Reproductive biology of Syagrus coronata (Arecaceae): sex‐biased insect visitation and the unusual case of scent emission by peduncular bracts

• Several monoecious species of palms have developed complex strategies to promote cross‐pollination, including the production of large quantities of floral resources and the emission of scents that are attractive to pollinators. Syagrus coronata constitutes an interesting model with which to understand the evolution of plant reproductive strategies in a monoecious species adapted to seasonally dry forests.
• We monitored blooming phenology over 1 year, during which we also collected and identified floral visitors and putative pollinators. We identified potential floral visitor attractants by characterizing the scent composition of inflorescences as well as of peduncular bracts, during both male and female phases, and the potential for floral thermogenesis.
• Syagrus coronata produces floral resources throughout the year. Its inflorescences are predominantly visited by a diverse assortment of small‐sized beetles, whose richness and abundance vary throughout the different phases of anthesis. We did not find evidence of floral thermogenesis. A total of 23 volatile compounds were identified in the scent emitted by the inflorescences, which did not differ between male and female phases; whereas the scent of the peduncular bracts was composed of only 4‐methyl guaiacol, which was absent in inflorescences.
• The composition of floral scent chemistry indicates that this palm has evolved strategies to be predominantly pollinated by small‐sized weevils. Our study provides rare evidence of a non‐floral scent emitting structure involved in pollinator attraction, only the second such case specifically in palms. The peculiarities of the reproductive strategy of S. coronata might play an important role in the maintenance of pollination services and pollen dispersion.

     

Diversity of pollination ecology in the Schismatoglottis Calyptrata Complex Clade (Araceae)

• Field studies integrating pollination investigations with an assessment of floral scent composition and thermogenesis in tropical aroids are rather few. Thus, this study aimed to investigate the pollination biology of nine species belonging to Schismatoglottis Calyptrata Complex Clade. The flowering mechanism, visiting insect activities, reproductive system, thermogenesis and floral scent composition were examined.
• Anthesis for all species started at dawn and lasted 25–29 h. Colocasiomyia (Diptera, Drosophilidae) are considered the main pollinators for all the investigated species. Cycreon (Coleoptera, Hydrophilidae) are considered secondary pollinators as they are only present in seven of the nine host plants, despite the fact that they are the most effective pollen carrier, carrying up to 15 times more pollen grains than Colocasiomyia flies. However, the number of Colocasiomyia individuals was six times higher than Cycreon beetles. Chaloenus (Chrysomelidae, Galeuricinae) appeared to be an inadvertent pollinator. Atheta (Coleoptera, Staphylinidae) is considered a floral visitor in most investigated species of the Calyptrata Complex Clade in Sarawak, but a possible pollinator in S. muluensis. Chironomidae midges and pteromalid wasps are considered visitors in S. calyptrata.
• Thermogenesis in a biphasic pattern was observed in inflorescences of S. adducta, S. calyptrata, S. giamensis, S. pseudoniahensis and S. roh. The first peak occurred during pistillate anthesis; the second peak during staminate anthesis. Inflorescences of all investigated species of Calyptrata Complex Clade emitted four types of ester compound, with methyl ester‐3‐methyl‐3‐butenoic acid as a single major VOC (volatile organic compound). The appendix, pistillate zone, staminate zone and spathe emitted all these compounds.
• A mixed fly–beetle pollination system is considered an ancestral trait in the Calyptrata Complex Clade, persisting in Sarawak taxa, whereas the marked reduction of interpistillar staminodes in taxa from Peninsular Malaysia and especially, Ambon, Indonesia, is probably linked to a shift in these taxa to a fly‐pollinated system.

     

Floral scent composition predicts bee pollination system in five butterfly bush (Buddleja,Scrophulariaceae) species

Traditionally, plant–pollinator interactions have been interpreted as pollination syndrome. However, the validity of pollination syndrome has been widely doubted in modern studies of pollination ecology. The pollination ecology of five Asian Buddleja species, B. asiatica, B. crispa, B. forrestii, B. macrostachya and B. myriantha, in the Sino‐Himalayan region in Asia, flowering in different local seasons, with scented inflorescences were investigated during 2011 and 2012. These five species exhibited diverse floral traits, with narrow and long corolla tubes and concealed nectar. According to their floral morphology, larger bees and Lepidoptera were expected to be the major pollinators. However, field observations showed that only larger bees (honeybee/bumblebee) were the primary pollinators, ranging from 77.95% to 97.90% of total visits. In this study, floral scents of each species were also analysed using coupled gas chromatography and mass spectrometry (GC‐MS). Although the five Buddleja species emitted differentiated floral scent compositions, our results showed that floral scents of the five species are dominated by substances that can serve as attractive signals to bees, including species‐specific scent compounds and principal compounds with larger relative amounts. This suggests that floral scent compositions are closely associated with the principal pollinator assemblages in these five species. Therefore, we conclude that floral scent compositions rather than floral morphology traits should be used to interpret plant–pollinator interactions in these Asian Buddleja species.     

First records of flower biology and pollination in African Annonaceae: Isolona, Piptostigma, Uvariodendron, Monodora and Uvariopsis

Seven species from five genera of Annonaceae were studied with regard to their flower biology and pollination in the Southwest Province of Cameroon, West Africa. They have protogynous hermaphroditic flowers, with exception of Uvariopsis species, which are monoecious. Fused petals of Isolona campanulata remain apically spreading and open during anthesis but form a deep basal urceolate tube around the reproductive organs. At anthesis the yellow pendent flowers emit a fruit-like scent and attracted small beetles, the likely pollinators. Piptostigma sp. flowers also emit a fruit-like scent but provide a closed pollination chamber formed by the three inner petals. Small staphylinid beetles attracted during the female stage of anthesis are released from the flowers at the end of the male stage 2-3 days later. Both species have diurnal anthesis, attracting and releasing the flower visitors during daytime. In contrast, Uvariodendron connivens and U. calophyllum have nocturnal anthesis with floral thermogenesis, produce spicy, aromatic and fruity scents and attract large Scarabaeidae beetles, the pollinators, along with many curculionid beetles, which were principally predators of the thick petals. The very large flowers of Monodora tenuifolia have yellowish petals which are spotted with dark red markings. Together with the sweetish, slightly disagreeable scent the flowers attract flies, principally dung flies. The two investigated Uvariopsis species are monoecious with pistillate and staminate flowers being functional at the same time. The violet red flowers of U. bakeriana visually seem to mimic the fruiting body of certain stinkhorn fungi (Phallaceae) although without producing their strong unpleasant carcass stench. Flower-visiting dung flies were rare. Conversely, U. congolana has a strong fungus-like scent, its flowers are presented at litter height and dung flies living in the litter were the flower visitors, albeit sporadic. The 4-5 days lasting anthesis of both Uvariopsis species appears to be an evolutionary consequence of their diffuse pollinator spectra. The studied African Annonaceae therefore have either cantharophilous or myiophilous/sapromyiophilous flowers with, in part, respectively, remarkably long anthesis, thermogenesis, and widely open, large flowers - all attributes unknown or rare in the hitherto better studied Neotropical Annonaceae.     

Evolutionary breakdown of distyly to homostyly is accompanied by reductions of floral scent in Primula oreodoxa

Reproductive traits that function in pollinator attraction may be reduced or lost during evolutionary transitions from outcrossing to selfing. Although floral scent plays an important role in attracting pollinators in outcrossing species, few studies have investigated associations between floral scent variation and intraspecific mating system transitions. The breakdown of distyly to homostyly represents a classic example of a shift from outcrossing to selfing and provides an opportunity to test whether floral fragrances have become reduced and/or changed in composition with increased selfing. Here, we evaluate this hypothesis by quantifying floral volatiles using gas chromatography-mass spectrometry in two distylous and four homostylous populations of Primula oreodoxa Franchet, a perennial herb from SW China. Our analysis revealed significant variation of volatile organic compounds (VOCs) among populations of P. oreodoxa. Although there was no difference in VOCs between floral morphs in distylous populations as predicted, we detected a substantial reduction in VOC emissions and the average number of scent compounds in homostylous compared with distylous populations. A total of 12 compounds, mainly monoterpenoids and sesquiterpenoids, distinguished homostylous and distylous morphs; of these, (E)-β-ocimene was the most important in contributing to the difference in volatiles, with significantly lower emissions in homostyles. Our findings support the hypothesis that the transition from outcrossing to selfing is accompanied by the loss of floral volatiles. The modification to floral fragrances in P. oreodoxa associated with mating system change might occur because high selfing rates in homostylous populations result in relaxed selection for floral attractiveness.     

Specialised protagonists in a plant–pollinator interaction: the pollination of Blumenbachia insignis (Loasaceae)

• Analyses of resource presentation, floral morphology and pollinator behaviour are essential for understanding specialised plant‐pollinator systems. We investigated whether foraging by individual bee pollinators fits the floral morphology and functioning of Blumenbachia insignis, whose flowers are characterised by a nectar scale‐staminode complex and pollen release by thigmonastic stamen movements.
• We described pollen and nectar presentation, analysed the breeding system and the foraging strategy of bee pollinators. We determined the nectar production pattern and documented variations in the longevity of floral phases and stigmatic pollen loads of pollinator‐visited and unvisited flowers.
• Bicolletes indigoticus (Colletidae) was the sole pollinator with females revisiting flowers in staminate and pistillate phases at short intervals, guaranteeing cross‐pollen flow. Nectar stored in the nectar scale‐staminode complex had a high sugar concentration and was produced continuously in minute amounts (~0.09 μl·h^?1). Pushing the scales outward, bees took up nectar, triggering stamen movements and accelerating pollen presentation. Experimental simulation of this nectar uptake increased the number of moved stamens per hour by a factor of four. Flowers visited by pollinators received six‐fold more pollen on the stigma than unvisited flowers, had shortened staminate and pistillate phases and increased fruit and seed set.
• Flower handling and foraging by Bicolletes indigoticus were consonant with the complex flower morphology and functioning of Blumenbachia insignis. Continuous nectar production in minute quantities but at high sugar concentration influences the pollen foraging of the bees. Partitioning of resources lead to absolute flower fidelity and stereotyped foraging behaviour by the sole effective oligolectic bee pollinator.

     

Scent glands in legume flowers

Scent glands, or osmophores, are predominantly floral secretory structures that secrete volatile substances during anthesis, and therefore act in interactions with pollinators. The Leguminosae family, despite being the third largest angiosperm family, with a wide geographical distribution and diversity of habits, morphology and pollinators, has been ignored with respect to these glands. Thus, we localised and characterised the sites of fragrance production and release in flowers of legumes, in which scent plays an important role in pollination, and also tested whether there are relationships between the structure of the scent gland and the pollinator habit: diurnal or nocturnal. Flowers in pre‐anthesis and anthesis of 12 legume species were collected and analysed using immersion in neutral red, olfactory tests and anatomical studies (light and scanning electron microscopy). The main production site of floral scent is the perianth, especially the petals. The scent glands are distributed in a restricted way in Caesalpinia pulcherrima, Anadenanthera peregrina, Inga edulis and Parkia pendula, constituting mesophilic osmophores, and in a diffuse way in Bauhinia rufa, Hymenaea courbaril, Erythrostemon gilliesii, Poincianella pluviosa, Pterodon pubescens, Platycyamus regnellii, Mucuna urens and Tipuana tipu. The glands are comprised of cells of the epidermis and mesophyll that secrete mainly terpenes, nitrogen compounds and phenols. Relationships between the presence of osmophores and type of anthesis (diurnal and nocturnal) and the pollinator were not found. Our data on scent glands in Leguminosae are original and detail the type of diffuse release, which has been very poorly studied.     

Pollination in Geonoma macrostachys and Three Congeners,G. acaulis,G. gracilis,and G. interrupta

A continuous 15 month study of the floral ecology of four syntopic understorey palm species of Genoma was conducted in Amazonian Peru lowland rainforest. The spicate inflorescences of G. macrostachys, G. acaulis and G. gracilis are strictly protandrous and the plants are functionally dioecious. Data suggest that in G. macrostachys and G. acaulis pollination is based on a mimicry system, the pistillate flowers mimicking the staminate ones in colour, shape and scent. Pollen-collecting meliponine bees (Hymenoptera, Apidae, Meliponinae) and pollen-feeding syrphid flies (Diptera, Syrphidae) which visit inflorescences during both sexual stages are the pollinators of G. macrostachys. Geonoma acaulis is pollinated by small pollen-feeding weevils (Coleoptera, Curculionidae, Derelomini) that visit male and female spikes. Additionally, in G. macrostachys another pollinator type, viz. euglossine bees (Hymenoptera, Apidae, Euglossinae), which are attracted and rewarded by both types of flowers may account for long-distance pollination. The palm G. gracilis shows a very distinct pollination system. Although opportunistic insect visitors are attracted to the inflorescences of this species it seems to be mainly anemophilous because pollen becomes powdery during an thesis. The branched inflorescences of G. interrupta are also protandrous, but unlike the other species of Geonoma observed, staminate and pistillate anthesis of individual flowers are, for the most, overlapping. A broad spectrum of visitors is attracted (bees, wasps, flies, and beetles), which all may act as pollinators. Outcrossing is especially encouraged during the purely female phase at the end of the flowering cycle when there are no more staminate flowers in the inflorescence. Effects on the reproductive biology and population structure of different pollination systems and breeding system are discussed.     

Diel fragrance pattern correlates with olfactory preferences of diurnal and nocturnal flower visitors in Salix caprea (Salicaceae)

Floral scent, often a complex mixture of several volatile organic compounds (VOCs), has generally been interpreted as an adaptation to attract pollinators. However, not many studies have analysed which VOCs are functionally relevant for the reproductive success of a plant. Here, we show that, in Salix caprea (Salicaceae), temporal changes in floral scent emission during the day and night attract two different types of flower visitor: bees during the day and moths during the evening and night. We analysed the contribution of the two flower visitor groups to the reproductive success of the plant. The differences in scent emitted during the peak activity times of flower visitors (day versus night) were quantified and the response of 13 diurnal/nocturnal pollinator taxa to the floral scents was tested using gas chromatographic and electroantennographic techniques. Many of the c. 40 identified scent compounds were physiologically active, and bees and moths responded to nearly identical sets of compounds, although the response strengths differed. In bioassays, bees preferred the most abundant 1,4‐dimethoxybenzene over lilac aldehyde, a compound with increased emission at night, whereas moths preferred lilac aldehyde over 1,4‐dimethoxybenzene. Pollination by wind plus nocturnal pollinators (mainly moths) or by wind alone contributed less to seed set than pollination by wind plus diurnal pollinators (mainly bees). This suggests that the emission of scent during the night and attracting moths have no significant effect on reproductive success. It is possible that the emission of lilac aldehydes and other compounds at night is s result of phylogenetic constraints. Future studies should investigate whether moths may produce a marginal fitness gain in some years and/or some populations. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 624–640.     

Parallel chemical switches underlying pollinator isolation in Asian Mitella

Floral scents are among the key signals used by pollinators to navigate to specific flowers. Thus, evolutionary changes in scents should have strong impacts on plant diversification, although scent‐mediated plant speciation through pollinator shifts has rarely been demonstrated, despite being likely. To examine whether and how scent‐mediated plant speciation may have occurred, we investigated the Asimitellaria plant lineage using multidisciplinary approaches including pollinator observations, chemical analyses of the floral scents, electroantennographic analyses and behavioural bioassays with the pollinators. We also performed phylogenetically independent contrast analyses of the pollinator/floral scent associations. First, we confirmed that the pairs of the sympatric, cross‐fertile Asimitellaria species in three study sites consistently attract different pollinators, namely long‐tongued and short‐tongued fungus gnats. We also found that a stereoisomeric set of floral volatiles, the lilac aldehydes, could be responsible for the pollinator specificity. This is because the compounds consistently elicited responses in the antennae of the long‐tongued fungus gnats and had contrasting effects on the two pollinators, that is triggering the nectaring behaviour of long‐tongued fungus gnats while repelling short‐tongued fungus gnats in a laboratory experiment. Moreover, we discovered that volatile composition repeatedly switched in Asimitellaria between species adapted to long‐tongued and short‐tongued fungus gnats. Collectively, our results support the idea that recurrent scent‐mediated speciation has taken place in the Asimitellaria–fungus gnat system.     

Heterodichogamy and nitidulid beetle pollination in Anaxagorea prinoides, an early divergent Annonaceae

Heterodichogamy in a natural population of an Annonaceae species from the rainforests of French Guiana is described for the first time. Anaxagorea prinoides had bisexual flowers and two floral morphs within the studied population were protogynous. The population under study comprised 7 mature trees belonging to one morph and 12 to the other. Statistical analyses showed that the two morphs were in a 50:50% ratio, and therefore the temporal sexual pattern of heterodichogamy is given. When anthesis of flowers in the male stage ended in one morph, anthesis started with flowers in the female stage in the complementary morph. Approximately 1?h before the end of anthesis in one morph, flowers of the reciprocal morph started to emit a fruit-like scent. The temporal separation of the female and the male stages of the two different morphs lasted only approximately 1?h. Six of the seven identified compounds in the banana-like floral scent were esters and one was an alcohol. The main compounds examined are known to be components of fruit scents. Nitidulidae beetles of the genus Colopterus were the pollinators of A. prinoides and during flowering were maintained within the population of this species. This was not only due to the fact that the beetles remained sheltered in the pollination chamber of the flowers, but also because upon release from individuals of male-stage flowers at the end of flower anthesis they were attracted by the odoriferous female-stage flowers of other individuals of the same population. Heterodichogamy of A. prinoides appears to be a means by which reproductive success is augmented. Attraction of beetle pollinators by ??fruit-imitating?? floral scent is not restricted to species of Anaxagorea, but occurs in many representatives of the Annonaceae.     

Pollinator shifts between Ophrys sphegodes populations: might adaptation to different pollinators drive population divergence?

Local adaptation to different pollinators is considered one of the possible initial stages of ecological speciation as reproductive isolation is a by‐product of the divergence in pollination systems. However, pollinator‐mediated divergent selection will not necessarily result in complete reproductive isolation, because incipient speciation is often overcome by gene flow. We investigated the potential of pollinator shift in the sexually deceptive orchids Ophrys sphegodes and Ophrys exaltata and compared the levels of floral isolation vs. genetic distance among populations with contrasting predominant pollinators. We analysed floral hydrocarbons as a proxy for floral divergence between populations. Floral adoption of pollinators and their fidelity was tested using pollinator choice experiments. Interpopulation gene flow and population differentiation levels were estimated using AFLP markers. The Tyrrhenian O. sphegodes population preferentially attracted the pollinator bee Andrena bimaculata, whereas the Adriatic O. sphegodes population exclusively attracted A. nigroaenea. Significant differences in scent component proportions were identified in O. sphegodes populations that attracted different preferred pollinators. High interpopulation gene flow was detected, but populations were genetically structured at species level. The high interpopulation gene flow levels independent of preferred pollinators suggest that local adaptation to different pollinators has not (yet) generated detectable genome‐wide separation. Alternatively, despite extensive gene flow, few genes underlying floral isolation remain differentiated as a consequence of divergent selection. Different pollination ecotypes in O. sphegodes might represent a local selective response imposed by temporal variation in a geographical mosaic of pollinators as a consequence of the frequent disturbance regimes typical of Ophrys habitats.     

Scent emissions and floral nutrients of Carludovicoideae (Cyclanthaceae) and their importance for associated beetles

To better understand the complex pollination biology of Carludovicoideae (Cyclanthaceae), four species from French Guiana were investigated in both the field and laboratory. The pistillate flowers of all species of the subfamily have long staminodes up to 10 cm long or more, which emit scent and apparently attract beetles during anthesis. Scent was collected by standard headspace methods and analyzed in the laboratory by GC/MS. The histology of staminodes, measurements of inflorescence temperatures, and analyses of the floral nutrients were performed. The staminodes have two ducts with a mucilage-like liquid containing sugar, which provides nourishment for beetle visitors. In Evodianthus funifer, four of the six beetle morphospecies (Curculionidae: Acalyptini) were pollinators and oviposited in staminate flowers. The remaining pair of morphospecies were non-pollinators, and avoided entering the inflorescence, while only cutting the staminodes for possible oviposition on the ground. Staminate flowers and staminodes have a high energy content, providing for larval development of the beetles. Our findings revealed that the staminodes released aromatic components, miscellaneous cyclic components, and terpenoids. Some of the major scent compounds of E. funifer and Ludovia lancifolia, i.e., (E,E)-α-farnesene-2(3),9(10)-diepoxid and 3-methylen-2-(pent-2(Z)-enyl)-cyclopentanol, are new to science. Also, Carludovicoideae are a subfamily of plants that attract beetle pollinators through highly specific scent compounds, making them comparable to species of Araceae, Magnoliaceae, and Annonaceae.     

Contrasting floral biology of Artabotrys species (Annonaceae): Implications for the evolution of pollinator trapping

Artabotrys comprises a species-poor early divergent grade (EDG) and a main species-rich clade (“main Artabotrys clade,” MAC). All members of the MAC exhibit a remarkably well-conserved floral Bauplan characterized by a tightly enclosed floral chamber with an elaborate rim between the inner petal blade and claw. Conversely, EDG resembles the sister genus Xylopia in lacking the inner petal rim, with one species (Artabotrys brachypetalus) bearing petals that lack the distinction between blade and claw altogether. The floral phenology and pollination ecology of two exemplar species—Artabotrys brachypetalus from the EDG and Artabotrys blumei from the MAC—are compared, including assessment of effective pollinators and their activity patterns, scent chemistry and thermogenesis. Our study revealed that Artabotrys blumei exhibits abbreviated anthesis (c. 27 hr), with a floral phenology and morphology that are clearly consistent with pollinator trapping, and may be pollinated by small beetles. Artabotrys brachypetalus has a typical anthetic duration (c. 45 hr), lacks a pollinator trapping mechanism, and is pollinated by honey bees and curculionid beetles. The “xylopioid” traits of the EDG are likely to be plesiomorphic, whereas the tightly enclosed floral chamber is likely to be apomorphic for the MAC and functionally significant in trapping pollinators.     

Variation in scent emission among floral parts and inflorescence developmental stages in beetle-pollinated Protea species (Proteaceae)

Floral fragrances are an important component for pollinator attraction in beetle-pollinated flowers. Several genera in the Proteaceae contain beetle-pollinated species. However, there is no information on the floral scent chemistry of beetle-pollinated members of the family. In this paper we report on the spatial variation and differences between developmental stages in emission of inflorescence (flowerhead) volatiles of four South African Protea species (P. caffra, P. dracomontana, P. simplex, and P. welwitschii) that are pollinated by cetoniine beetles. The scents from different inflorescence parts (bracts, perianth, styles, and nectar) and from successive anthesis stages of whole inflorescences were sampled using dynamic headspace collection and identified using GC–MS. Although the four species shared many scent compounds, possibly reflecting their close phylogenetic relationships and common pollinators, they showed significant differences in overall scent composition due to various species-specific compounds, such as the unique tiglate esters found in the scent of P. welwitschii. The strongest emissions and largest number of volatiles, especially monoterpenes, were from inflorescences at full pollen dehiscence. Senescing inflorescences of two species and nectars of all species emitted proportionally high amounts of acetoin (3-hydroxy-2-butanone) and aromatic alcohols, typical fermentation products. As a consequence, the scent composition of nectar was much more similar among species than was the scent composition of other parts of the inflorescence. These results illustrate how the blends of compounds that make up the overall floral scent are a dynamic consequence of emissions from various plant parts.     

THE INTRAFLORAL PHENOLOGY OF STREPTANTHUS TORTUOSUS (BRASSICACEAE)

The sequence of floral events during anthesis was examined in Streptanthus tortuosus to determine the relationship between the male and female floral phases. The flowers are strongly protandrous. In the staminate phase, the anthers mature sequentially over a 3–4-day period. Because pollinators quickly remove pollen from the anthers, sequential anther maturation prolongs the male phase relative to what it would be if anthers did not mature sequentially. Pollen applied to the stigma during the staminate phase does not adhere readily and does not germinate. The length of the pistillate phase depends on pollinator activity, as pollination accelerates the abscission of floral parts. Unpollinated flowers remain pistillate for 3–4 days, during which time stigmatic receptivity declines gradually. In the field, 72% to 80% of flowers are staminate at any time, indicating that the staminate phase is three times longer than the pistillate phase when pollinators have access to the flowers. The consequences of the relative length of the floral phases and the schedule of stigmatic receptivity are discussed in terms of outcrossing mechanism, floral longevity, and sexual selection models.     

Pollination biology of Aristolochia bianorii Sennen & Pau: promoting cross-pollination but assuring reproductive success in island ecosystems

• Deceptive pollination has been reported in the genus Aristolochia, but the floral biology and pollination strategy of A. bianorii, an endemic of the Balearic Islands, have not yet been studied. Here, we investigated floral anthesis, mating system, pollinators and volatile organic compounds (VOCs) emitted by its flowers.
• Flower buds were marked and monitored daily to define floral stages and their duration. Experimental bagging and hand-pollination were performed to test for autonomous self-pollination, induced self-pollination and cross-pollination. Flowers were collected to analyse the presence of entrapped pollinators. VOCs emitted by flowers were evaluated by means of solid phase microextraction followed by immediate GC–MS.
• Anthesis lasted between 63 and 96 h, and the species exhibited autonomous self-pollination with moderate inbreeding depression. Pollinators were mainly females of Oscinomorpha longirostris (Diptera; Chloropidae). The number of pollinators inside flowers was affected by floral stage and time of flowering. The most common VOCs were alkanes, oximes, esters, alkenes, cyclic unsaturated hydrocarbons, isocyanates, amides and carboxylic acids.
• Aristolochia bianorii can set seed by autonomous self-pollination, in contrast to other Aristolochia species, in which both protogyny and herkogamy prevent autonomous self-pollination. However, the species may encourage cross-pollination by attracting female chloropid flies though emission of floral scents that may mimic an oviposition site and, possibly, freshly killed true bugs (i.e. Heteroptera). In conclusion, A. bianorii promotes cross-pollination, but delayed autonomous self-pollination assures reproductive success in the putative absence of pollinators.

     

A test of phenotypic selection on petal form in the wild carnation,Dianthus inoxianus

Floral phenotypes are considered a product of pollinator‐mediated selection, which also has the side effect of decreasing floral variation within species. Correlates of flower visibility and function were studied in a carnation species (Dianthus inoxianus), which has crepuscular anthesis and scent‐based pollination by the hawkmoth Hyles livornica. We also assessed constancy of flower form in nature and in cultivation and, using fruit set as an estimate of plant relative fitness, tested whether the main pollinator exerted phenotypic selection on floral traits. Petal claw, which is roughly equivalent to the average depth at which an insect's proboscis must be inserted to reach nectar, was remarkably constant among wild plants (coefficient of variation 8%). In contrast, the area of the visible part of the petal, and the intensity of a coloured dot pattern on the petal was very variable (respectively CV = 34% and 102%). Cultivation in a common environment revealed significant variation among genotypes as regards petal area, degree of laciniation and extension of the dot pattern, but not petal claw length, which remained steady. Petal area, shape and colour did not affect relative fitness during the year of study, but plants with intermediate petal claws (i.e. floral tubes) set significantly more fruit. Results are compatible with low response of the main pollinator to variation in visual traits (petal area, laciniation, colour) and high responsiveness to variation in other aspects (tube length). Inconsistent phenotypic selection by pollinators may add to other causes of floral variation in the genus Dianthus, the causes of which are discussed.     

Absence of pollinator-mediated premating barriers in mixed-ploidy populations of <Emphasis Type="Italic">Gymnadenia conopsea</Emphasis> s.l. (Orchidaceae)

Polyploidy has played a key role in plant evolution and diversification. Despite this, the processes governing reproductive isolation among cytotypes growing in mixed-ploidy populations are still largely unknown. Theoretically, coexistence of diploid and polyploid individuals in sympatric populations is unlikely unless cytotypes are prezygotically isolated through assortative pollination. Here, we investigated the pre-mating barriers involved in the maintenance of three co-occurring cytotypes from the genus Gymnadenia (Orchidaceae): tetraploid and octoploid G. conopsea and tetraploid G. densiflora. We assessed differences in flowering phenology, floral morphology, and visual and olfactory cues, which could lead to assortative mating. Gas chromatography coupled with electroantennographic detection was used to identify scent compounds with physiological activity in the two main pollinators, Deilephila porcellus and Autographa gamma. The importance of olfactory cues was also assessed in the field by analysing the moths’ responses to the olfactory display of the plants, and by following the pollinator’s behaviour on artificial arrays. Our complex approach demonstrated that the coexistence of Gymnadenia cytotypes in mixed-ploidy populations was only partly explained by differences in floral phenology, as cytotypes with overlapping flowering (i.e., octoploid G. conopsea and tetraploid G. densiflora) might freely exchange pollen due to only 1 mm differences in spur lengths and the lack of assortative behaviour of pollinators. While floral colour among the cytotypes was similar, floral scent differed significantly. Though both pollinator species seemed to physiologically detect these differences, and the floral scent alone was sufficient to attract them, pollinators did not use this cue to discriminate the cytotypes in the field. The absence of pre-mating barriers among cytotypes, except partial temporal segregation, suggests the existence of other mechanisms involved in the cytotypes’ coexistence. The genetic differences in ITS sequences among cytotypes were used to discuss the cytotype’s origin.