The morphology, molecular development and ecological function of pseudonectaries on Nigella damascena (Ranunculaceae) petals.

Pseudonectaries, or false nectaries, the glistening structures that resemble nectaries or nectar droplets but do not secrete nectar, show considerable diversity and play important roles in plant-animal interactions. The morphological nature, optical features, molecular underpinnings and ecological functions of pseudonectaries, however, remain largely unclear. Here, we show that pseudonectaries of Nigella damascena (Ranunculaceae) are tiny, regional protrusions covered by tightly arranged, non-secretory polygonal epidermal cells with flat, smooth and reflective surface, and are clearly visible even under ultraviolet light and bee vision. We also show that genes associated with cell division, chloroplast development and wax formation are preferably expressed in pseudonectaries. Specifically, NidaYABBY5, an abaxial gene with ectopic expression in pseudonectaries, is indispensable for pseudonectary development: knockdown of it led to complete losses of pseudonectaries. Notably, when flowers without pseudonectaries were arrayed beside those with pseudonectaries, clear differences were observed in the visiting frequency, probing time and visiting behavior of pollinators (i.e., honey bees), suggesting that pseudonectaries serve as both visual attractants and nectar guides.

Lack of spatial structure for phenotypic and genetic variation despite high self-fertilization in Aquilegia canadensis (Ranunculaceae).

By reducing genetically effective population size and gene flow, self-fertilization should lead to strong spatial genetic structure (SGS). Although the short-lived plant Aquilegia canadensis produces large, complex, nectar-rich flowers, 75% of seed, on average, are self-fertilized. Previous experimental results are consistent with the fine-scale SGS expected in selfing populations. In contrast, key floral traits show no evidence of SGS, despite a significant genetic basis to phenotypic variation within populations. In this study, we attempt to resolve these contradictory results by hierarchically sampling plants from two plots nested within each of seven rock outcrops distributed over several km, and comparing the spatial pattern of phenotypic variation in four floral traits with neutral genetic variation at 10 microsatellite loci. For both floral and microsatellite variation, we detected only weak hierarchical structuring and no isolation by distance. The spatial pattern of variation in floral traits was on par with microsatellite polymorphisms. These results suggest regular long-distance gene flow via pollen. At much finer spatial scales within plots, estimates of relatedness were higher (albeit very low) between nearest neighbors than random plants, and declined with increasing distance between neighbors, which is consistent with highly localized seed dispersal. High selfing should yield SGS, but strong inbreeding depression in A. canadensis likely erodes SGS so that reproductive plants exhibit weak structure typical of outcrossers, especially given that outcrossing and consequent gene flow in this species are mediated by strong-flying hummingbirds and bumble bees.

Relationship between the species-representative phenotype and intraspecific variation in Ranunculaceae floral organ and Asteraceae flower numbers.

BACKGROUND AND AIMS: Phenotypic variation in floral morphologies contributes to speciation by testing various morphologies that might have higher adaptivity, leading eventually to phylogenetic diversity. Species diversity has been recognized, however, by modal morphologies where the variation is averaged out, so little is known about the relationship between the variation and the diversity. METHODS: We analysed quantitatively the intraspecific variation of the organ numbers within flowers of Ranunculaceae, a family which branched near the monocot-eudicot separation, and the numbers of flowers within the capitula of Asteraceae, one of the most diverse families of eudicots. We used four elementary statistical quantities: mean, standard deviation (s.d.), degree of symmetry (skewness) and steepness (kurtosis). KEY RESULTS: While these four quantities vary among populations, we found a common relationship between s.d. and the mean number of petals and sepals in Ranunculaceae and number of flowers per capitulum in Asteraceae. The s.d. is equal to the square root of the difference between the mean and specific number, showing robustness: for example, 3 in Ficaria sepals, 5 in Ranunculus petals and Anemone tepals, and 13 in Farfugium ray florets. This square-root relationship was not applicable to Eranthis petals which show little correlation between the s.d. and mean, and the stamens and carpels of Ranunculaceae whose s.d. is proportional to the mean. The specific values found in the square-root relationship provide a novel way to find the species-representative phenotype among varied morphologies. CONCLUSIONS: The representative phenotype is, in most cases, unique to the species or genus level, despite intraspecific differences of average phenotype among populations. The type of variation shown by the statistical quantities indicates not only the robustness of the morphologies but also how flowering plants changed during evolution among representative phenotypes that eventually led to phylogenetic diversification.

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.

Chromatographic fingerprint analysis of the floral parts of Trollius chinensis.

This study provided a practical procedure, for the first time, to compare the component difference of the floral parts of Trollius chinensis and identify the characteristic peaks of each floral part using the high-performance liquid chromatographic fingerprint technique followed by similarity analysis. The results showed that the constituents of different floral parts exhibited lower similarity than those of the same part. It can be concluded that the procedure established herein is useful for analysis of variability in constituent distribution of herbal drugs, and the components are unevenly distributed in the floral parts of T. chinensis.

Variation in the reproductive performance of the Trollius-Chiastocheta mutualism at the edge of its range in north-east Germany.

Empirical studies into obligate pollination mutualisms which elucidate the variation in reproductive performance of shrinking populations within human-altered environments are rare. This study focuses on the obligate pollination mutualism between Trollius europaeus (Ranunculaceae) and fly species of the genus Chiastocheta which act both as the plant's main pollinators and as predators in that their larvae eat a fraction of the developing seeds. The study area is situated in the lowlands of north-east Germany. Many populations of T. europaeus have become comparatively small and scattered in this region as a consequence of agricultural land use intensification. We studied the plant's reproductive fitness in populations ranging in size from 7 to 12,000 flowers. In a field experiment, we applied four pollination treatments and also recorded fly density in 28 natural T. europaeus populations. The fitness of the offspring from 19 populations was studied in a common garden experiment. In both approaches, a reduction in the fitness of small host plant populations could be demonstrated. Fitness loss can be put down to the quantitative and qualitative limitation of pollen caused by inbreeding and the negative feedback on relative seed set caused by the reduced ability of small plant populations to support a sufficiently large fly population. Although increases in fly density are associated with rising predation costs, the plant species' net benefit is a positive function of its population size. Our study highlights the reproductive variability of the Trollius-Chiastocheta interaction along a population size gradient in a marginal region of its range, thus contributing to the understanding of the overall variability of this mutualism.

The seasonal effect in one-dimensional Daisyworld.

We have studied the effects of seasonal Solar Radiation Forcing (SRF) on the climate self-regulatory capability of life, using a latitudinal-dependent Daisyworld model. Because the seasonal polarity of SRF increases poleward, habitable conditions exist in the equatorial regions year round, whereas, in the high latitudes, harsh winters cause annual extinction of life, and only the summers are inhabited or regulated by life. Seasonality affects climate regulation by two major mechanisms: (1) the cold winter conditions in the high latitudes reduce the global temperature below the optimal temperature; (2) during summer, life experiences higher SRF anomalies and, therefore, shifts to higher albedo when compared to annual mean SRF. In turn, a full capacity for temperature regulation is reached at lower SRF, and the range of SRF over which life regulates climate is significantly reduced. Lastly, initiation/extinction of life at low/highly-perturbed SRF occurs at the poles. Therefore, an irreversible global extinction occurs once life passes its regulatory capacity in the poles. We conduct extensive sensitivity analyses on various model parameters (latitudinal heat diffusion, heat capacity, and population death rate), strengthening the generality/robustness of the above net seasonal effects. Applications to other SRF fluctuation, as Milankovitch cycles are discussed.

Specialized nursery pollination mutualisms as evolutionary traps stabilized by antagonistic traits.

We examine the conditions for the transition from antagonism to mutualism between plants and their specialists nursery pollinators in a reference case which is the Trollius europaeus-Chiastocheta interaction. The mechanistic model we developed shows that a specialization of T. europaeus on Chiastocheta could be the result of an attempt to escape over-exploitation by closing its flower. The pressure for such an escape increases with the parasite's frequency and its pollination efficiency but decreases in the presence of alternative pollinators. The resulting specialization is a priori an unstable one, leading either to strong evolutionary oscillations, or to evolutionary suicide due to over-exploitation of the plants. It becomes stable if the plants develop a defense mechanism to regulate their parasite's population size and limit seed-exploitation. The development of a counter-measure by the latter can destabilize the mutualism depending on the costs linked to such a trait. On the other hand, we find that a specialization on a purely mutualistic basis would require a preexisting high diversity of flower-opening within the population.

Optimal pollinator attraction strategies in Trollius ranunculoides Hemsl. (Ranunculaceae) at different altitudes: increased floral display or promotion of nectar output?

For alpine plant species, patterns of resource allocation to functional floral traits for pollinator attraction can be highly significant in adaptation to low pollinator abundance and consequent pollen limitation. Increased pollination can be achieved either through a larger floral display or production of more pollen rewards. In this study, variation in resource allocation to different components for pollinator attraction was studied along an altitudinal gradient in Trollius ranunculoides, an obligate self-incompatible out-crosser of the Qinghai-Tibet Plateau. We compared resource allocation to conspicuous yellow sepals (which mainly provide visual attraction) and degenerate petals (which provide the major nectar reward) between populations at four altitudes. Furthermore, we investigated the contribution of sepals and petals to pollinator attraction and female reproductive success in an experiment with sepal or petal removal at sites at different altitudes. At the level of single flowers, resource allocation increased to sepals but decreased to petals with increasing altitude. Consistent with these results, sepals contributed much more to visitation rate and seed set than petals, as confirmed in the sepal or petal removal experiment. Sepals and petals contributed to female reproductive success by ensuring visitation rate rather than visitation duration. To alleviate increasing pollen limitation with increasing altitude, resource allocation patterns of T. ranunculoides altered to favour development of sepals rather than petals. This strategy may improve pollination and reproductive success through visual attraction (sepal) rather than nectar reward (petal) over a gradient of decreasing pollinator abundance.

The effect of flower position on variation and covariation in floral traits in a wild hermaphrodite plant.

BACKGROUND: Floral traits within plants can vary with flower position or flowering time. Within an inflorescence, sexual allocation of early produced basal flowers is often female-biased while later produced distal flowers are male-biased. Such temporal adjustment of floral resource has been considered one of the potential advantages of modularity (regarding a flower as a module) in hermaphrodites. However, flowers are under constraints of independent evolution of a given trait. To understand flower diversification within inflorescences, here we examine variation and covariation in floral traits within racemes at the individual and the maternal family level respectively in an alpine herb Aconitum gymnandrum (Ranunculaceae). RESULTS: We found that floral traits varied significantly with flower position and among families, and position effects were family-specific. Most of the variance of floral traits was among individuals rather than among flowers within individuals or among families. Significant phenotypic correlations between traits were not affected by position, indicating trait integration under shared developmental regulation. In contrast, positive family-mean correlations in floral traits declined gradually from basal to distal flowers (nine significant correlations among floral traits in basal flowers and only three in distal flowers), showing position-specificity. Therefore, the pattern and magnitude of genetic correlations decreased with flower position. CONCLUSIONS: This finding on covariation pattern in floral reproductive structures within racemes has not been revealed before, providing insights into temporal variation and position effects in floral traits within plants and the potential advantages of modularity in hermaphrodites.

Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae).

BACKGROUND AND AIMS: Asexual organisms are more widespread in previously glaciated areas than their sexual relatives ('geographical parthenogenesis'). In plants, this pattern is probably dependent on reproductive isolation and stability of cytotypes within their respective distribution areas. Both partial apomixis and introgressive hybridization potentially destabilize the spatial separation of sexual and apomictic populations. The wide distribution of apomicts may be further enhanced by uniparental reproduction which is advantageous for colonization. These factors are studied in the alpine species Ranunculus kuepferi. METHODS: Geographical distribution, diversity and mode of reproduction of cytotypes were assessed using flow cytometry and flow cytometric seed screening on samples from 59 natural populations of Ranunculus kuepferi. Seed set of cytotypes was compared in the wild. KEY RESULTS: Diploid sexuals are confined to the south-western parts of the Alps, while tetraploid apomicts dominate in previously glaciated and in geographically isolated areas despite a significantly lower fertility. Other cytotypes (3x, 5x and 6x) occur mainly in the sympatric zone, but without establishing populations. The tetraploids are predominantly apomictic, but also show a partial apomixis via an uncoupling of apomeiosis and parthenogenesis in the seed material. Both pseudogamy and autonomous endosperm formation are observed which may enhance uniparental reproduction. CONCLUSIONS: Diploids occupy a glacial relic area and resist introgression of apomixis, probably because of a significantly higher seed set. Among the polyploids, only apomictic tetraploids form stable populations; the other cytotypes arising from partial apomixis fail to establish, probably because of minority cytotype disadvantages. Tetraploid apomicts colonize previously devastated and also distant areas via long-distance dispersal, confirming Baker's law of an advantage of uniparental reproduction. It is concluded that stability of cytotypes and of modes of reproduction are important factors for establishing a pattern of geographical parthenogenesis.

Plant chemical defence: a partner control mechanism stabilising plant--seed-eating pollinator mutualisms.

BACKGROUND: Mutualisms are inherently conflictual as one partner always benefits from reducing the costs imposed by the other. Despite the widespread recognition that mutualisms are essentially reciprocal exploitation, there are few documented examples of traits that limit the costs of mutualism. In plant/seed-eating pollinator interactions the only mechanisms reported so far are those specific to one particular system, such as the selective abortion of over-exploited fruits. RESULTS: This study shows that plant chemical defence against developing larvae constitutes another partner sanction mechanism in nursery mutualisms. It documents the chemical defence used by globeflower Trollius europaeus L. (Ranunculaceae) against the seed-eating larvae of six pollinating species of the genus Chiastocheta Pokorny (Anthomyiidae). The correlative field study carried out shows that the severity of damage caused by Chiastocheta larvae to globeflower fruits is linked to the accumulation in the carpel walls of a C-glycosyl-flavone (adonivernith), which reduces the larval seed predation ability per damaged carpel. The different Chiastocheta species do not exploit the fruit in the same way and their interaction with the plant chemical defence is variable, both in terms of induction intensity and larval sensitivity to adonivernith. CONCLUSION: Adonivernith accumulation and larval predation intensity appear to be both the reciprocal cause and effect. Adonivernith not only constitutes an effective chemical means of partner control, but may also play a key role in the sympatric diversification of the Chiastocheta genus.

Evolutionary analysis of a key floral trait in aquilegia canadensis (ranunculaceae): genetic variation in herkogamy and its effect on the mating system.

The mating system of flowering plant populations evolves through selection on genetically based phenotypic variation in floral traits. The physical separation of anthers and stigmas within flowers (herkogamy) is expected to be an important target of selection to limit self-fertilization. We investigated the pattern of phenotypic and genetic variation in herkogamy and its effect of self-fertilization in a broad sample of natural populations of Aquilegia canadensis, a species that is highly selfing despite strong inbreeding depression. Within natural populations, plants exhibit substantial phenotypic variation in herkogamy caused primarily by variation in pistil length rather than stamen length. Compared to other floral traits, herkogamy is much more variable and a greater proportion of variation is distributed among rather than within individuals. We tested for a genetic component of this marked phenotypic variation by growing naturally pollinated seed families from five populations in a common greenhouse environment. For three populations, we detected a significant variation in herkogamy among families, and a positive regression between parental herkogamy measured in the field and progeny herkogamy in the greenhouse, suggesting that there is often genetic variation in herkogamy within natural populations. We estimated levels of self-fertilization for groups of flowers that differed in herkogamy and show that, as expected, herkogamy was associated with reduced selfing in 13 of 19 populations. In six of these populations, we performed floral emasculations to show that this decrease in selfing is due to decreased autogamy (within-flower selfing), the mode of selfing that herkogamy should most directly influence. Taken together, these results suggest that increased herkogamy should be selected to reduce the production of low-quality selfed seed. The combination of high selfing and substantial genetic variation for herkogamy in A. canadensis is enigmatic, and reconciling this observation will require a more integrated analysis of how herkogamy influences not only self-fertilization, but also patterns of outcross pollen import and export.

Geographical and within-population variation in the globeflower-globeflower fly interaction: the costs and benefits of rearing pollinator's larvae.

Interspecific interactions can vary within and among populations and geographical locations, and this variation can influence the nature of the interaction (e.g. mutualistic versus antagonistic) and its evolutionary stability. Globeflowers are exclusively pollinated by flies whose larvae feed only on their seeds. Here we document geographical variability in costs and benefits in globeflowers in sustaining their pollinating flies throughout the range of this arctic-alpine European plant over several years. A total of 1,710 flower heads from 38 populations were analysed for their carpel, egg and seed contents. Individual and population analyses control for the confounding influences of variation in both: (1) population traits, such as fly density and egg distribution among flower heads; and (2) individuals traits, such as carpel and egg numbers per flower head. Despite considerable variation in ecological conditions and pollinator densities across populations, large proportions (range 33-58%) of seeds are released after predation, with a benefit-to-cost ratio of 3, indicating that the mutualism is stable over the whole globeflower geographical range. The stability of the mutualistic interaction relies on density-dependent competition among larvae co-developing in a flower head. This competition is revealed by a sharp decrease in the number of seeds eaten per larva with increasing larval number, and is intensified by non-uniform egg distribution among globeflowers within a population. Carpel number is highly variable across globeflowers (range 10-69), and flies lay more eggs in large flowers. Most plants within a population contribute to the rearing of pollinators, but some pay more than others. Large globeflowers lose more seed to pollinator larvae, but also release more seed than smaller plants. The apparent alignment of interests between fly and plant (positive relationship between numbers of seeds released and destroyed) is shown to hide a conflict of interest found when flower size is controlled for.

Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): parallels to Ranunculaceae and Poaceae.

Apomixis in Crataegus is primarily aposporous and requires pollination. The embryo sac is of the Polygonum type. A combination of meiotically unreduced embryo sacs with apparently reduced pollen would violate the usual requirement for a 2 : 1 ratio of maternal to paternal contributions to the endosperm. We therefore investigated the origin of endosperm in seeds of sexual diploids and apomictic polyploids of the sister genera Crataegus and Mespilus. Flow-cytometric DNA measurements from embryo and endosperm in mature seeds were converted to ploidy levels using leaf-tissue information. The diploids had triploid endosperm. In c. 60% of seed from polyploids, one sperm apparently contributes to the endosperm, while 25% or more may involve two sperm. Additional results suggest that trinucleate central cells also occur. Fertilization of meiotically unreduced eggs is indicated. The ratio of maternal to paternal contributions to the endosperm in these apomictic Crataegus is not constrained to 2 : 1. They thus resemble some Sorbus (Pyreae) and very distantly related Ranunculus (Ranunculaceae). It is suggested that Paspalum (Poaceae) may have similarly flexible endosperm ploidy levels.

Geographic and within-population variation in the globeflower-globeflower fly interaction: the costs and benefits of rearing pollinators' larvae.

Interspecific interactions can vary within and among populations and geographic locations, and this variation can influence the nature of the interaction (e.g. mutualistic vs. antagonistic) and its evolutionary stability. Globeflowers are exclusively pollinated by flies, whose larvae feed only on their seeds. Here we document geographic variability in costs and benefits in globeflowers in sustaining their pollinating flies throughout the range of this arctic-alpine European plant over several years. A total of 1,710 flower heads from 38 populations were analysed for their carpel, egg and seed contents. Individual and population analyses control for the confounding influences of variation in both: (1) population traits, such as fly density and egg distribution among flower heads; and (2) individuals traits, such as carpel and egg numbers per flower head. Despite considerable variation in ecological conditions and pollinator densities across populations, large proportions (range 33-58%) of seeds were released after predation, with a benefit-to-cost ratio of 3, indicating that the mutualism is stable over the whole globeflower geographical range. The stability of the mutualistic interaction relies on density-dependent competition among larvae co-developing in a flower head. This competition is revealed by a sharp decrease in the number of seeds eaten per larva with increasing larval number, and is intensified by non-uniform egg distribution among globeflowers within a population. Carpel number is highly variable across globeflowers (range 10-69), and flies lay more eggs in large flowers. Most plants within a population contribute to the rearing of pollinators, but the costs are greater for some than for others. Large globeflowers lose more seed to pollinator larvae, but also release more seed than smaller plants. The apparent alignment of interests between fly and plants (positive relationship between numbers of seed released and destroyed) is shown to hide a conflict of interest found when flower size is controlled for.

Sex and pollen: the role of males in stabilising a plant-seed eater pollinating mutualism.

Some plants are exclusively pollinated by an insect whose larvae feed on their seeds. The net outcome of a single visit for the plant depends on the number of ovules fertilised by the visitor, the number of eggs laid, and the number of seeds eaten by each larva. Unlike other known plant-seed eater pollinating mutualisms, the globeflower-globeflower fly mutualism (Trollius europaeus-Chiastocheta spp.) is unique in that not only females but also males visit flowers, and both sexes are potential pollinators. I analysed the relative efficiency of Chiastocheta males versus females in transporting pollen and fertilising globeflower ovules. I show that there is no sex-specific morphological adaptation or behaviour to enhance pollen collection and transportation in Chiastocheta flies, and that males contribute to pollination. However, because of their smaller body size, males transport significantly less pollen than females. Less seeds are produced after a visit from a male than after a visit from a female. A single female visit contributes to about 12% of total seed production, and a single male visit to only 5.4%. Females tend to spend more time inside the flower than males, and the number of ovules fertilised is significantly correlated with the time insects spent inside the closed corolla. The lower efficiency of ovule fertilisation by a male's single visit is compensated for by the higher rate of flower visitation by males: a flower receives about twice as many visits from males as from females during a time unit. The contribution of males to pollination is of major importance with respect to understanding the evolutionary stability of the globeflower-globeflower fly mutualism, as males satiate pollen requirement of flowers, masking the antagonistic effect of ovipositing females.

Allocation to floral structures in Thalictrum pubescens (Ranunculaceae), a cryptically dioecious species.

Females of Thalictrum pubescens produce stamens that contain sterile pollen, whereas males are both functionally and morphologically unisexual. This study examines the investment in stamen production by females of T. pubescens by comparing the female structures with those of their fully functional male counterparts. Stamens from females had the same biomass and contained the same amount of nitrogen and phosphorus as stamens from males. Anther size was the same in males and females, but filaments were longer in stamens from males. Females produced more pollen per anther than males, and pollen size was the same in both sexes. Within flowers, there was a positive correlation between the amount of pollen per anther and the length of anthers in males, but not in females. This would be expected if males growing in better environmental conditions or with greater vigour invested more resources in pollen production, thereby increasing fitness. Females, who receive no fitness benefits from increased pollen production, did not show this pattern. There was also evidence of a trade-off within female flowers between the number of stamens and the number of pistils. This trade-off was noted in conditions when variance among plants was reduced, namely in the field during a year when flower size was particularly small and in a previous glasshouse study. Therefore, it appears that when environmental variance is low, stamens are produced at the expense of producing more pistils, and hence seeds. In conclusion, stamen production does not appear to be inconsequential to females of Thalictrum pubescens.