Geographic variation in cone volatiles and pollinators in the thermogenic African cycad Encephalartos ghellinckii Lem

• Heat and odour production can have profound effects on pollination in cycads. It is therefore expected that these traits would co‐vary geographically with pollinator assemblages. Such intraspecific variation, may lead to the evolution of pollination ecotypes, which can be an early stage of pollinator‐mediated speciation.
• We measured cone temperatures using miniature temperature data loggers and examined the composition of cone volatile odours using headspace sampling and analysis with gas chromatography‐mass spectrometry in four populations spanning the range of the African cycad Encephalartos ghellinckii. Pollinator assemblages were also investigated in three populations.
• Male and female cones were thermogenic at pollen shed and receptive stages, respectively, but patterns of thermogenesis did not vary among populations. Scent emissions from cones in populations in the Drakensberg Mountains were characterised by cis‐β‐ocimene, β‐myrcene and (3E)‐1,3‐octadiene, while camphene and α‐pinene were characteristic of scent emissions from cones in populations closer to the coast. These differences in volatile blends corresponded with differences in insect assemblages.
• These results confirm intraspecific variation in volatile emissions of E. ghellinckii and support the predictions that intraspecific variation in volatile emissions will be associated with shifts in pollinator assemblages. While further work needs to be done to test for local adaptation in this system, this preliminary evidence is consistent with the formation of pollination ecotypes in the E. ghellinckii species complex.

     

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.     

Philodendron adamantinum (Araceae) lures its single cyclocephaline scarab pollinator with specific dominant floral scent volatiles

Cyclocephline scarabs and their host plants are documented as highly specialized plant–pollinator associations, with various fine‐tuned adaptations. We studied the association between Philodendron adamantinum, a species endemic to the Espinhaço Range in Minas Gerais, South‐East Brazil, and its exclusive pollinators. We focused on the pollination mechanism and reproductive success of P. adamantinum, analysed its floral scent composition, and performed field bioassays to verify the scent‐mediated attraction of pollinators. The reproductive success of P. adamantinum depends on the presence of Erioscelis emarginata (Scarabaeidae, Cyclocephalini), its sole pollinator. At dusk, the inflorescences heat up to 18 °C above the surrounding ambient air temperature and give off a strong sweet odour, from which 32 volatile compounds were isolated. Dihydro‐β‐ionone, the major constituent in the floral scent bouquet, lures individuals of E. emarginata when applied to scented artificial decoys, either alone or blended with methyl jasmonate. We attribute the low fruit set of P. adamantinum at our study sites to pollinator limitation of small and isolated populations and geitonogamic pollen flow of vegetatively generated clonal plant groups. The interaction between P. adamantinum and E. emarginata shows common traits typical of the known plant–pollinator associations involving cyclocephaline scarabs: the asymmetrical dependence of plants on their pollinators, and the scent‐mediated interaction between flowers and beetles. In addition to updating the current catalogue of active compounds of cantharophilous pollination systems, further experimental studies should elucidate the role of the specific chemical compounds that attract pollinators along different time and biogeographic scales. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 679–691.     

Ecological and genetic evidence for cryptic ecotypes in a rare sexually deceptive orchid,Drakaea elastica

Species with specialized ecological interactions present significant conservation challenges. In plants that attract pollinators with pollinator‐specific chemical signals, geographical variation in pollinator species may indicate the presence of cryptic plant taxa. We investigated this phenomenon in the rare sexually deceptive orchid Drakaea elastica using a molecular phylogenetic analysis to resolve pollinator species boundaries, pollinator choice experiments and a population genetic study of the orchid. Pollinator choice experiments demonstrated the existence of two ecotypes within D. elastica, each attracting their own related but phylogenetically distinct pollinator species. Despite the presence of ecotypes, population genetic differentiation was low across populations at six microsatellite loci (F_ST = 0.026). However, Bayesian STRUCTURE analysis revealed two genetic clusters, broadly congruent with the ecotype distributions. These ecotypes may represent adaptation to regional variation in pollinator availability and perhaps the early stages of speciation, with pronounced morphological and genetic differences yet to evolve. Resolution of the taxonomic status of the D. elastica ecotypes is required as this has implications for conservation efforts and allocation of management funding. Furthermore, any reintroduction programmes must incorporate knowledge of ecotype distribution and pollinator availability to ensure reproductive success in restored populations. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 124–140.     

Evolutionary consequences of ecological factors: pollinator reliability predicts mating‐system traits of a perennial plant

The reproductive‐assurance hypothesis predicts that mating‐system traits will evolve towards increased autonomous self‐pollination in plant populations experiencing unreliable pollinator service. We tested this long‐standing hypothesis by assessing geographic covariation among pollinator reliability, outcrossing rates, heterozygosity and relevant floral traits across populations of Dalechampia scandens in Costa Rica. Mean outcrossing rates ranged from 0.16 to 0.49 across four populations, and covaried with the average rates of pollen arrival on stigmas, a measure of pollinator reliability. Across populations, genetically based differences in herkogamy (anther–stigma distance) were associated with variation in stigmatic pollen loads, outcrossing rates and heterozygosity. These observations are consistent with the hypothesis that, when pollinators are unreliable, floral traits promoting autonomous selfing evolve as a mechanism of reproductive assurance. Extensive covariation between floral traits and mating system among closely related populations further suggests that floral traits influencing mating systems track variation in adaptive optima generated by variation in pollinator reliability.     

Multiple parapatric pollinators have radiated across a continental fig tree displaying clinal genetic variation

The ways that plant‐feeding insects have diversified are central to our understanding of terrestrial ecosystems. Obligate nursery pollination mutualisms provide highly relevant model systems of how plants and their insect associates have diversified and the over 800 species of fig trees (Ficus) allow comparative studies. Fig trees can have one or more pollinating fig wasp species (Agaonidae) that breed within their figs, but factors influencing their number remain to be established. In some widely distributed fig trees, the plants form populations isolated by large swathes of sea, and the different populations are pollinated by different wasp species. Other Ficus species with continuous distributions may present genetic signatures of isolation by distance, suggesting more limited pollinator dispersal, which may also facilitate pollinator speciation. We tested the hypothesis that Ficus hirta, a species for which preliminary data showed genetic isolation by distance, would support numerous pollinator species across its range. Our results show that across its range F. hirta displays clinal genetic variation and is pollinated by nine parapatric species of Valisia. This is the highest number of pollinators reported to date for any Ficus species, and it is the first demonstration of the occurrence of parapatric pollinator species on a fig host displaying continuous genetic structure. Future comparative studies across Ficus species should be able to establish the plant traits that have driven the evolution of pollinator dispersal behaviour, pollinator speciation and host plant spatial genetic structure.     

Phenotypic selection on flowering phenology and pollination efficiency traits between Primula populations with different pollinator assemblages

Floral traits have largely been attributed to phenotypic selection in plant–pollinator interactions. However, the strength of this link has rarely been ascertained with real pollinators. We conducted pollinator observations and estimated selection through female fitness on flowering phenology and floral traits between two Primula secundiflora populations. We quantified pollinator‐mediated selection by subtracting estimates of selection gradients of plants receiving supplemental hand pollination from those of plants receiving open pollination. There was net directional selection for an earlier flowering start date at populations where the dominant pollinators were syrphid flies, and flowering phenology was also subjected to stabilized quadratic selection. However, a later flowering start date was significantly selected at populations where the dominant pollinators were legitimate (normal pollination through the corolla tube entrance) and illegitimate bumblebees (abnormal pollination through nectar robbing hole which located at the corolla tube), and flowering phenology was subjected to disruptive quadratic selection. Wider corolla tube entrance diameter was selected at both populations. Furthermore, the strength of net directional selection on flowering start date and corolla tube entrance diameter was stronger at the population where the dominant pollinators were syrphid flies. Pollinator‐mediated selection explained most of the between‐population variations in the net directional selection on flowering phenology and corolla tube entrance diameter. Our results suggested the important influence of pollinator‐mediated selection on floral evolution. Variations in pollinator assemblages not only resulted in variation in the direction of selection but also the strength of selection on floral traits.     

Variation in native pollinators in the absence of honeybees: implications for reproductive success of an Australian generalist-pollinated herb Trachymene incisa (Apiaceae)

Most plants are generalist in terms of pollination, with the potential for significant spatial and temporal variation in their pollinators. Few studies have investigated how variable pollinators are in a generalist pollination system or how this variation affects plant reproduction. We investigated the degree of variation in pollinators and resulting reproductive success among populations of a widespread generalist-pollinated herb, Trachymene incisa (Apiaceae). The European honeybee was unexpectedly absent from the pollinator assemblages, providing the unique opportunity to study the native Australian pollinators. Insect visitation rates and the taxonomic composition of the pollinator assemblage varied significantly across populations, indicating that populations of T. incisa are not equally serviced and are not equally generalist. This highlights that sampling one population would not characterize the extent of species generalization. There was no positive correlation between insect visitation rate and reproductive success, with the Agnes Banks population receiving the highest visitation rate but producing the lowest reproductive output, and the Myall Lakes population receiving the lowest visitation rate and producing the highest seedling emergence. This study shows that variation in pollinators can have measurable effects on populations of generalist-pollinated plants, therefore there is potential for large-scale change in all plant–pollinator interactions.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 479–490.     

Genic rather than genome‐wide differences between sexually deceptive Ophrys orchids with different pollinators

High pollinator specificity and the potential for simple genetic changes to affect pollinator attraction make sexually deceptive orchids an ideal system for the study of ecological speciation, in which change of flower odour is likely important. This study surveys reproductive barriers and differences in floral phenotypes in a group of four closely related, coflowering sympatric Ophrys species and uses a genotyping‐by‐sequencing (GBS) approach to obtain information on the proportion of the genome that is differentiated between species. Ophrys species were found to effectively lack postpollination barriers, but are strongly isolated by their different pollinators (floral isolation) and, to a smaller extent, by shifts in flowering time (temporal isolation). Although flower morphology and perhaps labellum coloration may contribute to floral isolation, reproductive barriers may largely be due to differences in flower odour chemistry. GBS revealed shared polymorphism throughout the Ophrys genome, with very little population structure between species. Genome scans for F_ST outliers identified few markers that are highly differentiated between species and repeatable in several populations. These genome scans also revealed highly differentiated polymorphisms in genes with putative involvement in floral odour production, including a previously identified candidate gene thought to be involved in the biosynthesis of pseudo‐pheromones by the orchid flowers. Taken together, these data suggest that ecological speciation associated with different pollinators in sexually deceptive orchids has a genic rather than a genomic basis, placing these species at an early phase of genomic divergence within the ‘speciation continuum’.     

Altitudinal flower size variation correlates with local pollinator size in a bumblebee‐pollinated herb,Prunella vulgaris L. (Lamiaceae)

The influence of locally different species interactions on trait evolution is a focus of recent evolutionary studies. However, few studies have demonstrated that geographically different pollinator‐mediated selection influences geographic variation in floral traits, especially across a narrow geographic range. Here, we hypothesized that floral size variation in the Japanese herb Prunella vulgaris L. (Lamiaceae) is affected by geographically different pollinator sizes reflecting different pollinator assemblages. To evaluate this hypothesis, we posed two questions. (1) Is there a positive correlation between floral length and the proboscis length of pollinators (bumblebees) across altitude in a mountain range? (2) Does the flower–pollinator size match influence female and male plant fitness? We found geographic variation in the assemblage of pollinators of P. vulgaris along an altitudinal gradient, and, as a consequence, the mean pollinator proboscis length also changed altitudinally. The floral corolla length of P. vulgaris also varied along an altitudinal gradient, and this variation strongly correlated with the local pollinator size but did not correlate with altitude itself. Furthermore, we found that the size match between the floral corolla length and bee proboscis length affected female and male plant fitness and the optimal size match (associated with peak fitness) was similar for the female and male fitness. Collectively, these results suggest that pollinator‐mediated selection influences spatial variation in the size of P. vulgaris flowers at a fine spatial scale.     

Geographical matching of volatile signals and pollinator olfactory responses in a cycad brood-site mutualism

Brood-site mutualisms represent extreme levels of reciprocal specialization between plants and insect pollinators, raising questions about whether these mutualisms are mediated by volatile signals and whether these signals and insect responses to them covary geographically in a manner expected from coevolution. Cycads are an ancient plant lineage in which almost all extant species are pollinated through brood-site mutualisms with insects. We investigated whether volatile emissions and insect olfactory responses are matched across the distribution range of the African cycad Encephalartos villosus. This cycad species is pollinated by the same beetle species across its distribution, but cone volatile emissions are dominated by alkenes in northern populations, and by monoterpenes and a pyrazine compound in southern populations. In reciprocal choice experiments, insects chose the scent of cones from the local region over that of cones from the other region. Antennae of beetles from northern populations responded mainly to alkenes, while those of beetles from southern populations responded mainly to pyrazine. In bioassay experiments, beetles were most strongly attracted to alkenes in northern populations and to the pyrazine compound in southern populations. Geographical matching of cone volatiles and pollinator olfactory preference is consistent with coevolution in this specialized mutualism.     

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.     

Modeling the two‐locus architecture of divergent pollinator adaptation: how variation in SAD paralogs affects fitness and evolutionary divergence in sexually deceptive orchids

Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n‐alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species‐specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator‐mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two‐locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator‐mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator‐driven ecological speciation.     

Pollinator‐mediated selection in a specialized pollination system: matches and mismatches across populations

Most studies on pollinator‐mediated selection have been performed in generalized rather than specialized pollination systems. This situation has impeded evaluation of the extent to which selection acts on attraction or specialized key floral traits involved in the plant‐pollinator phenotypic interphase. We studied pollinator‐mediated selection in four populations of Nierembergia linariifolia, a self‐incompatible and oil‐secreting plant pollinated exclusively by oil‐collecting bees. We evaluated whether floral traits experience variable selection among populations and whether attraction and fit traits are heterogeneously selected across populations. Populations differed in every flower trait and selection was consistently observed for corolla size and flower shape, two traits involved in the first steps of the pollination process. However, we found no selection acting on mechanical‐fit traits. The observation that selection occurred upon attraction rather than mechanical‐fit traits, suggests that plants are not currently evolving fine‐tuned morphological adaptations to local pollinators and that phenotypic matching is not necessarily an expected outcome in this specialized pollination system.     

Speciation in sexually deceptive orchids: pollinator-driven selection maintains discrete odour phenotypes in hybridizing species

Ophrys orchids mimic the female sex pheromones of their pollinator species to attract males for pollination. Reproductive isolation in Ophrys is based on the selective attraction of only a single pollinator species. A change of floral odour can result in the attraction of a new pollinator species that acts as an isolation barrier towards other sympatrically occurring Ophrys species. Ophrys lupercalis, Ophrys bilunulata, and Ophrys fabrella grow sympatrically and bloom consecutively on Majorca and are pollinated by three species of Andrena. We investigated variation of phenotypic and genotypic flower traits, aiming to study the role of the floral odour for reproductive isolation and speciation. Using chemical and electrophysiology (gas chromatography coupled with an electroantennographic detector) methods, we show that the three Ophrys species use the same odour compounds for pollinator attraction, but in different proportions. A comparison of the floral odour bouquets in a multivariate analysis revealed a clear grouping of plants from the same species, although with an overlap between species. A comparison of the same plants using molecular markers gave a contrasting result. Although O. lupercalis and O. fabrella were genetically well separated, plants of O. bilunulata did not form a distinct group but were similar to either O. lupercalis or O. fabrella. Our data indicate gene flow and hybridization to occur between O. bilunulata and O. lupercalis as well as between O. bilunulata and O. fabrella. All plants of O. bilunulata, despite having different genotypes, showed a very similar floral odour. This reflects a strong selective pressure by the pollinating males. The overlap of genotypes of O. bilunulata and O. fabrella supports our hypothesis that O. fabrella diverged from O. bilunulata by scent variation and the attraction of a new pollinator species, Andrena fabrella. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 439–451.     

Geographical variation in floral traits of the tree tobacco in relation to its hummingbird pollinator fauna

Nicotiana glauca , a hummingbird pollinated plant, exhibits geographical variation in several floral traits. We examined whether geographical differentiation occurred for different flower characters and if this differentiation could be explained, at least in part, by the existence or abundance of different hummingbird species in the respective pollinator assemblages. The comparison between five populations showed significant variation in six floral traits and two female fitness measures. The traits that better discriminated between populations were corolla length and corolla width. There were metric correlations between corolla length and style length in all populations studied and, in four of the five populations, both corolla length and width were also correlated. Among plants in each population, seed weight was correlated positively and significantly with style exertion, suggesting that fruit quality is dependent on the degree of cross pollination. Assemblages of hummingbirds differed between populations in species composition, visitation frequencies, and bill length. Linear regression involving bill length of the more frequent hummingbird pollinators and corolla length yielded positive and significant relationships. Thus, there appears to be an adjustment between pollinators and flowers traits that have high incidence in the among population variation.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 657–667.     

Clinal variability of oil and nectar rewards in Monttea aphylla (Plantaginaceae): relationships with pollinators and climatic factors in the Monte Desert

Monttea aphylla flowers simultaneously produce oil and nectar, rewards known to differentially attract ecologically and functionally distinct pollinators. We examined whether geographical differentiation occurred for rewards, and whether this could be explained by spatially heterogeneous pollinator guilds and climate. Rewards were measured across the entire species range. Geographical patterns of reward quantity and their relationships with biotic and abiotic factors were examined using uni‐ and multivariate analyses. Latitude significantly explained population variation in nectar and oils, although in contrasting ways. Pollinator assemblages showed a prevalence of oil‐collecting Centris bees. Centris vardyorum, specialized on M. aphylla, showed a latitudinal pattern of visits. Oil production was not higher where plants associated with ecologically specialized bees (i.e. C. vardyorum), but this occurred instead where they were less frequent and co‐occurred with other bees that used many floral sources, including other plant species that produce oil. The multivariate analysis showed that the prevalence of C. vardyorum was the factor that contributed most significantly to the combined patterns of rewards. We suggest that large‐scale variation in rewards involves local optima throughout the species range, related to processes that operate in each ecoregion with their particular biotic and abiotic scenarios. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 314–328.     

Floral integration,phenotypic covariance structure and pollinator variation in bumblebee‐pollinated Helleborus foetidus

By analysing patterns of phenotypic integration and multivariate covariance structure of five metric floral traits in nine Iberian populations of bumblebee‐pollinated Helleborus foetidus (Ranunculaceae), this paper attempts to test the general hypothesis that pollinators enhance floral integration and selectively modify phenotypic correlations between functionally linked floral traits. The five floral traits examined exhibited significant phenotypic integration at all populations, and both the magnitude and the pattern of integration differed widely among populations. Variation in extent and pattern of integration was neither distance‐dependent nor significantly related to between‐population variation in taxonomical composition and morphological diversity of the pollinator assemblage. Patterns of floral integration were closer to expectations derived from consideration of developmental affinities between floral whorls than to expectations based on a pollinator‐mediated adaptive hypothesis. Taken together, results of this study suggest that between‐population differences in magnitude and pattern of floral integration in H. foetidus are probably best explained as a consequence of random genetic sampling in the characteristically small and ephemeral populations of this species, rather than reflecting the selective action of current pollinators.     

Patterns of odour emission,thermogenesis and pollinator activity in cones of an African cycad: what mechanisms apply?

Background and Aims

Ontogenetic patterns of odour emissions and heating associated with plant reproductive structures may have profound effects on insect behaviour, and consequently on pollination. In some cycads, notably Macrozamia, temporal changes in emission of specific odour compounds and temperature have been interpreted as a ‘push–pull’ interaction in which pollinators are either attracted or repelled according to the concentration of the emitted volatiles. To establish which mechanisms occur in the large Encephalartos cycad clade, the temporal patterns of volatile emissions, heating and pollinator activity of cones of Encephalartos villosus in the Eastern Cape (EC) and KwaZulu Natal (KZN) of South Africa were investigated.

Methods and Key Results

Gas chromatography–mass spectrometry (GC-MS) analyses of Encephalartos villosus cone volatiles showed that emissions, dominated by eucalyptol and 2-isopropyl-3-methoxypyrazine in EC populations and (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene in the KZN populations, varied across developmental stages but did not vary significantly on a daily cycle. Heating in male cones was higher at dehiscence than during pre- and post-dehiscence, and reached a maximum at about 1830 h when temperatures were between 7·0 and 12·0 °C above ambient. Daily heating of female cones was less pronounced and reached a maximum at about 1345 h when it was on average between 0·9 and 3·0 °C above ambient. Insect abundance on male cones was higher at dehiscence than at the other stages and significantly higher in the afternoon than in the morning and evening.

Conclusions

There are pronounced developmental changes in volatile emissions and heating in E. villosus cones, as well as strong daily changes in thermogenesis. Daily patterns of volatile emissions and pollinator abundance in E. villosus are different from those observed in some Macrozamia cycads and not consistent with the push–pull pattern as periods of peak odour emission do not coincide with mass exodus of insects from male cones.     

Selection by Pollinators on Floral Traits in Generalized Trollius ranunculoides (Ranunculaceae) along Altitudinal Gradients

Abundance and visitation of pollinator assemblages tend to decrease with altitude, leading to an increase in pollen limitation. Thus increased competition for pollinators may generate stronger selection on attractive traits of flowers at high elevations and cause floral adaptive evolution. Few studies have related geographically variable selection from pollinators and intraspecific floral differentiation. We investigated the variation of Trollius ranunculoides flowers and its pollinators along an altitudinal gradient on the eastern Qinghai-Tibet Plateau, and measured phenotypic selection by pollinators on floral traits across populations. The results showed significant decline of visitation rate of bees along altitudinal gradients, while flies was unchanged. When fitness is estimated by the visitation rate rather than the seed number per plant, phenotypic selection on the sepal length and width shows a significant correlation between the selection strength and the altitude, with stronger selection at higher altitudes. However, significant decreases in the sepal length and width of T. ranunculoides along the altitudinal gradient did not correspond to stronger selection of pollinators. In contrast to the pollinator visitation, mean annual precipitation negatively affected the sepal length and width, and contributed more to geographical variation in measured floral traits than the visitation rate of pollinators. Therefore, the sepal size may have been influenced by conflicting selection pressures from biotic and abiotic selective agents. This study supports the hypothesis that lower pollinator availability at high altitude can intensify selection on flower attractive traits, but abiotic selection is preventing a response to selection from pollinators.