'Bleeding' flowers of Ceropegia gerrardii (Apocynaceae-Asclepiadoideae) mimic wounded insects to attract kleptoparasitic fly pollinators.

Kleptomyiophily, where flowers imitate wounded insects to attract 'kleptoparasitic' flies as pollinators, is one of the most specialized types of floral mimicry and often involves physical trapping devices. However, the diversity of pollinators and functional floral traits involved in this form of mimicry remain poorly understood. We report a novel example of kleptomyiophily in the nontrapping flowers of Ceropegia gerrardii and explore the floral traits responsible for attracting pollinators. The pollinators, reproductive biology and floral traits (including epidermal surfaces, spectral reflectance and the composition of nectariferous petal secretions and scent) were investigated. Attractive volatiles were identified using electrophysiological and behavioural experiments. Ceropegia gerrardii was predominantly pollinated by kleptoparasitic Desmometopa spp. (Milichiidae) flies. The flower corollas extrude a protein- and sugar-containing secretion, similar to the haemolymph of wounded insects, on which the flies feed. Floral scent was chemically similar to that of injured honey bees. Four out of 24 electrophysiologically active compounds, all released by injured honey bees, were identified as key players in pollinator attraction. Our results suggest that C. gerrardii flowers chemically mimic wounded honey bees to attract kleptoparasitic flies and reward them with a secretion similar to the haemolymph on which they would normally feed.

The enantiostylous floral polymorphism of Barberetta aurea (Haemodoraceae) facilitates wing pollination by syrphid flies.

BACKGROUND AND AIMS: Sexual polymorphisms of flowers have traditionally been interpreted as devices that promote cross-pollination, but they may also represent adaptations for exploiting particular pollination niches in local environments. The cross-pollination function of enantiostyly, characterized by flowers having either left- or right-deflected styles, has been uncertain in some lineages, such as the Haemodoraceae, because the positioning of stamens and styles is not always completely reciprocal among morphs. METHODS: We examined the floral biology of populations of the poorly known species Barberetta aurea (Haemodoraceae) across its native range in South Africa to establish the general features of its enanatiostylous reproductive system and the agents and mechanism of pollen transfer. RESULTS: We confirmed that B. aurea has a system of dimorphic enantiostyly. Style morph ratios varied among populations sampled, but with an overall tendency to being equal. Crossing experiments demonstrated that B. aurea is fully self-compatible, that intra- and inter-morph crosses are equally fertile and that it is wholly dependent on pollinator visits for seed production. Pollination is mainly by syrphid flies that transfer the sticky pollen via their wings, which contact the anthers and stigma precisely as they hover during approach and feeding. The majority of syrphid fly visitors feed on a film of highly concentrated nectar situated at the base of ultraviolet-absorbent 'nectar guides'. Because one of the three stamens is deflected in the same direction as the style, we predicted a high likelihood of intra-morph pollination, and this was corroborated by patterns of transfer of coloured dye particles in cage experiments involving syrphid flies. CONCLUSIONS: Barbaretta aurea exhibits dimorphic enantiostyly and, in contrast to most enantiostylous species, which are pollinated by bees, its flowers are specialized for pollination by syrphid flies. The lack of complete reciprocity of the enantiostylous arrangement of sexual organs facilitates both inter- and intra-morph pollen transfer on the wings of these flies.

Observations on the pollination and breeding systems of two Corybas species (Diurideae; Orchidaceae) by fungus gnats (Mycetophilidae) in southwestern Yunnan, China.

Modes of floral presentation in some angiosperms attract flies that eat and/or oviposit on seasonal fruiting bodies of fungi. Mushroom mimesis by orchid flowers has been speculated in the geoflorous, Indo-Malaysian-Australasian, genus Corybas s.l. for decades but most studies remain fragmentary and are often inconclusive. Here we report the roles of fungus gnats as pollinators of Corybas geminigibbus and C. shanlinshiensis in southwestern Yunnan, China, combining results of field observations, lab analyses, and manipulative experiments. Hand pollination experiments suggested both species were self-compatible but incapable of mechanical self-pollination, thereby requiring pollinators for fruit production. A female of a Phthinia sp. (Mycetophilidae) carried a pollinarium of C. geminigibbus dorsally on its thorax. Two females and one male of Exechia sp. (Mycetophilidae) visiting flowers of C. shanlinshiensis carried dorsal depositions of pollinaria on their thoraces. Mycetophilid eggs were not found in the flowers of either species. The comparative fragrance analyses of these flowers and three co-fruiting mushroom species did not suggest that either orchid species was a brood-site mimic. This is the first confirmation of the dispersal of pollinaria of Corybas species by fungus gnats in subtropical-temperate Asia.

Pre-pollination barriers between two sympatric Arisaema species in northern Shikoku Island, Japan.

PREMISE: The genus Arisaema (Araceae) has rapidly diversified in Japan, and multiple species often coexist in the field. Although Japanese Arisaema species hybridize from artificial crossing, hybrid individuals are rare in mixed populations; suggesting the presence of effective pre-pollination barriers. We examined the following reproductive barriers between A. sikokianum and A. tosaense: habitat, phenology, and pollinator isolations. METHODS: Habitat isolation was examined by interspecific comparisons of microhabitat conditions at a mixed site and of altitude at the sampling site of herbarium specimens. Phenological isolation was evaluated by comparing seasonal transition in apparent spathe condition and frequency of insect visitation. Pollinator isolation was examined by comparing floral visitor assemblages between the two Arisaema species. To avoid overestimation of pollinator isolation due to seasonal changes in insect assemblages, we also compared visitor assemblages between natural and late-flowering A. sikokianum, where the latter was experimentally introduced and blooming with a natural A. tosaense population. RESULTS: Microhabitat conditions and sampling elevations of herbarium specimens overlapped between the two Arisaema species. At the population level, A. sikokianum and A. tosaense flowered for 39 and 52 days, respectively, with 13 days overlap. Insect visitation in A. sikokianum decreased before the seasonal overlap. Floral visitor assemblages differed between the two Arisaema species, while the difference between natural and late-flowering A. sikokianum was less distinct. CONCLUSIONS: Phenological and pollinator isolation contribute to reproductive isolation between the two Arisaema species and should enable the two species to coexist in this area.

Flies as pollinators of melittophilous Salvia species (Lamiaceae).

UNLABELLED: • PREMISE OF THE STUDY: Floral adaptation to a functional pollinator group does not necessarily mean close specialization to a few pollinator species. For the more than 950 species of Salvia, only bee and bird pollinations are known. Restriction to these pollinators is mainly due to the specific flower construction (lever mechanism). Nevertheless, it has been repeatedly suggested that Salvia flowers might also be pollinated by flies. Are flies able to handle the lever mechanism? Are they functionally equivalent pollinators? In this study, we compared and quantified pollen transfer by bees and flies to test whether flies are true pollinators in Salvia.• METHODS: We identified pollinators using field observations and photos. Video documentation of the visitation rate and the site of pollen placement on the pollinator body, morphometric measurements, quantification of pollen placement, pollen load, handling time, and stigma contact ratio were analyzed.• KEY RESULTS: Field investigations revealed that 19 insect species pollinated S. virgata and four pollinated S. verticillata, including 16 bee species from seven genera of the Apidae and three fly species from three genera of the Nemestrinidae and Tabanidae.• CONCLUSIONS: Flies have been found to be pollinators in primarily bee-pollinated Salvia species. This result demonstrates the potential of a given "melittoid" flower construction to broaden the range of pollinators to guarantee successful pollination and seed production. Though bees, particularly Bombus terrestris, were more efficient than flies, the study shows that flies significantly contribute to pollen transfer in Salvia.

Floral mechanisms promote pollination success and reduce the incidence of self-pollination in a fly-pollinated self-incompatible orchid.

Among flowering plants, self-incompatibility is considered the most efficient system for avoiding self-fertilization. However, many self-incompatible plants have also evolved floral mechanisms to reduce sexual conflict. In China, some studies of Bulbophyllum have been reported to be self-incompatible and no fruit sets. However, we have observed relatively high fruit sets in Bulbophyllum funingense. Therefore, we speculated that if B. funingense is also self-incompatible, and it might present a floral mechanism to avoid sexual conflict. Natural fruit sets, pollinia removal and deposition rates were determined and breeding system was tested in a hand-pollination experiment. The pollination process and visiting frequency of pollinators and their behavior after escape from access were observed and recorded. Floral traits associated with pollination and pollinator size were measured. B. funingense was completely self-incompatible, the fruit sets of cross-pollination in 2 years were all more than 70%, and the natural fruit sets for 2 years were 1.70 ± 4.31% and 6.63 ± 5.29%, respectively. B. funingense did not produce strong odor or nectar, but produced a kind of secretions from its labellum that attracted flies. Calliphora vicina (Calliphoridae) was its only effective pollinator. When C. vicina licked the secretions, they were stuck in the access for a long time. Thus, when they escaped from access, they almost always flew quickly away from the inflorescence removing pollinia most of the times. In B. funingense, a floral mechanism improves pollinia transfer efficiency, reduces pollinia waste, promotes pollination success, reduces the incidence of self-pollination, and avoids sexual conflict to a certain extent.

Pollination and mating systems of Apodanthaceae and the distribution of reproductive traits in parasitic angiosperms.

PREMISE OF THE STUDY: The most recent reviews of the reproductive biology and sexual systems of parasitic angiosperms were published 17 yr ago and reported that dioecy might be associated with parasitism. We use current knowledge on parasitic lineages and their sister groups, and data on the reproductive biology and sexual systems of Apodanthaceae, to readdress the question of possible trends in the reproductive biology of parasitic angiosperms. • METHODS: Fieldwork in Zimbabwe and Iran produced data on the pollinators and sexual morph frequencies in two species of Apodanthaceae. Data on pollinators, dispersers, and sexual systems in parasites and their sister groups were compiled from the literature. • KEY RESULTS: With the possible exception of some Viscaceae, most of the ca. 4500 parasitic angiosperms are animal-pollinated, and ca. 10% of parasites are dioecious, but the gain and loss of dioecy across angiosperms is too poorly known to infer a statistical correlation. The studied Apodanthaceae are dioecious and pollinated by nectar- or pollen-foraging Calliphoridae and other flies. • CONCLUSIONS: Sister group comparisons so far do not reveal any reproductive traits that evolved (or were lost) concomitant with a parasitic life style, but the lack of wind pollination suggests that this pollen vector may be maladaptive in parasites, perhaps because of host foliage or flowers borne close to the ground.

Labellum Features and Chemical Composition of Floral Scent in Bulbophyllum carunculatum Garay, Hamer & Siegrist (Section Lepidorhiza Schltr., Bulbophyllinae Schltr., Orchidaceae Juss.).

The vast majority of fly-pollinated Bulbophyllum species use a combination of visual and olfactory clues to mimic food sources and brood/oviposition sites of pollinators. The aims of the present work were to characterize the floral secretory tissue and the floral scent and compare them with those previously described in B. echinolabium. Based on the histochemical results, the labellar secretion in B. carunculatum is the protein-rich mucilage. The adaxial epidermal cells of the labellum showed typical features of secretory activity. Plastids contained plastoglobuli, which are thought to be the places for scent production in osmophores. Juxtaposed with FeCl3 staining, the presence of dihydroxyphenolic globules in the cytoplasm of the epidermis and sub-epidermis was confirmed. Phenolic derivatives were also described with GC/MS analysis of the floral scent. The number of aromatic compounds and hydrocarbons was indicated in the floral scent of B. carunculatum. Moreover, pregnane-3,20-dione, occurring in the highest percentage in the floral fragrance of B. carunculatum, is a biologically active, 5-alpha-reduced metabolite of plasma progesterone. Progesterone is a mammalian gonadal hormone, but, like other steroid hormones, has been found in plants as intermediates in different biosynthetic pathways. The research on biosynthesis and functions of progesterone and its derivatives in flowers is still lacking.

Intraspecific variation in morphology of spiny pollen grains along an altitudinal gradient in an insect-pollinated shrub.

Intraspecific variations in pollen morphological traits are poorly studied. Interspecific variations are often associated with pollination systems and pollinator types. Altitudinal environmental changes, which can influence local pollinator assemblages, provide opportunities to explore differentiation in pollen traits of a single species over short distances. The aim of this study is to examine intraspecific variations in pollen traits of an insect-pollinated shrub, Weigela hortensis (Caprifoliaceae), along an altitudinal gradient. Pollen spine phenotypes (length, number and density), pollen diameter, lipid mass (pollenkitt) around pollen grains, pollen production per flower and pollinator assemblages were compared at four sites at different altitudes. Spine length and the spine length/diameter ratio of pollen grains were greater at higher altitudes but not correlated with flower or plant size. Spine number and density increased as flower size increased, and pollen lipid mass decreased as plant size increased. Bees were the predominant pollinators at low-altitude sites whereas flies, specifically Oligoneura spp. (Acroceridae), increased in relative abundance with increasing altitude. The results of this study suggest that the increase in spine length with altitude was the result of selection favouring longer spines at higher-altitude sites and/or shorter spines at lower-altitude sites. The altitudinal variation in selection pressure on spine length could reflect changes in local pollinator assemblages with altitude.

Scent chemistry and pollinators in the holoparasitic plant Cynomorium songaricum (Cynomoriaceae).

Holoparasitic plants are interesting heterotrophic angiosperms. However, carrion- or faeces-mimicking is rarely described for such plants. There is no information on the pollination biology of Cynomoriaceae, despite the fact that these plants are rare and vulnerable. This is the first study to reveal pollination in a member of this family, Cynomorium songaricum, a root holoparasite with a distinctive and putrid floral odour. From 2016 to 2018, we studied the floral volatiles, floral visitors and pollinators, behavioural responses of visitors to floral volatiles, breeding system, flowering phenology and floral biology of two wild populations of C. songaricum in Alxa, Inner Mongolia, China. A total of 42 volatiles were identified in inflorescences of C. songaricum. Among these volatiles are compounds known as typical carrion scents, such as p-cresol, indole, dimethyl disulphide and 1-octen-3-ol. C. songaricum is pollinated by various Diptera, such as Musca domestica, M. stabulans (Muscidae), Delia setigera, D. platura (Anthomyiidae), Lucilia sericata, L. caesar (Calliphoridae), Wohlfahrtia indigens, Sarcophaga noverca, S. crassipalpis and Sarcophila meridionalis (Sarcophagidae). The inflorescence scent of C. songaricum attracted these pollinators. The plants significantly benefit from insect pollination, although wind can be a pollen vector in the absence of pollinators. C. songaricum is a cross-pollinated, self-incompatible plant. Our findings suggest that C. songaricum releases malodorous volatiles to attract Diptera to achieve pollination. This new example lays the foundation for further comparative studies in other members of this plant group and contributes to a better understanding of fly-pollinated, carrion mimicking plants.

Floral Scent Evolution in the Genus Jaborosa (Solanaceae): Influence of Ecological and Environmental Factors.

Floral scent is a key communication channel between plants and pollinators. However, the contributions of environment and phylogeny to floral scent composition remain poorly understood. In this study, we characterized interspecific variation of floral scent composition in the genus Jaborosa Juss. (Solanaceae) and, using an ecological niche modelling approach (ENM), we assessed the environmental variables that exerted the strongest influence on floral scent variation, taking into account pollination mode and phylogenetic relationships. Our results indicate that two major evolutionary themes have emerged: (i) a 'warm Lowland Subtropical nectar-rewarding clade' with large white hawkmoth pollinated flowers that emit fragrances dominated by oxygenated aromatic or sesquiterpenoid volatiles, and (ii) a 'cool-temperate brood-deceptive clade' of largely fly-pollinated species found at high altitudes (Andes) or latitudes (Patagonian Steppe) that emit foul odors including cresol, indole and sulfuric volatiles. The joint consideration of floral scent profiles, pollination mode, and geoclimatic context helped us to disentangle the factors that shaped floral scent evolution across "pollinator climates" (geographic differences in pollinator abundance or preference). Our findings suggest that the ability of plants in the genus Jaborosa to colonize newly formed habitats during Andean orogeny was associated with striking transitions in flower scent composition that trigger specific odor-driven behaviors in nocturnal hawkmoths and saprophilous fly pollinators.

Fly Pollination of Kettle Trap Flowers of Riocreuxiatorulosa (Ceropegieae-Anisotominae): A Generalized System of Floral Deception.

Elaborated kettle trap flowers to temporarily detain pollinators evolved independently in several angiosperm lineages. Intensive research on species of Aristolochia and Ceropegia recently illuminated how these specialized trap flowers attract particular pollinators through chemical deception. Morphologically similar trap flowers evolved in Riocreuxia; however, no data about floral rewards, pollinators, and chemical ecology were available for this plant group. Here we provide data on pollination ecology and floral chemistry of R. torulosa. Specifically, we determined flower visitors and pollinators, assessed pollen transfer efficiency, and analysed floral scent chemistry. R. torulosa flowers are myiophilous and predominantly pollinated by Nematocera. Pollinating Diptera included, in order of decreasing abundance, male and female Sciaridae, Ceratopogonidae, Scatopsidae, Chloropidae, and Phoridae. Approximately 16% of pollen removed from flowers was successfully exported to conspecific stigmas. The flowers emitted mainly ubiquitous terpenoids, most abundantly linalool, furanoid (Z)-linalool oxide, and (E)-ß-ocimene-compounds typical of rewarding flowers and fruits. R. torulosa can be considered to use generalized food (and possibly also brood-site) deception to lure small nematocerous Diptera into their flowers. These results suggest that R. torulosa has a less specific pollination system than previously reported for other kettle trap flowers but is nevertheless specialized at the level of Diptera suborder Nematocera.

Flower Diversification Across "Pollinator Climates": Sensory Aspects of Corolla Color Evolution in the Florally Diverse South American Genus Jaborosa (Solanaceae).

Flower phenotype may diverge within plant lineages when moving across "pollinator climates" (geographic differences in pollinator abundance or preference). Here we explored the potential importance of pollinators as drivers of floral color diversification in the nightshade genus Jaborosa, taking into account color perception capabilities of the actual pollinators (nocturnal hawkmoths vs. saprophilous flies) under a geographic perspective. We analyzed the association between transitions across environments and perceptual color axes using comparative methods. Our results revealed two major evolutionary themes in Jaborosa: (1) a "warm subtropical sphingophilous clade" composed of three hawkmoth-pollinated species found in humid lowland habitats, with large white flowers that clustered together in the visual space of a model hawkmoth (Manduca sexta) and a "cool-temperate brood-deceptive clade" composed of largely fly-pollinated species with small dark flowers found at high altitudes (Andes) or latitudes (Patagonian Steppe), that clustered together in the visual space of a model blowfly (Lucilia sp.) and a syrphid fly (Eristalis tenax). Our findings suggest that the ability of plants to colonize newly formed environments during Andean orogeny and the ecological changes that followed were concomitant with transitions in flower color as perceived by different pollinator functional groups. Our findings suggest that habitat and pollination mode are inextricably linked in the history of this South American plant lineage.

Labellum structure of Bulbophyllum echinolabium J.J. Sm. (section Lepidorhiza Schltr., Bulbophyllinae Schltr., Orchidaceae Juss.).

This micromorphological, chemical and ultrastructural study is a continuation of research conducted on the section Lepidorhiza. The Bulbophyllum echinolabium flowers comprised features that characterize a sapromyophilous syndrome, having large, glistening parts that emit an intense scent of rotten meat. The secretory activity was described in the hypochile (nectary in longitudinal groove and in the prickles) and the epichile (putative osmophore). The ultrastructural studies revealed a dense cytoplasm in the epidermis and subepidermal tissue with large nuclei and numerous mitochondria, the profiles of SER and RER, and dictyosomes. Large amounts of heterogeneous residues of secreted material (possibly phenolic) were present on the cuticle surface, similar to the unusual prominent periplasmic space with flocculent secretory material. The chemical analysis (GC/MS) of the scent profile of lips comprised carbohydrates and their derivatives (29.55% of all compounds), amino acids (1.66%), lipids (8.04%) and other organic compounds (60.73%). A great number of identified compounds are Diptera attractants (mainly Milichiidae, Tephritidae, Drosophilidae, Muscidae, Sarcophagidae, Tachinidae). The examination of visual and olfactory features indicates correlation between colour of flowers and the type of olfactory mimicry, where a dark colour labellum emits strong smell of rotten waste.

Colour evolution within orchids depends on whether the pollinator is a bee or a fly.

Orchids are a classic angiosperm model for understanding biotic pollination. We studied orchid species within two species-rich herbaceous communities that are known to have either hymenopteran or dipteran insects as the dominant pollinators, in order to understand how flower colour relates to pollinator visual systems. We analysed features of the floral reflectance spectra that are significant to pollinator visual systems and used models of dipteran and hymenopteran colour vision to characterise the chromatic signals used by fly-pollinated and bee-pollinated orchid species. In contrast to bee-pollinated flowers, fly-pollinated flowers had distinctive points of rapid reflectance change at long wavelengths and a complete absence of such spectral features at short wavelengths. Fly-pollinated flowers also had significantly more restricted loci than bee-pollinated flowers in colour space models of fly and bee vision alike. Globally, bee-pollinated flowers are known to have distinctive, consistent colour signals. Our findings of different signals for fly pollination is consistent with pollinator-mediated selection on orchid species that results from the distinctive features of fly visual systems.

Psychophysics of the hoverfly: categorical or continuous color discrimination?

There is increasing interest in flies as potentially important pollinators. Flies are known to have a complex visual system, including 4 spectral classes of photoreceptors that contribute to the perception of color. Our current understanding of how color signals are perceived by flies is based on data for the blowfly Lucilia sp., which after being conditioned to rewarded monochromatic light stimuli, showed evidence of a categorical color visual system. The resulting opponent fly color space has 4 distinct categories, and has been used to interpret how some fly pollinators may perceive flower colors. However, formal proof that flower flies (Syrphidae) only use a simple, categorical color process remains outstanding. In free-flying experiments, we tested the hoverfly Eristalis tenax, a Batesian mimic of the honeybee, that receives its nutrition by visiting flowers. Using a range of broadband similar-dissimilar color stimuli previously used to test color perception in pollinating hymenopteran species, we evaluated if there are steep changes in behavioral choices with continuously increasing color differences as might be expected by categorical color processing. Our data revealed that color choices by the hoverfly are mediated by a continuous monotonic function. Thus, these flies did not use a categorical processing, but showed evidence of a color discrimination function similar to that observed in several bee species. We therefore empirically provide data for the minimum color distance that can be discriminated by hoverflies in fly color space, enabling an improved understanding of plant-pollinator interactions with a non-model insect species.

Breeding systems in Balsaminaceae in relation to pollen/ovule ratio, pollination syndromes, life history and climate zone.

Pollen/ovule (P/O) ratios are often used as proxy for breeding systems. Here, we investigate the relations between breeding systems and P/O ratios, pollination syndromes, life history and climate zone in Balsaminaceae. We conducted controlled breeding system experiments (autonomous and active self-pollination and outcrossing tests) for 65 Balsaminaceae species, analysed pollen grain and ovule numbers and evaluated the results in combination with data on pollination syndrome, life history and climate zone on a phylogenetic basis. Based on fruit set, we assigned three breeding systems: autogamy, self-compatibility and self-incompatibility. Self-pollination led to lower fruit set than outcrossing. We neither found significant P/O differences between breeding systems nor between pollination syndromes. However, the numbers of pollen grains and ovules per flower were significantly lower in autogamous species, but pollen grain and ovule numbers did not differ between most pollination syndromes. Finally, we found no relation between breeding system and climate zone, but a relation between climate zone and life history. In Balsaminaceae reproductive traits can change under resource or pollinator limitation, leading to the evolution of autogamy, but are evolutionary rather constant and not under strong selection pressure by pollinator guild and geographic range changes. Colonisation of temperate regions, however, is correlated with transitions towards annual life history. Pollen/ovule-ratios, commonly accepted as good indicators of breeding system, have a low predictive value in Balsaminaceae. In the absence of experimental data on breeding system, additional floral traits (overall pollen grain and ovule number, traits of floral morphology) may be used as proxies.

Geographic patterns and pollination ecotypes in Claytonia virginica.

Geographical variation in pollinators visiting a plant can produce plant populations adapted to local pollinator environments. We documented two markedly different pollinator climates for the spring ephemeral wildflower Claytonia virginica: in more northern populations, the pollen-specialist bee Andrena erigeniae dominated, but in more southern populations, A. erigeniae visited rarely and the bee-fly Bombylius major dominated. Plants in the northern populations experienced faster pollen depletion than plants in southern populations. We also measured divergent pollen-related plant traits; plants in northern populations produced relatively more pollen per flower and anther dehiscence was more staggered than plants in southern populations. These plant traits might function to increase pollen dispersal via the different pollen vectors.