Community-wide patterns in pollen and ovule production, their ratio (P/O), and other floral traits along an elevation gradient in southwestern China.

BACKGROUND: As the male and female gametophytes of flowering plants, pollen and ovules largely determine the upper and lower boundaries of plant reproductive success. It is commonly predicted that pollen and ovule number per flower should increase, and pollen-ovule ratio (P/O) per flower should decrease with increasing elevation in response to a more stochastic pollination environment. Here, we aimed to determine the response of pollen number, ovule number, and P/O to other floral traits and elevation gradients for 84 insect-pollinated herbaceous flowering plant species in five sub-alpine and alpine communities (2709 to 3896 m a.s.l.) on Yulong Snow Mountain, southwestern China. RESULTS: Six floral traits, including P/O, floral display area, flower number, tube depth, flower shape, and pollen presentation, were highly correlated with pollen and ovule number per flower. With increasing elevation, pollen number and P/O per flower increased marginally and significantly, respectively; ovule number per individual, flower number per individual, stigma stamen separation, and inflorescence height decreased significantly. However, ovule number per flower and other floral traits (i.e., floral display area, tube depth, stigma height, stamen height, and pollen and P/O per individual) did not change with elevation. We detected significant phylogenetic signals for pollen number, ovule number, and P/O, suggesting that these traits may be highly conserved and with limited response to changing environmental conditions. CONCLUSIONS: Results revealed patterns of plant reproductive character evolution along elevation gradients and the potential factors governing their spatial variation in high-elevation environments. Plant species at high elevations are more likely adapted to cross-pollination, indicated by increased P/O per flower at high elevations on Yulong Mountain. Combined effects of phylogenetic history and plant-pollinator interactions should determine plant trait evolution.

Changes in floral nectar are unlikely adaptive responses to pollinator flight sound.

Under noiseless experimental conditions, sugar concentration of secreted floral nectar may increase after flower exposure to nearby sounds of pollinator flight (Veits et al. 2019). However, we reject the argument that this represents adaptive plant behaviour, and consider that the appealing analogy between a flower and human ear is unjustified.

Interaction intensity and pollinator-mediated selection.

In animal-pollinated plants, the opportunity for selection and the strength of pollinator-mediated selection are expected to increase with the degree of pollen limitation. However, whether differences in pollen limitation can explain variation in pollinator-mediated and net selection among animal-pollinated species is poorly understood. In the present study, we quantified pollen limitation, variance in relative fitness and pollinator-mediated selection on five traits important for pollinator attraction (flowering start, plant height, flower number, flower size) and pollination efficiency (spur length) in natural populations of 12 orchid species. Pollinator-mediated selection was quantified by subtracting estimates of selection gradients for plants receiving supplemental hand-pollination from estimates obtained for open-pollinated control plants. Mean pollen limitation ranged from zero to 0.96. Opportunity for selection, pollinator-mediated selection and net selection were all positively related to pollen limitation, whereas nonpollinator-mediated selection was not. Opportunity for selection varied five-fold, strength of pollinator-mediated selection varied three-fold and net selection varied 1.5-fold among species. Supplemental hand-pollination reduced both opportunity for selection and selection on floral traits. The results show that the intensity of biotic interactions is an important determinant of the selection regime, and indicate that the potential for pollinator-mediated selection and divergence in floral traits is particularly high in species that are strongly pollen-limited.

Herbivory Increases Fruit Set in Silene latifolia: A Consequence of Induced Pollinator-Attracting Floral Volatiles?

Although the effect of herbivory on plant reproduction has been investigated in some detail, little is known about how herbivores affect floral signalling. Here, we investigated the effect of foliar herbivory by the African Cotton Leafworm (Spodoptera littoralis) on floral signalling and fruit set in the White Campion (Silene latifolia). We found no effects of herbivory on floral traits involved in visual signalling (flower number, corolla diameter, calyx length, petal length) or in amount of nectar produced. However, Spodoptera-infested plants emitted higher amounts of the two floral volatiles, (Z)-3-hexenyl acetate and ß-ocimene, than control plants. Open pollinated, infested plants also were found to produce more fruits than control plants, but only with nocturnal pollinators. Experimental addition of the two induced floral volatiles to non-infested Silene flowers also led to the production of more fruits with nocturnal pollination. This suggests that higher fruit production in herbivore-infested plants was caused by increased nocturnal pollinator attraction, mediated by the induced floral emission of these two volatiles. Our results show that the effects of herbivory on plant reproductive success are not necessarily detrimental, as plants can compensate herbivory with increased investment in pollinator attraction.

Flowering phenology differs among wet and dry sub-alpine meadows in southwestern China.

The effect of floral traits, floral rewards and plant water availability on plant-pollinator interactions are well-documented; however, empirical evidence of their impact on flowering phenology in high-elevation meadows remains scarce. In this study, we assessed three levels of flowering phenology, i.e. population-, individual- and flower-level (floral longevity), in two nearby but contrasting (wet versus dry) sub-alpine meadows on Yulong Snow Mountain, southwestern China. We also measured a series of floral traits (pollen number, ovule number, and the ratio of pollen to ovule number per flower, i.e. pollen:ovule ratio [P/O]) and floral rewards (nectar availability and pollen presentation) as plausible additional sources of variation for each phenological level. Floral longevity in the wet meadow was significantly longer than that for the dry meadow, whereas population- and individual-flowering duration were significantly shorter. Our results showed a significant positive relationship between flowering phenology with pollen number and P/O per flower; there was no relationship with ovule number per flower. Further, we found a significant effect of flowering phenology on nectar availability and pollen presentation. Our findings suggest that shorter floral longevity in dry habitats compared to wet might be due to water-dependent maintenance costs of flowers, where the population- and individual-level flowering phenology may be less affected by habitats. Our study shows how different levels of flowering phenology underscore the plausible effects of contrasting habitats on reproductive success.

Separating selection by diurnal and nocturnal pollinators on floral display and spur length in Gymnadenia conopsea.

Most plants attract multiple flower visitors that may vary widely in their effectiveness as pollinators. Floral evolution is expected to reflect interactions with the most important pollinators, but few studies have quantified the contribution of different pollinators to current selection on floral traits. To compare selection mediated by diurnal and nocturnal pollinators on floral display and spur length in the rewarding orchid Gymnadenia conopsea, we manipulated the environment by conducting supplemental hand-pollinations and selective pollinator exclusions in two populations in central Norway. In both populations, the exclusion of diurnal pollinators significantly reduced seed production compared to open pollination, whereas the exclusion of nocturnal pollinators did not. There was significant selection on traits expected to influence pollinator attraction and pollination efficiency in both the diurnal and nocturnal pollination treatment. The relative strength of selection among plants exposed to diurnal and nocturnal visitors varied among traits and populations, but the direction of selection was consistent. The results suggest that diurnal pollinators are more important than nocturnal pollinators for seed production in the study populations, but that both categories contribute to selection on floral morphology. The study illustrates how experimental manipulations can link specific categories of pollinators to observed selection on floral traits, and thus improve our understanding of how species interactions shape patterns of selection.

Reproductive Isolation Among Three Nocturnal Moth-Pollinated Sympatric Habenaria Species (Orchidaceae).

Comparison and quantification of multiple pre- and post-pollination barriers to interspecific hybridization are important to understand the factors promoting reproductive isolation. Such isolating factors have been studied recently in many flowering plant species which seek after the general roles and relative strengths of different pre- and post-pollination barriers. In this study, we quantified six isolating factors (ecogeographic isolation, phenological isolation, pollinator isolation, pollinia-pistil interactions, fruit production, and seed development) that could possibly be acting as reproductive barriers at different stages among three sympatric Habenaria species (H. limprichtii, H. davidii, and H. delavayi). These three species overlap geographically but occupy different microhabitats varying in soil water content. They were isolated through pollinator interactions both ethologically (pollinator preference) and mechanically (pollinia attachment site), but to a variable degree for different species pairs. Interspecific crosses between H. limprichtii and H. davidii result in high fruit set, and embryo development suggested weak post-pollination barriers, whereas bidirectional crosses of H. delavayi with either of the other two species fail to produce fruits. Our results revealed that pollinators were the most important isolating barrier including both ethological and mechanical mechanisms, to maintain the boundaries among these three sympatric Habenaria species. Our study also highlights the importance of a combination of pre-and post-pollination barriers for species co-existence in Orchidaceae.

Flower Color Evolution and the Evidence of Pollinator-Mediated Selection.

The evolution of floral traits in animal-pollinated plants involves the interaction between flowers as signal senders and pollinators as signal receivers. Flower colors are very diverse, effect pollinator attraction and flower foraging behavior, and are hypothesized to be shaped through pollinator-mediated selection. However, most of our current understanding of flower color evolution arises from variation between discrete color morphs and completed color shifts accompanying pollinator shifts, while evidence for pollinator-mediated selection on continuous variation in flower colors within populations is still scarce. In this review, we summarize experiments quantifying selection on continuous flower color variation in natural plant populations in the context of pollinator interactions. We found that evidence for significant pollinator-mediated selection is surprisingly limited among existing studies. We propose several possible explanations related to the complexity in the interaction between the colors of flowers and the sensory and cognitive abilities of pollinators as well as pollinator behavioral responses, on the one hand, and the distribution of variation in color phenotypes and fitness, on the other hand. We emphasize currently persisting weaknesses in experimental procedures, and provide some suggestions for how to improve methodology. In conclusion, we encourage future research to bring together plant and animal scientists to jointly forward our understanding of the mechanisms and circumstances of pollinator-mediated selection on flower color.

Bimodal activity of diurnal flower visitation at high elevation.

Successful pollination in animal-pollinated plants depends on the temporal overlap between flower presentation and pollinator foraging activity. Variation in the temporal dimension of plant-pollinator networks has been investigated intensely across flowering seasons. However, over the course of a day, the dynamics of plant-pollinator interactions may vary strongly due environmental fluctuations. It is usually assumed there is a unimodal, diurnal, activity pattern, while alternative multimodal types of activity patterns are often neglected and deserve greater investigation. Here, we quantified the daily activity pattern of flower visitors in two different habitats contrasting high elevation meadows versus forests in Southwest China to investigate the role of abiotic conditions in the temporal dynamics of plant-pollinator interactions. We examined diurnal activity patterns for the entire pollinator community. Pollinator groups may differ in their ability to adapt to habitats and abiotic conditions, which might be displayed in their patterns of activity. We hypothesized that (a) pollinator communities show multimodal activity patterns, (b) patterns differ between pollinator groups and habitat types, and (c) abiotic conditions explain observed activity patterns. In total, we collected 4,988 flower visitors belonging to six functional groups. There was a bimodal activity pattern when looking at the entire pollinator community and in five out of six flower visitor groups (exempting solitary bees) regardless of habitat types. Bumblebees, honeybees, dipterans, lepidopterans, and other insects showed activity peaks in the morning and afternoon, whereas solitary bees were most active at midday. Activity of all six pollinator groups increased as solar radiation increased and then decreased after reaching a certain threshold. Our findings suggest that in habitats at higher elevations, a bimodal activity pattern of flower visitation is commonly employed across most pollinator groups that are diurnal foragers. This pattern may be caused by insects avoiding overheating due to elevated temperatures when exposed to high solar radiation at midday.

Manipulation of trait expression and pollination regime reveals the adaptive significance of spur length.

Understanding the mechanisms of adaptive population differentiation requires that both the functional and adaptive significance of divergent traits are characterized in contrasting environments. Here, we (a) determined the effects of floral spur length on pollen removal and receipt using plants with artificial spurs representing the species-wide variation in length, and (b) quantified pollinator-mediated selection on spur length and three traits contributing to floral display in two populations each of the short-spurred and the long-spurred ecotype of the orchid Platanthera bifolia. Both pollen receipt and removal reached a maximum at 28-29 mm long spurs in a short-spurred population visited by short-tongued moths. In contrast, pollen receipt increased linearly across the tested range (4-52 mm) and pollen removal was unrelated to spur length in a long-spurred population predominantly visited by a long-tongued moth. The experimentally documented effects on pollen transfer were not reflected in pollinator-mediated selection through female fitness or pollen removal indicating that the natural within-population variation in spur length was insufficient to result in detectable variation in pollen limitation. Our study illustrates how combining trait manipulation with analysis of causes and strength of phenotypic selection can illuminate the functional and adaptive significance of trait expression when trait variation is limited.

Salvage of floral resources through re-absorption before flower abscission.

Plants invest floral resources, including nectar and pigment, with likely consequent reproductive costs. We hypothesized that plants, whose flowers abscise with age, reabsorb nectar and pigment before abscission. This was tested with flowers of Rhododendron decorum, which has large, conspicuous white flowers that increasingly abscise corollas as flowers age. As this species is pollinated by bees, we also hypothesized that nectar concentration would be relatively high (i.e., > 30% wt/vol) and petals would contain UV-absorbing pigment. Floral nectar volume and concentration were sampled on successive days until abscission (up to ten days old, peak at five days) and for sub-sample of four-day-old flowers. Flowers just abscised were similarly sampled. Flower colours were measured using a modified camera, with recordings of spectral reflectance for abscised and open non-abscised flowers. Pigment content was summed values of red, green, blue channels of false color photos. As expected, flowers reabsorbed almost all nectar before abscission, separately reabsorbing nectar-sugar and nectar-water, and petals contained UV-absorbing pigment. However, flowers did not reabsorb pigment and nectar-concentration was < 30% wt/vol. That flowers reabsorb nectar, not pigment, remains unexplained, though possibly pigment reabsorption is uneconomical. Understanding floral resource reabsorption therefore requires determination of biochemical mechanisms, plus costs/benefits for individual plants.

Nectar mimicry: a new phenomenon.

Nectar is the most common floral reward for flower-visiting flies, bees, bats and birds. Many flowers hide nectar in the floral tube and preclude sensing of nectar by flower-visitors from a distance. Even in those flowers that offer easily accessible nectar, the nectaries are mostly inconspicuous to the human eye and the amount of nectar is sparse. It is widely accepted that many flowers display nectar guides in order to direct flower-visitors towards the nectar. Using false colour photography, covering ultraviolet, blue and green ranges of wavelength, revealed a yet unknown conspicuousness of nectar, nectaries and false nectaries for bees due to concordant reflection in the ultraviolet range of wavelength. Nectars, many nectaries and false nectaries have glossy surfaces and reflect all incident light including UV-light. In most cases, this is not particularly conspicuous to the human eye, but highly visible for UV-sensitive insects, due to the fact that the glossy areas are often positioned in UV-absorbing central flower parts and thus produce a strong UV-signal. The optical contrast produced by the glossiness of small smooth areas in close proximity to nectar holders represents a widespread yet overlooked floral cue that nectarivorous flower-visitors might use to locate the floral nectar.

The independent and combined effects of floral traits distinguishing two pollination ecotypes of a moth-pollinated orchid.

Identifying traits and agents of selection involved in local adaptation is important for understanding population divergence. In southern Sweden, the moth-pollinated orchid Platanthera bifolia occurs as a woodland and a grassland ecotype that differ in dominating pollinators. The woodland ecotype is taller (expected to influence pollinator attraction) and produces flowers with longer spurs (expected to influence efficiency of pollen transfer) compared to the grassland ecotype. We examined whether plant height and spur length affect pollination and reproductive success in a woodland population, and whether effects are non-additive, as expected for traits influencing two multiplicative components of pollen transfer. We reduced plant height and spur length to match trait values observed in the grassland ecotype and determined the effects on pollen removal, pollen receipt, and fruit production. In addition, to examine the effects of naturally occurring variation, we quantified pollinator-mediated selection through pollen removal and seed production in the same population. Reductions of plant height and spur length decreased pollen removal, number of flowers receiving pollen, mean pollen receipt per pollinated flower, and fruit production per plant, but no significant interaction effect was detected. The selection analysis demonstrated pollinator-mediated selection for taller plants via female fitness. However, there was no current selection mediated by pollinators on spur length, and pollen removal was not related to plant height or spur length. The results show that, although both traits are important for pollination success and female fitness in the woodland habitat, only plant height was sufficiently variable in the study population for current pollinator-mediated selection to be detected. More generally, the results illustrate how a combination of experimental approaches can be used to identify both traits and agents of selection.

Strong pollinator-mediated selection for increased flower brightness and contrast in a deceptive orchid.

Contrasting flower color patterns that putatively attract or direct pollinators toward a reward are common among angiosperms. In the deceptive orchid Anacamptis morio, the lower petal, which makes up most of the floral display, has a light central patch with dark markings. Within populations, there is pronounced variation in petal brightness, patch size, amount of dark markings, and contrast between patch and petal margin. We tested whether pollinators mediate selection on these color traits and on morphology (plant height, number of flowers, corolla size, spur length), and whether selection is consistent with facilitated or negative frequency-dependent pollination. Pollinators mediated strong selection for increased petal brightness (Δßpoll = 0.42) and contrast (Δßpoll = 0.51). Pollinators also tended to mediate stabilizing selection on brightness (Δγpoll = -0.27, n.s.) favoring the most common phenotype in the population. Selection for reduced petal brightness among hand-pollinated plants indicated a fitness cost associated with brightness. The results demonstrate that flower color traits influence pollination success and seed production in A. morio, indicating that they affect attractiveness to pollinators, efficiency of pollen transfer, or both. The documented selection is consistent with facilitated pollination and selection for color convergence toward cooccurring rewarding species.