Floral scent evolution in the section Pseudophrys: pollinator‐mediated selection or phylogenetic constraints?

• Sexually deceptive orchid species from the Mediterranean genus Ophrys usually interact with one or a few pollinator species by means of specific floral scents. In this study, we investigated the respective role of pollinator‐mediated selection and phylogenetic constraints in the evolution of floral scents in the section Pseudophrys.
• We built a phylogenetic tree of 19 Pseudophrys species based on three nuclear loci; we gathered a dataset on their pollination interactions from the literature and from our own field data; and we extracted and analysed their floral scents using solid phase microextraction and gas chromatography‐mass spectrometry. We then quantified the phylogenetic signal carried by floral scents and investigated the link between plant–pollinator interactions and floral scent composition using phylogenetic comparative methods.
• We confirmed the monophyly of the section Pseudophrys and demonstrated the existence of three main clades within this section. We found that floral scent composition is affected by both phylogenetic relationships among Ophrys species and pollination interactions, with some compounds (especially fatty acid esters) carrying a significant phylogenetic signal and some (especially alkenes and alkadienes) generating dissimilarities between closely related Pseudophrys pollinated by different insects.
• Our results show that in the section Pseudophrys, floral scents are shaped both by pollinator‐mediated selection and by phylogenetic constraints, but that the relative importance of these two evolutionary forces differ among compound classes, probably reflecting distinct selective pressures imposed upon behaviourally active and non‐active compounds.

     

Floral scent in bat-pollinated plants: a case of convergent evolution

The chemical composition of floral scent in eight bat-pollinated species belonging to six different plant families was investigated. Floral scent was collected by headspace trapping using porous adsorbents and the chemical composition determined by coupled gas chromatography-mass spectrometry. In all species except one the floral scent was found to include sulphur-containing compounds, of which several are reported for the first time in floral scents. Three species contained mushroom-like smelling fatty acid derivatives with a C₈-skeleton. Such flowers may be recognized by pollinators as humid environments in otherwise dry surroundings. The presence of similar or chemically closely related sulphur containing compounds in floral scent of bat-pollinated plant species from differing families may represent a case of convergent evolution in scent composition and an adaptation to attract this specific group of pollinators with similar sensory preferences.     

Scent wars: the chemobiology of competitive signalling in mice

Many mammals use scent marks to advertise territory ownership, but only recently have we started to understand the complexity of these scent signals and the types of information that they convey. Whilst attention has generally focused on volatile odorants as the main information molecules in scents, studies of the house mouse have now defined a role for a family of proteins termed major urinary proteins (MUPs) which are, of course, involatile. MUPs bind male signalling volatiles and control their release from scent marks. These proteins are also highly polymorphic and the pattern of polymorphic variants provides a stable ownership signal that communicates genome-derived information on the individual identity of the scent owner. Here we review the interaction between the chemical basis of mouse scents and the dynamics of their competitive scent marking behaviour, demonstrating how it is possible to provide reliable signals of the competitive ability and identity of individual males.     

Biologically meaningful scents: a framework for understanding predator–prey research across disciplines

Fear of predation is a universal motivator. Because predators hunt using stealth and surprise, there is a widespread ability among prey to assess risk from chemical information – scents – in their environment. Consequently, scents often act as particularly strong modulators of memory and emotions. Recent advances in ecological research and analytical technology are leading to novel ways to use this chemical information to create effective attractants, repellents and anti‐anxiolytic compounds for wildlife managers, conservation biologists and health practitioners. However, there is extensive variation in the design, results, and interpretation of studies of olfactory‐based risk discrimination. To understand the highly variable literature in this area, we adopt a multi‐disciplinary approach and synthesize the latest findings from neurobiology, chemical ecology, and ethology to propose a contemporary framework that accounts for such disparate factors as the time‐limited stability of chemicals, highly canalized mechanisms that influence prey responses, and the context within which these scents are detected (e.g. availability of alternative resources, perceived shelter, and ambient physical parameters). This framework helps to account for the wide range of reported responses by prey to predator scents, and explains, paradoxically, how the same individual predator scent can be interpreted as either safe or dangerous to a prey animal depending on how, when and where the cue was deposited. We provide a hypothetical example to illustrate the most common factors that influence how a predator scent (from dingoes, Canis dingo) may both attract and repel the same target organism (kangaroos, Macropus spp.). This framework identifies the catalysts that enable dynamic scents, odours or odorants to be used as attractants as well as deterrents. Because effective scent tools often relate to traumatic memories (fear and/or anxiety) that cause future avoidance, this information may also guide the development of appeasement, enrichment and anti‐anxiolytic compounds, and help explain the observed variation in post‐traumatic‐related behaviours (including post‐traumatic stress disorder, PTSD) among diverse terrestrial taxa, including humans.     

Floral scent in bird- and beetle-pollinated Protea species (Proteaceae): Chemistry,emission rates and function

Evolutionary shifts between pollination systems are often accompanied by modifications of floral traits, including olfactory cues. We investigated the implications of a shift from passerine bird to beetle pollination in Protea for floral scent chemistry, and also explored the functional significance of Protea scent for pollinator attraction. Using headspace sampling and gas chromatography–mass spectrometry, we found distinct differences in the emission rates and chemical composition of floral scents between eight bird- and four beetle-pollinated species. The amount of scent emitted from inflorescences of beetle-pollinated species was, on average, about 10-fold greater than that of bird-pollinated species. Floral scent of bird-pollinated species consists mainly of small amounts of “green-leaf volatiles” and benzenoid compounds, including benzaldehyde, anisole and benzyl alcohol. The floral scent of beetle-pollinated species is dominated by emissions of linalool, a wide variety of other monoterpenes and the benzenoid methyl benzoate, which imparts a fruity odour to the human nose. The number of compounds recorded in the scent of beetle-pollinated species was, on average, greater than in bird-pollinated species (45 versus 29 compounds, respectively). Choice experiments using a Y-maze showed that a primary pollinator of Protea species, the cetoniine beetle Atrichelaphinis tigrina, strongly preferred the scent of inflorescences of the beetle-pollinated Protea simplex over those of the bird-pollinated sympatric congener, Protea roupelliae. This study shows that a shift from passerine bird- to insect-pollination can be associated with marked up-regulation and compositional changes in floral scent emissions.     

The predation risks of interspecific eavesdropping: weasel–vole interactions

Competing species benefit from eavesdropping on each other's signals by learning about shared resources or predators. But conspicuous signals are also open to exploitation by eavesdropping predators and should also pose a threat to other sympatric prey species. In western Finland, sibling voles Microtus rossiameridionalis and field voles M. agrestis compete for food and space, and both species rely upon scent marks for intraspecific communication. Both vole species are prey to a range of terrestrial scent hunting predators such as least weasels, however, the competitively superior sibling voles are taken preferentially. We tested in large out‐door enclosures whether field voles eavesdrop on the signals of its competitor, and whether they behave as though this eavesdropping carries a risk of predation. We presented field voles with scent marks from unknown conspecifics and sibling voles and measured their visitation, activity and scent marking behaviours at these scents under high (weasel present) and low (weasel absent) predation risk. Field voles readily visited both field and sibling vole scents under both high and low predation risk; however their activity at sibling vole scent marks declined significantly under increased predation risk. In contrast, predation risk did not affect field voles’ activity at conspecific scents. Thus, field voles were compelled to maintain eavesdropping on heterospecific scents under an increased risk of predation, however they compensated for this additional risk by reducing their activity at these risky scents. Scent marking rates declined significantly under high predation risk. Our results therefore reveal a hidden complexity in the use of social signals within multi‐species assemblages that is clearly sensitive to the potential for increased predation risk. The predation risks of interspecific eavesdropping demonstrated here represents a significant generalisation of the concept of associational susceptibility.     

Comparative analyses of flower scent in Sileneae reveal a contrasting phylogenetic signal between night and day emissions

Flower scents are complex blends of volatile compounds often shaped by selection pressures exerted by mutualistic and antagonistic interaction partners, but also by phylogenetic constraints. So far, little is known about the relative effect of selection and phylogenetic signal on scent patterns, and no study to date analyzed the phylogenetic signal in multivariate semiquantitative scent patterns. We analyzed the phylogenetic signal in qualitative and semi‐quantitative patterns of flower scents in 47 Sileneae (Caryophyllaceae) species using phylogenetic principal component analysis (pPCA) and several indices of univariate and multivariate phylogenetic signal. As previous results showed that Sileneae species are visited by diurnal and nocturnal pollinators and flower scents also vary along the day, we compared the phylogenetic signal between night and day. Multivariate pPCA analyses identified compounds that correlate with the phylogeny at both night and day; however, multivariate Bloomberg′s K detected phylogenetic signal in the dataset of night scents, but not of day scents. In multivariate qualitative datasets, phylogenetic signal was neither found for day nor for night scents. In univariate analyses, phylogenetic signal was detected for some compounds both for day and night scents. Overall, we found that the phylogenetic signal is stronger in night compared to day scents, which might be owed to the different guilds of pollinators at day and night. At day, the phylogenetic resemblance of Sileneae scents might be masked by or disappear due to divergent selective pressures exerted by a diverse guild of pollinators on the different species. In contrast, we hypothesize that the nocturnal moth pollinators exert similar selective pressures; thus, the phylogenetic similarity of scent profiles might be conserved. Future studies of scent phylogenetic signal must consider not only the usage of qualitative measures but also semiquantitative analyses.     

Dogs discriminate identical twins

Earlier studies have shown variation among experimental attempts to establish whether human monozygotic twins that are genetically identical also have identical individual scents. In none of the cases were the dogs able to distinguish all the individual scents of monozygotic twins living in the same environment if the scents were presented to them separately. Ten specially trained police German Shepherd dogs of three Czech Republic Police Regional Headquarters were used for scent identification in our study. The dogs were supposed to match scents of two monozygotic pairs (5 and 7 years old) and two dizygotic twin pairs (8 and 13 years old). Scents were collected on cotton squares stored in glass jars. Dog handlers were blind to the experiment details. In each trial (line-up), one scent was used as a starting scent and the dog was then sent to determine if any of the 7 presented glass jars contained a matching scent. Scents of children of similar ages were used as distractors. In the matching procedure, the dogs matched correctly the scent of one twin with the other, as well as two scents collected from every single identical and non-identical twin to prove their efficacy and likewise, the presence of the matching twin scent in any given glass jar. All dogs in all trials distinguished correctly the scents of identical as well as non-identical twins. All dogs similarly matched positively two scents collected from the same individuals. Our findings indicated that specially trained German Shepherd dogs are able to distinguish individual scents of identical twins despite the fact that they live in the same environment, eat the same food and even if the scents are not presented to them simultaneously.     

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.     

Petunia flowers solve the defence/apparency dilemma of pollinator attraction by deploying complex floral blends

Flowers recruit floral visitors for pollination services by emitting fragrances. These scent signals can be intercepted by antagonists such as florivores to locate host plants. Hence, as a consequence of interactions with both mutualists and antagonists, floral bouquets likely consist of both attractive and defensive components. While the attractive functions of floral bouquets have been studied, their defensive function has not, and field‐based evidence for the deterrence of floral‐scent constituents is lacking. In field and glasshouse experiments with five lines of transgenic Petunia x hybrida plants specifically silenced in their ability to release particular components of their floral volatile bouquet, we demonstrate that the emission of single floral‐scent compounds can dramatically decrease damage from generalist florivores. While some compounds are used in host location, others prevent florivory. We conclude that the complex blends that comprise floral scents are likely sculpted by the selective pressures of both pollinators and herbivores.     

Floral scent emissions from Asarum yaeyamense and related species

The flowers of Asarum are usually regarded as scentless or sometimes to have a foul odor. Recently, we noticed that Asarum yaeyamense, endemic to Iriomote Island, Japan, has a floral fragrance with a distinct “fruity note.” To determine the chemical characteristics of this fragrance and whether “non-scented” Asarum species emit any volatiles, we collected floral scents of A. yaeyamense and related species (A. lutchuense, A. hypogynum, A. fudsinoi, A dissitum, A. tokarense, and A. senkakuinsulare) using headspace methods and analyzed these scents by gas chromatography–mass spectrometry (GC–MS). The results indicated that A. yaeyamense mainly emitted α-cedrene (tentatively identified), an unidentified sesquiterpene, methyl tiglate, and manoyl oxide (tentatively identified). Methyl tiglate may be a source of the “fruity note” in the A. yaeyamense fragrance. We also detected emissions of volatiles, mainly sesquiterpenes, from some “non-scented” Asarum species. This study constitutes a rare case of the detection of the emission of a diterpene (manoyl oxide) as a floral scent volatile.     

Complex memories in honeybees: can there be more than two?

Foraging honeybees are likely to learn visual and chemical cues associated with many different food sources. Here, we explore how many such sources can be memorized and recalled. Marked bees were trained to visit two (or three) sugar feeders, each placed at a different outdoor location and carrying a different scent. We then tested the ability of the bees to recall these locations and fly to them, when the training scents were blown into the hive, and the scents and food at the feeders were removed. When trained on two feeder locations, each associated with a different scent, the bees could correctly recall the location associated with each scent. However, this ability broke down when the number of scents and feeder locations was increased to three. Performance was partially restored when each of the three training feeders was endowed with an additional cue, namely, a distinct colour. Our results suggest that bees can recall a maximum of two locations when each is associated with a different scent. However, this number can be increased if the scent cues are augmented by visual cues. These findings have implications for the ways in which associations are established and laid down in honeybee memory.     

Food Deprivation Suppresses a Preference for the Top-Scent Mark of an Over-Mark in Meadow Voles (Microtus pennsylvanicus)

Food availability affects whether mammals communicate their interest in interacting with opposite‐sex conspecifics. This study examined the responses of voles to over‐marks, and factors that influence the formation and maintenance of a preference for the top‐scent in an over‐mark. Specifically, we investigated how food deprivation affected the amount of time male and female voles exposed to an over‐mark, later responded to the marks of the top‐ and bottom‐scent donors when subsequently presented with the two scents side by side. Males and females that were not food deprived and males that were food deprived 6 h before exposure to an over‐mark later maintained a preference for the donor of the top‐scent mark compared with the donor of the bottom‐scent mark of the over‐mark. Females that were food deprived for 6 h before or after exposure of the over‐mark and males food deprived 6 h after the exposure to the over‐mark showed no preference for the top‐scent mark donor. Re‐feeding females that were food deprived for 6 h before exposure to an over‐mark was sufficient to restore their preference for the mark of the top‐scent male over that of the bottom‐scent male. The different responses of food‐deprived male and female voles to over‐marks of opposite‐sex conspecifics may be associated with differences in their tactics for interacting with potential mates and the higher energetic costs of reproduction in female voles than in male voles.     

Predator‐Naïve Brown Trout (Salmo trutta) Show Antipredator Behaviours to Scent from an Introduced Piscivorous Mammalian Predator Fed Conspecifics

Introduced mammalian predators may pose a high risk for native and naïve prey populations, but little is known about how native fish species may recognize and respond to scents from introduced mammalian predators. We investigated the role of diet‐released chemical cues in facilitating predator recognition, hypothesizing that native brown trout (Salmo trutta) would exhibit antipredator behaviours to faeces scents from the introduced American mink (Neovision vison) fed conspecifics, but not to non‐trout diets. In treatments‐control and replicate stream tank experiments, brown trout showed significant antipredator responses to faeces scent from mink fed conspecifics, but not to faeces scent from mink fed a non‐trout diet (chicken), or the non‐predator food control, Eurasian beaver (Castor fiber). We conclude that native and naïve brown trout show relevant antipredator behaviours to an introduced mammalian predator, presumably based on diet‐released conspecific alarm cues and thereby estimate the predation risk.     

What smells? Developing in‐field methods to characterize the chemical composition of wild mammalian scent cues

Olfactory cues play an important role in mammalian biology, but have been challenging to assess in the field. Current methods pose problematic issues with sample storage and transportation, limiting our ability to connect chemical variation in scents with relevant ecological and behavioral contexts. Real‐time, in‐field analysis via portable gas chromatography–mass spectrometry (GC‐MS) has the potential to overcome these issues, but with trade‐offs of reduced sensitivity and compound mass range. We field‐tested the ability of portable GC‐MS to support two representative applications of chemical ecology research with a wild arboreal primate, common marmoset monkeys (Callithrix jacchus). We developed methods to (a) evaluate the chemical composition of marmoset scent marks deposited at feeding sites and (b) characterize the scent profiles of exudates eaten by marmosets. We successfully collected marmoset scent marks across several canopy heights, with the portable GC‐MS detecting known components of marmoset glandular secretions and differentiating these from in‐field controls. Likewise, variation in the chemical profile of scent marks demonstrated a significant correlation with marmoset feeding behavior, indicating these scents’ biological relevance. The portable GC‐MS also delineated species‐specific olfactory signatures of exudates fed on by marmosets. Despite the trade‐offs, portable GC‐MS represents a viable option for characterizing olfactory compounds used by wild mammals, yielding biologically relevant data. While the decision to adopt portable GC‐MS will likely depend on site‐ and project‐specific needs, our ability to conduct two example applications under relatively challenging field conditions bodes well for the versatility of in‐field GC‐MS.     

Geographic variation of floral scent in a highly specialized pollination mutualism

Floral scents are important signals for communication between plants and pollinators. Several studies have focused on interspecific variation of these signals, but little is known about intraspecific variation in flower scent, particularly for species with wide geographic distributions. In the highly specific mutualism between Ficus species and their pollinating wasps, chemical mediation is crucial for partner encounter. Several studies show that scents, i.e. blends of volatiles, are species-specific, but no studies address interpopulation variation of scents in fig pollination mutualisms, which often have broad geographic distributions. In this study, using absorption/desorption headspace techniques, we analyzed variation in floral scent composition among three populations of each of two widely distributed Asian Ficus species. We identified more than 100 different volatile organic compounds, predominantly terpenes. In both species, significant differences were found between scent bouquets of East Asian and Indian populations. These differences are discussed in relation to geographical barriers that could disrupt gene exchange between these two areas, thereby isolating Indian populations from those of Eastern Asia.     

植物花香代谢调节与基因工程研究进展

植物花香在吸引昆虫授粉、提高观赏价值和香精的商业价值方面具有重要的作用。随着分子生物学技术的发展,近年来植物花香基因被大量克隆,对花香化合物的合成与代谢的网络调控机制有了更深刻的认识,基因工程改良花香成为可能。对近年来植物花香的合成途径、花香的释放与基因调节、基因工程的研究进展进行了综述,并就存在的问题进行了分析,为花香的分子育种研究提供参考。     

Intensity and the ratios of compounds in the scent of snapdragon flowers affect scent discrimination by honeybees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Floral scent is used by pollinators during foraging to identify and discriminate among flowers. The ability to discriminate among scents may depend on both scent intensity and the ratios of the concentrations of the volatile compounds of a complex mixture rather than on the presence of a few compounds. We used four scent-emitting cultivars of snapdragon (Antirrhinum majus) to test this hypothesis by examining the ability of honeybees to differentiate among their scents. Each cultivar produced three monoterpenes (myrcene, E--ocimene, and linalool) and five phenylpropanoids (methylbenzoate, acetophenone, dimethoxytoluene, cis-methylcinnamate, and trans-methylcinnamate). Cultivars were reliably classified by their scents in a canonical discriminant analysis. Honeybees were unable to discriminate among the scents of flowers of the same cultivar in our assay. The ability of honeybees to discriminate among the scents of different cultivars was a function of the intensity of the floral scent. Discrimination was also correlated to the distance among the scents described by the discriminant analysis; the cultivars that had the greatest differences observed in the discriminant analysis were the easiest to discriminate. Our results show that honeybees are capable of using all of the floral volatiles to discriminate subtle differences in scent.