Fine-scale genetic structure in the orchid Gymnadenia conopsea is not associated with local density of flowering plants.

PREMISE: Density-dependent pollinator visitation can lead to density-dependent mating patterns and within-population genetic structure. In Gymnadenia conopsea, individuals in low-density patches receive more self pollen than individuals in high-density patches, suggesting higher relatedness at low density. Ongoing fragmentation is also expected to cause more local matings, potentially leading to biparental inbreeding depression. METHODS: To evaluate whether relatedness decreases with local density, we analyzed 1315 SNP loci in 113 individuals within two large populations. We quantified within-population genetic structure in one of the populations, recorded potential habitat barriers, and visualized gene flow using estimated effective migration surfaces (EEMS). We further estimated the magnitude of biparental inbreeding depression that would result from matings restricted to within 5 m. RESULTS: There was no significant relationship between local density and relatedness in any population. We detected significant fine-scale genetic structure consistent with isolation by distance, with positive kinship coefficients at distances below 10 m. Kinship coefficients were low, and predicted biparental inbreeding depression resulting from matings within the closest 5 m was a modest 1-3%. The EEMS suggested that rocks and bushes may act as barriers to gene flow within a population. CONCLUSIONS: The results suggest that increased self-pollen deposition in sparse patches does not necessarily cause higher selfing rates or that inbreeding depression results in low establishment success of inbred individuals. The modest relatedness suggests that biparental inbreeding depression is unlikely to be an immediate problem following fragmentation of large populations. The results further indicate that habitat structure may contribute to governing fine-scale genetic structure in G. conopsea.

Soil water and nutrient availability interactively modify pollinator-mediated directional and correlational selection on floral display.

The individual and combined effects of abiotic factors on pollinator-mediated selection on floral traits are not well documented. To examine potential interactive effects of water and nutrient availability on pollinator-mediated selection on three floral display traits of Primula tibetica, we manipulated pollination and nutrient availability in a factorial experiment, conducted at two common garden sites with different soil water content (natural vs addition). We found that both water and nutrient availability affected floral trait expression in P. tibetica and that hand pollination increased seed production most when both nutrient content and water content were high, indicating joint pollen and resource limitation. We documented selection on all floral traits, and pollinators contributed to directional and correlational selection on plant height and number of flowers. Soil water and nutrient availability interactively influenced the strength of both pollinator-mediated directional and correlational selection, with significant selection observed when nutrient or water availability was high, but not when none or both were added. The results suggest that resource limitation constrains the response of P. tibetica to among-individual variation in pollen receipt, that addition of nutrients or water leads to pollinator-mediated selection and that effects of the two abiotic factors are nonadditive.

Measuring, comparing and interpreting phenotypic selection on floral scent.

Natural selection on floral scent composition is a key element of the hypothesis that pollinators and other floral visitors drive scent evolution. The measure of such selection is complicated by the high-dimensional nature of floral scent data and uncertainty about the cognitive processes involved in scent-mediated communication. We use dimension reduction through reduced-rank regression to jointly estimate a scent composite trait under selection and the strength of selection acting on this trait. To assess and compare variation in selection on scent across species, time and space, we reanalyse 22 datasets on six species from four previous studies. The results agreed qualitatively with previous analyses in terms of identifying populations and scent compounds subject to stronger selection but also allowed us to evaluate and compare the strength of selection on scent across studies. Doing so revealed that selection on floral scent was highly variable, and overall about as common and as strong as selection on other phenotypic traits involved in pollinator attraction or pollen transfer. These results are consistent with an important role of floral scent in pollinator attraction. Our approach should be useful for further studies of plant-animal communication and for studies of selection on other high-dimensional phenotypes. In particular, our approach will be useful for studies of pollinator-mediated selection on complex scent blends comprising many volatiles, and when no prior information on the physiological responses of pollinators to scent compounds is available.

Extensive pollinator sharing does not promote character displacement in two orchid congeners.

Pollinator sharing between close relatives can be costly and can promote pollination niche partitioning and floral divergence. This should be reflected by a higher species divergence in sympatry than in allopatry. We tested this hypothesis in two orchid congeners with overlapping distributions and flowering times. We characterized floral traits and pollination niches and quantified pollen limitation in 15 pure and mixed populations, and we measured phenotypic selection on floral traits and performed controlled crosses in one mixed site. Most floral traits differed between species, yet pollinator sharing was extensive. Only the timing of scent emission diverged more in mixed sites than in pure sites, and this was not mirrored by the timing of pollinator visitation. We did not detect divergent selection on floral traits. Seed production was pollen limited in most populations but not more severely in mixed sites than in pure sites. Interspecific crosses produced the same or a higher proportion of viable seeds than intraspecific crosses. The two orchid species attract the same pollinator species despite showing divergent floral traits. However, this does not promote character displacement, implying a low cost of pollinator sharing. Our results highlight the importance of characterizing both traits and ecological niches in character displacement studies.

Divergent selection on flowering phenology but not on floral morphology between two closely related orchids.

Closely related species often differ in traits that influence reproductive success, suggesting that divergent selection on such traits contribute to the maintenance of species boundaries. Gymnadenia conopsea ss. and Gymnadenia densiflora are two closely related, perennial orchid species that differ in (a) floral traits important for pollination, including flowering phenology, floral display, and spur length, and (b) dominant pollinators. If plant-pollinator interactions contribute to the maintenance of trait differences between these two taxa, we expect current divergent selection on flowering phenology and floral morphology between the two species. We quantified phenotypic selection via female fitness in one year on flowering start, three floral display traits (plant height, number of flowers, and corolla size) and spur length, in six populations of G. conopsea s.s. and in four populations of G. densiflora. There was indication of divergent selection on flowering start in the expected direction, with selection for earlier flowering in two populations of the early-flowering G. conopsea s.s. and for later flowering in one population of the late-flowering G. densiflora. No divergent selection on floral morphology was detected, and there was no significant stabilizing selection on any trait in the two species. The results suggest ongoing adaptive differentiation of flowering phenology, strengthening this premating reproductive barrier between the two species. Synthesis: This study is among the first to test whether divergent selection on floral traits contribute to the maintenance of species differences between closely related plants. Phenological isolation confers a substantial potential for reproductive isolation, and divergent selection on flowering time can thus greatly influence reproductive isolation and adaptive differentiation.

Pollen limitation in a single year is not compensated by future reproduction.

Seed production is critical to the persistence of most flowering plant populations, but may be strongly pollen limited. To what extent long-lived plants can compensate pollen limitation by increasing future reproduction is poorly understood. We tested for compensation in two Dactylorhiza species that differ in reproductive investment by experimentally reducing and increasing pollination in two independent annual cohorts and monitoring demographic responses in the subsequent 2 years for the 2014 cohort and in 1 year for the 2015 cohort. Demographic rates in the second year were significantly affected by pollination treatment in both species, but specific responses differed both between species and years. There was no effect of pollination treatment on demographic responses in the third year. In sum, effects were too weak to make up for the lost reproduction; total fruit production across all 3 years was by far highest in the increased pollination treatment in both species. These results show that long-lived plants do not necessarily compensate for pollen limitation by increasing future reproduction. It further suggests that even periodic declines in pollination rates may have severe demographic consequences, particularly in populations where germination is not density dependent. This has implications for predicting plant population viability in response to changes in pollination intensity.

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.

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.

Conflicting selection on floral scent emission in the orchid Gymnadenia conopsea.

Floral scent is a crucial trait for pollinator attraction. Yet only a handful of studies have estimated selection on scent in natural populations and no study has quantified the relative importance of pollinators and other agents of selection. In the fragrant orchid Gymnadenia conopsea, we used electroantennographic data to identify floral scent compounds detected by local pollinators and quantified pollinator-mediated selection on emission rates of 10 target compounds as well as on flowering start, visual display and spur length. Nocturnal pollinators contributed more to reproductive success than diurnal pollinators, but there was significant pollinator-mediated selection on both diurnal and nocturnal scent emission. Pollinators selected for increased emission of two compounds and reduced emission of two other compounds, none of which were major constituents of the total bouquet. In three cases, pollinator-mediated selection was opposed by nonpollinator-mediated selection, leading to weaker or no detectable net selection. Our study demonstrates that minor scent compounds can be targets of selection, that pollinators do not necessarily favour stronger scent signalling, and that some scent compounds are subject to conflicting selection from pollinators and other agents of selection. Hence, including floral scent traits into selection analysis is important for understanding the mechanisms behind floral evolution.

Evolutionary ecology of nectar.

Background: Floral nectar is an important determinant of plant-pollinator interactions and an integral component of pollination syndromes, suggesting it is under pollinator-mediated selection. However, compared to floral display traits, we know little about the evolutionary ecology of nectar. Combining a literature review with a meta-analysis approach, we summarize the evidence for heritable variation in nectar traits and link this variation to pollinator response and plant fitness. We further review associations between nectar traits and floral signals and discuss them in the context of honest signalling and targets of selection. Scope: Although nectar is strongly influenced by environmental factors, heritable variation in nectar production rate has been documented in several populations (mean h2 = 0.31). Almost nothing is known about heritability of other nectar traits, such as sugar and amino acid concentrations. Only a handful of studies have quantified selection on nectar traits, and few find statistically significant selection. Pollinator responses to nectar traits indicate they may drive selection, but studies tying pollinator preferences to plant fitness are lacking. So far, only one study conclusively identified pollinators as selective agents on a nectar trait, and the role of microbes, herbivores, nectar robbers and abiotic factors in nectar evolution is largely hypothetical. Finally, there is a trend for positive correlations among floral cues and nectar traits, indicating honest signalling of rewards. Conclusions: Important progress can be made by studies that quantify current selection on nectar in natural populations, as well as experimental approaches that identify the target traits and selective agents involved. Signal-reward associations suggest that correlational selection may shape evolution of nectar traits, and studies exploring these more complex forms of natural selection are needed. Many questions about nectar evolution remain unanswered, making this a field ripe for future research.

A meta-analysis of the agents of selection on floral traits.

Floral traits are hypothesized to evolve primarily in response to selection by pollinators. However, selection can also be mediated by other environmental factors. To understand the relative importance of pollinator-mediated selection and its variation among trait and pollinator types, we analyzed directional selection gradients on floral traits from experiments that manipulated the environment to identify agents of selection. Pollinator-mediated selection was stronger than selection by other biotic factors (e.g., herbivores), but similar in strength to selection by abiotic factors (e.g., soil water), providing partial support for the hypothesis that floral traits evolve primarily in response to pollinators. Pollinator-mediated selection was stronger on pollination efficiency traits than on other trait types, as expected if efficiency traits affect fitness via interactions with pollinators, but other trait types also affect fitness via other environmental factors. In addition to varying among trait types, pollinator-mediated selection varied among pollinator taxa: selection was stronger when bees, long-tongued flies, or birds were the primary visitors than when the primary visitors were Lepidoptera or multiple animal taxa. Finally, reducing pollinator access to flowers had a relatively small effect on selection on floral traits, suggesting that anthropogenic declines in pollinator populations would initially have modest effects on floral evolution.

Drivers of vegetative dormancy across herbaceous perennial plant species.

Vegetative dormancy, that is the temporary absence of aboveground growth for ≥ 1 year, is paradoxical, because plants cannot photosynthesise or flower during dormant periods. We test ecological and evolutionary hypotheses for its widespread persistence. We show that dormancy has evolved numerous times. Most species displaying dormancy exhibit life-history costs of sprouting, and of dormancy. Short-lived and mycoheterotrophic species have higher proportions of dormant plants than long-lived species and species with other nutritional modes. Foliage loss is associated with higher future dormancy levels, suggesting that carbon limitation promotes dormancy. Maximum dormancy duration is shorter under higher precipitation and at higher latitudes, the latter suggesting an important role for competition or herbivory. Study length affects estimates of some demographic parameters. Our results identify life historical and environmental drivers of dormancy. We also highlight the evolutionary importance of the little understood costs of sprouting and growth, latitudinal stress gradients and mixed nutritional modes.

Response to Comment on "Precipitation drives global variation in natural selection".

The comment by Myers-Smith and Myers focuses on three main points: (i) the lack of a mechanistic explanation for climate-selection relationships, (ii) the appropriateness of the climate data used in our analysis, and (iii) our focus on estimating climate-selection relationships across (rather than within) taxonomic groups. We address these critiques in our response.

Diel pattern of floral scent emission matches the relative importance of diurnal and nocturnal pollinators in populations of Gymnadenia conopsea.

Background and Aims: Floral scent is considered an integral component of pollination syndromes, and its composition and timing of emission are thus expected to match the main pollinator type and time of activity. While floral scent differences among plant species with different pollination systems can be striking, studies on intraspecific variation are sparse, which limits our understanding of the role of pollinators in driving scent divergence. Methods: Here, we used dynamic headspace sampling to quantify floral scent emission and composition during the day and at night in the natural habitat of six Scandinavian populations of the fragrant orchid Gymnadenia conopsea. We tested whether diel scent emission and composition match pollinator type by comparing four populations in southern Sweden, where nocturnal pollinators are more important for plant reproductive success than are diurnal pollinators, with two populations in central Norway, where the opposite is true. To determine to what extent scent patterns quantified in the field reflected plasticity, we also measured scent emission in a common growth chamber environment. Key Results: Both scent composition and emission rates differed markedly between day and night, but only the latter varied significantly among populations. The increase in scent emission rate at night was considerably stronger in the Swedish populations compared with the Norwegian populations. These patterns persisted when plants were transferred to a common environment, suggesting a genetic underpinning of the scent variation. Conclusions: The results are consistent with a scenario where spatial variation in relative importance of nocturnal and diurnal pollinators has resulted in selection for different scent emission rhythms. Our study highlights the importance of adding a characterization of diel variation of scent emission rates to comparative studies of floral scent, which so far have often focused on scent composition only.

What Are the Environmental Determinants of Phenotypic Selection? A Meta-analysis of Experimental Studies.

Although many selection estimates have been published, the environmental factors that cause selection to vary in space and time have rarely been identified. One way to identify these factors is by experimentally manipulating the environment and measuring selection in each treatment. We compiled and analyzed selection estimates from experimental studies. First, we tested whether the effect of manipulating the environment on selection gradients depends on taxon, trait type, or fitness component. We found that the effect of manipulating the environment was larger when selection was measured on life-history traits or via survival. Second, we tested two predictions about the environmental factors that cause variation in selection. We found support for the prediction that variation in selection is more likely to be caused by environmental factors that have a large effect on mean fitness but not for the prediction that variation is more likely to be caused by biotic factors. Third, we compared selection gradients from experimental and observational studies. We found that selection varied more among treatments in experimental studies than among spatial and temporal replicates in observational studies, suggesting that experimental studies can detect relationships between environmental factors and selection that would not be apparent in observational studies.

Precipitation drives global variation in natural selection.

Climate change has the potential to affect the ecology and evolution of every species on Earth. Although the ecological consequences of climate change are increasingly well documented, the effects of climate on the key evolutionary process driving adaptation-natural selection-are largely unknown. We report that aspects of precipitation and potential evapotranspiration, along with the North Atlantic Oscillation, predicted variation in selection across plant and animal populations throughout many terrestrial biomes, whereas temperature explained little variation. By showing that selection was influenced by climate variation, our results indicate that climate change may cause widespread alterations in selection regimes, potentially shifting evolutionary trajectories at a global scale.

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.

Actuarial senescence in a long-lived orchid challenges our current understanding of ageing.

The dominant evolutionary theory of actuarial senescence-an increase in death rate with advancing age-is based on the concept of a germ cell line that is separated from the somatic cells early in life. However, such a separation is not clear in all organisms. This has been suggested to explain the paucity of evidence for actuarial senescence in plants. We used a 32 year study of Dactylorhiza lapponica that replaces its organs each growing season, to test whether individuals of this tuberous orchid senesce. We performed a Bayesian survival trajectory analysis accounting for reproductive investment, for individuals under two types of land use, in two climatic regions. The mortality trajectory was best approximated by a Weibull model, showing clear actuarial senescence. Rates of senescence in this model declined with advancing age, but were slightly higher in mown plots and in the more benign climatic region. At older ages, senescence was evident only when accounting for a positive effect of reproductive investment on mortality. Our results demonstrate actuarial senescence as well as a survival-reproduction trade-off in plants, and indicate that environmental context may influence senescence rates. This knowledge is crucial for understanding the evolution of demographic senescence and for models of plant population dynamics.

Experimental reduction in interaction intensity strongly affects biotic selection.

The link between biotic interaction intensity and strength of selection is of fundamental interest for understanding biotically driven diversification and predicting the consequences of environmental change. The strength of selection resulting from biotic interactions is determined by the strength of the interaction and by the covariance between fitness and the trait under selection. When the relationship between trait and absolute fitness is constant, selection strength should be a direct function of mean population interaction intensity. To test this prediction, we excluded pollinators for intervals of different length to induce five levels of pollination intensity within a single plant population. Pollen limitation (PL) increased from 0 to 0.77 across treatments, accompanied by a fivefold increase in the opportunity for selection. Trait-fitness covariance declined with PL for number of flowers, but varied little for other traits. Pollinator-mediated selection on plant height, corolla size, and spur length increased by 91%, 34%, and 330%, respectively, in the most severely pollen-limited treatment compared to open-pollinated plants. The results indicate that realized biotic selection can be predicted from mean population interaction intensity when variation in trait-fitness covariance is limited, and that declines in pollination intensity will strongly increase selection on traits involved in the interaction.

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.