts, and a study was conducted to test the attraction of bees to floral volatiles.

Key Results

Open-pollinated blueberry flowers had 32 % lower volatile emissions than pollinator-excluded flowers. In particular, cinnamyl alcohol, a major component of the floral blend that is emitted exclusively from petals, was emitted in lower quantities from open-pollinated flowers. Although, no differences in cinnamyl alcohol emissions were detected among three blueberry cultivars or at different times of day, some components of the blueberry floral blend were emitted in higher amounts from certain cultivars and at mid-day. Field observations showed that more bees visited bushes with pollinator-excluded flowers. Also, more honey bees were caught in traps baited with a synthetic blueberry floral blend than in unbaited traps.

Conclusions

Greater volatile emissions may help guide bees to unpollinated flowers, and thus increase plant fitness and bee energetic return when foraging in blueberries. Furthermore, the variation in volatile emissions from blueberry flowers depending on pollination status, plant cultivar and time of day suggests an adaptive role of floral signals in increasing pollination of flowers.

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.

Background and Aims

Dimorphism among floral traits can evolve through variation in selection intensity between female and male performance, especially when sex functions are separated between flowers on a plant (monoecy), or between individuals (dioecy). In animal-pollinated species, male floral traits are predicted to be larger because competition for pollinators should favour larger displays. Floral dimorphism may be greater in dioecious than monoecious populations because of trade-offs between female and male function and opportunities for selfing in hermaphrodites.

Methods

These predictions were tested by surveying flower size, total flowers per inflorescence and daily display size in the insect-pollinated Sagittaria latifolia (Alismataceae). This species is useful for comparative analysis because populations are mostly either monoecious or dioecious. We examined floral dimorphism in 13 monoecious and 16 dioecious populations in eastern North America.

Key Results

Male flowers were significantly larger than female flowers in monoecious and dioecious populations, but there was no evidence for greater flower size dimorphism in dioecious populations despite their larger flower sizes overall. Although inflorescences in both dioecious and monoecious populations produced more male flowers, daily floral displays were significantly larger for female than male function due to more synchronous female flower opening. Daily floral display dimorphism was significantly greater in dioecious populations, due to greater female daily floral displays. There was a positive relationship between mean flower size and total flowers per inflorescence for both sexes in dioecious populations, but no relationship for either sex function in monoecious populations. Flower size dimorphism was positively correlated with the frequencies of females in dioecious populations.

Conclusions

The increased size and number of male flowers and protracted male floral displays in S. latifolia are probably shaped by sexual selection for more effective pollen dispersal.

Most dioecious plant species are believed to derive from hermaphrodite ancestors. The regulatory pathways that have been modified during evolution of the hermaphrodite ancestors and led to the emergence of dioecious species (with separate sexes) still remain unknown. Silene latifolia is a dioecious plant species harbouring XY sex chromosomes. To identify the molecular mechanisms involved in female organ suppression in male flowers of S. latifolia, we looked for genes potentially involved in the establishment of floral organ and whorl boundaries. We identified Arabidopsis thaliana homologs of SHOOTMERISTEMLESS (STM) and CUP SHAPED COTYLEDON 1 (CUC1) and CUC2 genes in S. latifolia. Our phylogenetic analyses suggest that we identified true orthologs for both types of genes. Detailed expression analyses showed a conserved expression pattern for these genes between S. latifolia and A. thaliana, suggesting a conserved function of the corresponding proteins. Both orthologs showed clear differences in their expression pattern between males and females or hermaphrodites suggesting their possible involvement in the sex determination pathway in S. latifolia.

In southwestern USA, the jimsonweed Datura wrightii and the nocturnal moth Manduca sexta form a pollinator–plant and herbivore–plant association. Because the floral scent is probably important in mediating this interaction, we investigated the floral volatiles that might attract M. sexta for feeding and oviposition. We found that flower volatiles increase oviposition and include small amounts of both enantiomers of linalool, a common component of the scent of hawkmoth-pollinated flowers. Because (+)-linalool is processed in a female-specific glomerulus in the primary olfactory centre of M. sexta, we hypothesized that the enantiomers of linalool differentially modulate feeding and oviposition. Using a synthetic mixture that mimics the D. wrightii floral scent, we found that the presence of linalool was not necessary to evoke feeding and that mixtures containing (+)- and/or (−)-linalool were equally effective in mediating this behaviour. By contrast, females oviposited more on plants emitting (+)-linalool (alone or in mixtures) over control plants, while plants emitting (−)-linalool (alone or in mixtures) were less preferred than control plants. Together with our previous investigations, these results show that linalool has differential effects in feeding and oviposition through two neural pathways: one that is sexually isomorphic and non-enantioselective, and another that is female-specific and enantioselective.

Plants are sessile organisms, often characterized by limited dispersal. Seeds and pollen are the critical stages for gene flow. Here we investigate spatial genetic structure, gene dispersal and the relative contribution of pollen vs seed in the movement of genes in a stable metapopulation of the white campion Silene latifolia within its native range. This short-lived perennial plant is dioecious, has gravity-dispersed seeds and moth-mediated pollination. Direct measures of pollen dispersal suggested that large populations receive more pollen than small isolated populations and that most gene flow occurs within tens of meters. However, these studies were performed in the newly colonized range (North America) where the specialist pollinator is absent. In the native range (Europe), gene dispersal could fall on a different spatial scale. We genotyped 258 individuals from large and small (15) subpopulations along a 60 km, elongated metapopulation in Europe using six highly variable microsatellite markers, two X-linked and four autosomal. We found substantial genetic differentiation among subpopulations (global FST=0.11) and a general pattern of isolation by distance over the whole sampled area. Spatial autocorrelation revealed high relatedness among neighboring individuals over hundreds of meters. Estimates of gene dispersal revealed gene flow at the scale of tens of meters (5–30 m), similar to the newly colonized range. Contrary to expectations, estimates of dispersal based on X and autosomal markers showed very similar ranges, suggesting similar levels of pollen and seed dispersal. This may be explained by stochastic events of extensive seed dispersal in this area and limited pollen dispersal.

Background and Aims

Studies of floral scent evolution often attribute variation in floral scent to differences in pollinator behaviour, ignoring the potential for shared biochemistry between floral scent and floral colour to dictate patterns of phenotypic variation in scent production. To determine the relative effects of shared biochemistry and/or localized population-level phenomena on floral scent phenotype, floral scent composition and emission rate were examined in five wild populations of colour polymorphic Hesperis matronalis (Brassicaceae).

Methods

Floral scent was collected by in situ dynamic headspace extraction on purple and white colour morphs in each of five wild populations. Gas chromatography–mass spectroscopy of extracts allowed determination of floral scent composition and emission rate for all individuals, which were examined by non-metric multidimensional scaling and analysis of variance (ANOVA), respectively, to determine the contributions of floral colour and population membership to scent profile variation.

Key Results

Despite the fact that colour morph means were very similar in some populations and quite different in other populations, colour morphs within populations did not differ from each other in terms of scent composition or emission rate. Populations differed significantly from one another in terms of both floral scent composition and emission rate.

Conclusions

Shared biochemistry alone cannot explain the variation in floral scent phenotype found for H. matronalis. Such a result may suggest that the biochemical association between floral scent and floral colour is complex or dependent on genetic background. Floral scent does vary significantly with population membership; several factors, including environmental conditions, founder effects and genetics, may account for this differentiation and should be considered in future studies.

Background and Aims

Competition among genetically different pollen donors within one recipient flower may play an important role in plant populations, increasing offspring genetic diversity and vigour. However, under field conditions stochastic pollen arrival times may result in disproportionate fertilization success of the first-arriving pollen, even to the detriment of the recipient plant's and offspring fitness. It is therefore critical to evaluate the relative importance of arrival times of pollen from different donors in determining siring success.

Methods

Hand pollinations and genetic markers were used to investigate experimentally the effect of pollination timing on seed paternity, seed mass and stigmatic wilting in the the dioecious plant Silene latifolia. In this species, high prevalence of multiply-sired fruits in natural populations suggests that competition among different donors may often take place (at fertilization or during seed development); however, the role of variation due to pollen arrival times is not known.

Key Results

First-arriving pollen sired significantly more seeds than later-arriving pollen. This advantage was expressed already before the first pollen tubes could reach the ovary. Simultaneously with pollen tube growth, the stigmatic papillae wilted visibly. Individual seeds were heavier in fruits where one donor sired most seeds than in fruits where both donors had more even paternity shares.

Conclusions

In field populations of S. latifolia, fruits are often multiply-sired. Because later-arriving pollen had decreased chances of fertilizing the ovules, this implies that open-pollinated flowers often benefit from pollen carry-over or pollinator visits within short time intervals, which may contribute to increase offspring genetic diversity and fitness.

Background and Aims

A report is made on a new species of Clusia related to C. sellowiana that dominates the vegetation of the Nouragues inselberg in French Guiana. The focus is on the pollination biology and on the remarkable relationship of this plant species to Amazonina platystylata, its cockroach pollinator. This appears to be only the second record of pollination by cockroaches.

Methods

Pollination ecology was investigated by combining morphological studies, field observations and additional experiments. Floral scent was analysed by gas chromatography–mass spectrometry. The role of acetoin, the major component of the scent of this species of Clusia, in attracting pollinators was examined in field attraction experiments. The ability of cockroaches to perceive acetoin was investigated by electroantennography (EAG).

Key Results

The Clusia species studied produces seeds only sexually. Its nocturnal flowers are visited by crickets, ants, moths and cockroaches. A species of cockroach, Amazonina platystylata, is the principal pollinator. The reward for the visit is a liquid secretion produced by tissues at the floral apex and at the base of the ovary. Although the cockroaches have no structures specialized for pollen collection, their body surface is rough enough to retain pollen grains. The cockroaches show significant EAG reactions to floral volatiles and acetoin, suggesting that the floral scent is a factor involved in attracting the cockroaches to the flowers.

Conclusions

The results suggest that the plant–cockroach interaction may be quite specialized and the plant has probably evolved a specific strategy to attract and reward its cockroach pollinators. Acetoin is a substance involved in the chemical communication of several other cockroach species and it seems plausible that the plant exploits the sensitivity of cockroaches to this compound to attract them to the flowers as part of the pollination syndrome of this species.

Background and Aims

A comparative investigation was made of floral scent variation in the closely related, food-rewarding Anacamptis coriophora and the food-deceptive Anacamptis morio in order to identify patterns of variability of odour compounds in the two species and their role in pollinator attraction/avoidance learning.

Methods

Scent was collected from plants in natural populations and samples were analysed via quantitative gas chromatography and mass spectrometry. Combined gas chromatography and electroantennographic detection was used to identify compounds that are detected by the pollinators. Experimental reduction of scent variability was performed in the field with plots of A. morio plants supplemented with a uniform amount of anisaldehyde.

Key Results

Both orchid species emitted complex odour bouquets. In A. coriophora the two main benzenoid compounds, hydroquinone dimethyl ether (1,4-dimethoxybenzene) and anisaldehyde (methoxybenzaldehyde), triggered electrophysiological responses in olfactory neurons of honey-bee and bumble-bee workers. The scent of A. morio, however, was too weak to elicit any electrophysiological responses. The overall variation in scent was significantly lower in the rewarding A. coriophora than in the deceptive A. morio, suggesting pollinator avoidance-learning selecting for high variation in the deceptive species. A. morio flowers supplemented with non-variable scent in plot experiments, however, did not show significantly reduced pollination success.

Conclusions

Whereas in the rewarding A. coriophora stabilizing selection imposed by floral constancy of the pollinators may reduce scent variability, in the deceptive A. morio the emitted scent seems to be too weak to be detected by pollinators and thus its high variability may result from relaxed selection on this floral trait.

• Background and Aims Although numerous angraecoid orchids in Madagascar display typical sphingophilous syndrome (i.e. white, nectariferous, long-spurred flowers, producing a strong scent at the crepuscule that is attractive to moths), three species of Angraecum in Reunion, belonging to the endemic section Hadrangis, have atypical unscented and short-spurred flowers. The aim of the study was to investigate the implication of plant–pollinator interaction on the evolution of floral morphology of these peculiar island floral forms.

• Methods The flower morphology of A. striatum (one of the three section Hadrangis species) was investigated by performing a set of floral measures, and the reproductive biology was investigated by a set of hand pollination experiments. Natural pollinators were observed by means of a digital video camera. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified once a week in natural field conditions during the 2005 flowering season (i.e. from January to March).

• Key Results The orchid is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator was observed, the endemic white-eye Zosterops borbonicus (Zosteropidae). These birds perched on inflorescences, and probed most fresh-looking flowers on each plant for nectar. Nectar was both abundant (averaging 7·7 µL) and dilute (averaging 9·7 % sugar in sucrose equivalents). Birds were mostly active between 0830 and 0930 h. Visits to plants were extremely short, lasting from 9 to 27 s. At the study site, 60·9 % of flowers had pollen removed, and 46·4 % had pollinia deposited on stigmas. The proportion of flowers that initiated a fruit averaged 20·6 % in natural conditions.

• Conclusions For the first time, a bird-pollinated orchid is described from a sub-tribe that is mainly specialized for moth pollination. This study documents a morphological shift in flowers in response to pollinator adaptations in the insular context of the Mascarene Archipelago.

Background and Aims

Biological mutualisms rely on communication between partners, but also require protective measures against exploitation. Animal-pollinated flowers need to attract pollinators but also to avoid conflicts with antagonistic consumers. The view of flower visitors as mutualistic and antagonistic agents considers primarily the plants' interest. A classification emphasizing the consumer's point of view, however, may be more useful when considering animal's adaptations to flower visits which may include a tolerance against defensive floral scent compounds.

Methods

In a meta-analysis covering 18 studies on the responses of animals to floral scents, the animals were assigned to the categories of obligate and facultative flower visitors which considers their dependency on floral resources. Their responses on floral scents were compared.

Key Results

On average, obligate flower visitors, often corresponding to pollinators, were attracted to floral scent compounds. In contrast, facultative and mainly antagonistic visitors were strongly repelled by floral scents. The findings confirm that floral scents have a dual function both as attractive and defensive cues.

Conclusions

Whether an animal depends on floral resources determines its response to these signals, suggesting that obligate flower visitors evolved a tolerance against primarily defensive compounds. Therefore, floral scent bouquets in conjunction with nutritious rewards may solve the conflicting tasks of attracting mutualists while repelling antagonists.

The radiation of the angiosperms is often attributed to repeated evolutionary shifts between different pollinators, as this process drives diversification of floral forms and can lead to reproductive isolation. Floral scent is an important functional trait in many pollination systems but has seldom been implicated as a key mechanism in pollinator transitions. In this study, we suggest a role for sulphur compounds in mediating a shift between specialized carrion-fly and pompilid-wasp pollination systems in Eucomis (Hyacinthaceae). Flowers of closely related Eucomis species pollinated by carrion flies or pompilid wasps have very similar greenish-white flowers, but differ markedly in floral scent chemistry (determined by GC–MS analysis of headspace extracts). Comparison of the floral colours of the four Eucomis species in the visual systems of flies and wasps suggests that colour plays little role in pollinator discrimination. Nectar properties and morphology also do not differ strongly between fly- and wasp-pollinated flowers. By comparing floral scent bouquets and experimentally manipulating the scent of plants in the field, we demonstrate that shifts between wasp and fly pollination in these four congeners can depend on the production or suppression of sulphur compounds (dimethyl disulphide and dimethyl trisulphide) in the fragrance bouquet. This suggests that mutations affecting the production of particular scent compounds could precipitate shifts between pollinators, independently of floral morphology, colour or nectar properties.

Background and Aims

Sexually deceptive orchids achieve cross-pollination by mimicking the mating signals of female insects, generally hymenopterans. This pollination mechanism is often highly specific as it is based primarily on the mimicry of mating signals, especially the female sex pheromones of the targeted pollinator. Like many deceptive orchids, the Mediterranean species Ophrys arachnitiformis shows high levels of floral trait variation, especially in the colour of the perianth, which is either green or white/pinkinsh within populations. The adaptive significance of perianth colour polymorphism and its influence on pollinator visitation rates in sexually deceptive orchids remain obscure.

Methods

The relative importance of floral scent versus perianth colour in pollinator attraction in this orchid pollinator mimicry system was evaluated by performing floral scent analyses by gas chromatography-mass spectrometry (GC-MS) and behavioural bioassays with the pollinators under natural conditions were performed.

Key Results

The relative and absolute amounts of behaviourally active compounds are identical in the two colour morphs of O. arachnitiformis. Neither presence/absence nor the colour of the perianth (green versus white) influence attractiveness of the flowers to Colletes cunicularius males, the main pollinator of O. arachnitiformis.

Conclusion

Chemical signals alone can mediate the interactions in highly specialized mimicry systems. Floral colour polymorphism in O. arachnitiformis is not subjected to selection imposed by C. cunicularius males, and an interplay between different non-adaptive processes may be responsible for the maintenance of floral colour polymorphism both within and among populations.

The flowers and inflorescences of animal-pollinated dioecious plants are generally small and inconspicuous in comparison with outcrossing cosexual species. The net benefits of an attractive floral display may be different for dioecious compared to cosexual populations because dioecious species experience a more severe reduction in pollen delivery when pollinators forage longer on fewer individuals. Here, we develop a model that predicts the decrease in pollen delivery in dioecious relative to cosexual populations from female–female, female–male and male–male visit sequences as the number of individuals visited varies. To evaluate the predictions of our model we conducted a common garden experiment with dioecious and monoecious (cosexual) arrays of the insect-pollinated herb Sagittaria latifolia. We find that, although increasing the advertisements of floral rewards (i.e. increasing floral display) attracts more pollinators to individuals, the probability that these pollinators subsequently deliver pollen to neighbouring plants depends on sexual system. Because the number of individual plants visited per foraging trip did not increase significantly with floral display, the relative pollination success of dioecious versus monoecious populations decreases with increased floral display. We propose that this could explain why dioecy is strongly correlated with reduced floral display among angiosperm species.

Background and Aims

Among-population differentiation in phenotypic traits and allelic variation is expected as a consequence of isolation, drift, founder effects and local selection. Therefore, investigating molecular and quantitative genetic divergence is a pre-requisite for studies of local adaptation in response to selection under variable environmental conditions.

Methods

Among- and within-population variation were investigated in six geographically separated European populations of the white campion, Silene latifolia, both for molecular variation at six newly developed microsatellite loci and for quantitative variation in morphological and life-history traits. To avoid confounding effects of the maternal environment, phenotypic traits were measured on greenhouse-reared F1 offspring. Tests were made for clinal variation, and the correlations among molecular, geographic and phenotypic distances were compared with Mantel tests.

Key Results

The six populations of Silene latifolia investigated showed significant molecular and quantitative genetic differentiation. Geographic and phenotypic distances were significantly associated. Age at first flowering increased significantly with latitude and exhibited a Qst value of 0·17 in females and 0·10 in males, consistent with adaptation to local environmental conditions. By contrast, no evidence of isolation-by-distance and no significant association between molecular and phenotypic distances were found.

Conclusions

Significant molecular genetic divergence among populations of Silene latifolia, from the European native range is consistent with known limited seed and pollen flow distances, while significant quantitative genetic divergence among populations and clinal variation for age at first flowering suggest local adaptation.

Visual and olfactory floral signals are essential for the establishment of plant-pollinator mutualisms. Different batteries of floral features attract different pollinators and may achieve specific relationships that are essential for the immediate plant reproductive success, and at an evolutionary time scale have been of vital importance in the radiation of Angiosperms. We have found that mutualistic services by ants, insects traditionally considered ineffective pollinators, are essential for the pollination of Cytinus hypocistis (Cytinaceae), a Mediterranean root holoparasitic plant. Diverse floral signals, mainly nectar characteristics and floral scent could be playing a key role in the attraction of different species of ants, which pollinate effectively the flowers. Surprisingly, the abundance of other insects foraging in this parasite was very low and, although this scarcity could be due in part to the presence of ants, we suggest that different floral features exhibited by C. hypocistis could be evolving for attracting ants. Based on some current findings, we suspect that the study of floral signals in Cytinaceae is critical in the understanding the divergence of pollination systems in this fascinating family of parasitic plants.

Silene latifolia is dioecious, yet rare hermaphrodites have been found, and such natural mutants can provide valuable insight into genetic mechanisms. Here, we describe a hermaphrodite-inducing mutation that is almost certainly localized to the gynoecium-suppression region of the Y chromosome in S. latifolia. The mutant Y chromosome was passed through the megaspore, and the presence of two X chromosomes was not necessary for seed development in the parent. This result supports a lack of degeneration of the Y chromosome in S. latifolia, consistent with the relatively recent formation of the sex chromosomes in this species. When crossed to wild-type plants, hermaphrodites performed poorly as females, producing low seed numbers. When hermaphrodites were pollen donors, the sex ratio of offspring they produced through crosses was biased towards females. This suggests that hermaphroditic S. latifolia would fail to thrive and potentially explains the rarity of hermaphrodites in natural populations of S. latifolia. These results indicate that the Y chromosome in Silene latifolia remains very similar to the X, perhaps mostly differing in the primary sex determination regions.

Pollen fate can strongly affect the genetic structure of populations with restricted gene flow and significant inbreeding risk. We established an experimental population of inbred and outbred Silene latifolia plants to evaluate the effects of (i) inbreeding depression, (ii) phenotypic variation and (iii) relatedness between mates on male fitness under natural pollination. Paternity analysis revealed that outbred males sired significantly more offspring than inbred males. Independently of the effects of inbreeding, male fitness depended on several male traits, including a sexually dimorphic (flower number) and a gametophytic trait (in vitro pollen germination rate). In addition, full-sib matings were less frequent than randomly expected. Thus, inbreeding, phenotype and genetic dissimilarity simultaneously affect male fitness in this animal-pollinated plant. While inbreeding depression might threaten population persistence, the deficiency of effective matings between sibs and the higher fitness of outbred males will reduce its occurrence and counter genetic erosion.

Evidence for dosage compensation in Silene latifolia, a plant with 10-million-year-old sex chromosomes, reveals that dosage compensation can evolve rapidly in young XY systems and is not an animal-specific phenomenon.

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ∼10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.

Author Summary

The mammalian sex chromosomes originated from an ancestral pair of autosomes about 150 million years ago and the Y chromosome subsequently degenerated, losing most of its genes. During this process, a phenomenon called dosage compensation evolved to compensate for the gene loss on the Y chromosome and to equalize expression of X-linked genes in the two sexes. In humans, this is achieved by inactivating one of the two X chromosomes in females. Dosage compensation has also been reported in other animal XY systems such as fruit flies and worms, each 100 million years old or more. Here we studied dosage compensation in plants. We used high-throughput RNA sequencing in male and female Silene latifolia (white campion)—a dioecious plant whose XY chromosomes originated only about 10 million years ago—to identify hundreds of sex-linked genes. Analysis of their expression patterns in males and females revealed equal doses of sex-linked transcripts in both sexes, regardless of the degree of reduction of Y expression due to degeneration. Our results thus show that dosage compensation occurs in plants and is thus not an animal-specific phenomenon. They also reveal that proportionate dosage compensation can evolve rapidly de novo after the origin of sex chromosomes.

Background

Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis.

Results

The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives.

Conclusion

This systems biology program combined chemical analysis, genomics and bioinformatics to elucidate the scent biosynthesis pathway and identify the relevant genes. It integrates the forward and reverse genetic approaches to knowledge discovery by which researchers can study non-model plants.