Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers.

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Elevational divergence in pigmentation plasticity is associated with selection and pigment biochemistry.

Phenotypic plasticity is predicted to evolve in environmentally variable habitats, or those experiencing a high frequency of strong selection. The evolution of plasticity may however be constrained by costs or physiological limitations. In flowers, UV-absorbing pigmentation ameliorates UV damage to pollen, and is linked with elevated UV exposure. Whether plasticity contributes to this pattern remains unclear. Petals of Argentina anserina have larger UV-absorbing petal areas at high elevations where they experience higher and more variable UV exposure than low elevations. We measured UV-induced pigmentation plasticity in high- and low-elevation populations (hereafter, "high," "low"), and selection on pigmentation via male fitness. We dissected UV pigment biochemistry using metabolomics to explore biochemical mechanisms underlying plasticity. High displayed positive UV-induced pigmentation plasticity but low lacked plasticity. Selection favored elevated pigmentation under UV in high, supporting adaptive plasticity. In high, UV absorption was conferred by flavonoids produced in one flavonoid pathway branch. However, in low, UV absorption was associated with many compounds spanning multiple branches. Elevated plasticity was thus associated with reduced pigment diversity. These results are consistent with adaptive floral pigmentation plasticity in more extreme and variable environments. We discuss how biochemical underpinnings of pigmentation may permit or constrain the evolution of pigmentation plasticity.

Molecular Mechanism Involved in Carotenoid Metabolism in Post-Smolt Atlantic Salmon: Astaxanthin Metabolism During Flesh Pigmentation and Its Antioxidant Properties.

A better understanding of carotenoid dynamics (transport, absorption, metabolism, and deposition) is essential to develop a better strategy to improve astaxanthin (Ax) retention in muscle of Atlantic salmon. To achieve that, a comparison of post-smolt salmon with (+ Ax) or without (- Ax) dietary Ax supplementation was established based on a transcriptomic approach targeting pyloric, hepatic, and muscular tissues. Results in post-smolts showed that the pyloric caeca transcriptome is more sensitive to dietary Ax supplementation compared to the other tissues. Key genes sensitive to Ax supplementation could be identified, such as cd36 in pylorus, agr2 in liver, or fbp1 in muscle. The most modulated genes in pylorus were related to absorption but also metabolism of Ax. Additionally, genes linked to upstream regulation of the ferroptosis pathway were significantly modulated in liver, evoking the involvement of Ax as an antioxidant in this process. Finally, the muscle seemed to be less impacted by dietary Ax supplementation, except for genes related to actin remodelling and glucose homeostasis. In conclusion, the transcriptome data generated from this study showed that Ax dynamics in Atlantic salmon is characterized by a high metabolism during absorption at pyloric caeca level. In liver, a link with a potential of ferroptosis process appears likely via cellular lipid peroxidation. Our data provide insights into a better understanding of molecular mechanisms involved in dietary Ax supplementation, as well as its beneficial effects in preventing oxidative stress and related inflammation in muscle.

Ecological effects on female bill colour explain plastic sexual dichromatism in a mutually-ornamented bird.

Sex differences in ornamentation are common and, in species with conventional sex roles, are generally thought of as stable, due to stronger sexual selection on males. Yet, especially in gregarious species, ornaments can also have non-sexual social functions, raising the possibility that observed sex differences in ornamentation are plastic. For example, females may invest in costly ornamentation more plastically, to protect body and reproductive ability in more adverse ecological conditions. We tested this hypothesis with experimental work on the mutually-ornamented common waxbill (Estrilda astrild), supplementing their diets either with pigmentary (lutein, a carotenoid) or non-pigmentary (vitamin E) antioxidants, or alleviating winter cold temperature. We found that both lutein and vitamin E supplementation increased red bill colour saturation in females, reaching the same mean saturation as males, which supports the hypothesis that female bill colour is more sensitive to environmental or physiological conditions. The effect of vitamin E, a non-pigment antioxidant, suggests that carotenoids were released from their antioxidant functions. Alleviating winter cold did not increase bill colour saturation in either sex, but increased the stability of female bill colour over time, suggesting that female investment in bill colour is sensitive to cold-mediated stress. Together, results show that waxbill bill sexual dichromatism is not stable. Instead, sexual dichromatism can be modulated, and even disappear completely, due to ecology-mediated plastic adjustments in female bill colour.

Astaxanthin Z-isomer-rich diets enhance egg yolk pigmentation in laying hens compared to that in all-E-isomer-rich diets.

The effects of feeding diets containing astaxanthin with different Z-isomer ratios to laying hens on egg qualities, such as astaxanthin concentration in egg yolk and yolk color, were investigated. As the astaxanthin source, a natural microorganism Paracoccus carotinifaciens was used. Astaxanthin with different Z-isomer ratios was prepared by thermal treatment with different conditions and then added to the basal diet at a final astaxanthin concentration of 8 mg/kg. We found that, as the Z-isomer ratios of astaxanthin in the diet increased, the astaxanthin concentration in egg yolk and the yolk color fan score also increased significantly. Importantly, feeding a 50.6% Z-isomer ratio diet increased astaxanthin concentration in egg yolk by approximately fivefold and the color fan score by approximately 2 compared to that in hens fed an all-E-isomer-rich diet. Moreover, we showed that feeding Z-isomer-rich astaxanthin to laying hens increased plasma astaxanthin concentration by more than five times in comparison to that in hens fed an all-E-isomer-rich diet. These results indicate that Z-isomers of astaxanthin have higher bioavailability than that of the all-E-isomer and thus they exhibit greater egg yolk-accumulation efficiency.

Commercial Harvesting Has Driven the Evolution of Camouflage in an Alpine Plant.

Color in nature mediates numerous among and within species interactions,1 and anthropogenic impacts have long had major influences on the color evolution of wild animals.2 An under-explored area is commercial harvesting, which in animals can exert a strong selection pressure on various traits, sometimes greater even than natural selection or other human activities.3,4 Natural populations of plants that are used by humans have likely also suffered strong pressure from harvesting, yet the potential for evolutionary change induced by humans has received surprisingly little attention.5 Here, we show that the leaf coloration of a herb used in traditional Chinese medicine (Fritillaria delavayi) varies among populations, with leaves matching their local backgrounds most closely. The degree of background matching correlates with estimates of harvest pressure, with plants being more cryptic in heavily collected populations. In a human search experiment, the time it took participants to find plants was greatly influenced by target concealment. These results point to humans as driving the evolution of camouflage in populations of this species through commercial harvesting, changing the phenotype of wild plants in an unexpected and dramatic way.

Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in Salvia miltiorrhiza Bge. flowers.

BACKGROUND: The objectives of this study were to reveal the anthocyanin biosynthesis metabolic pathway in white and purple flowers of Salvia miltiorrhiza using metabolomics and transcriptomics, to identify different anthocyanin metabolites, and to analyze the differentially expressed genes involved in anthocyanin biosynthesis. RESULTS: We analyzed the metabolomics and transcriptomics data of S. miltiorrhiza flowers. A total of 1994 differentially expressed genes and 84 flavonoid metabolites were identified between the white and purple flowers of S. miltiorrhiza. Integrated analysis of transcriptomics and metabolomics showed that cyanidin 3,5-O-diglucoside, malvidin 3,5-diglucoside, and cyanidin 3-O-galactoside were mainly responsible for the purple flower color of S. miltiorrhiza. A total of 100 unigenes encoding 10 enzymes were identified as candidate genes involved in anthocyanin biosynthesis in S. miltiorrhiza flowers. Low expression of the ANS gene decreased the anthocyanin content but enhanced the accumulation of flavonoids in S. miltiorrhiza flowers. CONCLUSIONS: Our results provide valuable information on the anthocyanin metabolites and the candidate genes involved in the anthocyanin biosynthesis pathways in S. miltiorrhiza.

Comparison of Metabolome and Transcriptome of Flavonoid Biosynthesis Pathway in a Purple-Leaf Tea Germplasm Jinmingzao and a Green-Leaf Tea Germplasm Huangdan reveals Their Relationship with Genetic Mechanisms of Color Formation.

Purple-leaf tea is a phenotype with unique color because of its high anthocyanin content. The special flavor of purple-leaf tea is highly different from that of green-leaf tea, and its main ingredient is also of economic value. To probe the genetic mechanism of the phenotypic characteristics of tea leaf color, we conducted widely targeted metabolic and transcriptomic profiling. The metabolites in the flavonoid biosynthetic pathway of purple- and green-leaf tea were compared, and results showed that phenolic compounds, including phenolic acids, flavonoids, and tannins, accumulated in purple-leaf tea. The high expression of genes related to flavonoid biosynthesis (e.g., PAL and LAR) exhibits the specific expression of biosynthesis and the accumulation of these metabolites. Our result also shows that two CsUFGTs were positively related to the accumulation of anthocyanin. Moreover, genes encoding transcription factors that regulate flavonoids were identified by coexpression analysis. These results may help to identify the metabolic factors that influence leaf color differentiation and provide reference for future research on leaf color biology and the genetic improvement of tea.

Reduction of spermatophore melanization in Litopenaeus vannamei shrimp fed Ulva clathrata during a commercial hatchery production.

Male broodstock (Litopenaeus vannamei, 36 ± 7 g, n = 600) reproductive performance, spermatophores and reproductive tract melanization, prostaglandin concentrations and biochemical composition were evaluated after including 3.8% Ulva clathrata meal in the diet (dry base) of a commercial hatchery during 45 days. Males fed Ulva had less melanized spermatophores (120 compared with 233, P < 0.01), less bacteria in the ductus deferens (P < 0.01), more sperm cells in testicles (P < 0.05), and increased courtship activity (839 compared with 689, P < 0.01), with no effect on mortality. Ulva-fed males had more arachidonic acid (20:4n-6) in the spermatophores (P < 0.05) but this did not affect the prostaglandin concentrations in response to diet. Males fed Ulva had more carotenoids in the hepatopancreas (0.08 ± 0.02 compared with 0.01 ± 0.01 mg/g, P < 0.05), and phenolic compounds in hepatopancreas (6.1 ± 0.7 compared with 1.8 ± 0.7 mg eq. phloroglucinol/g, P < 0.05) and muscle (0.4 ± 0.3 compared with 0.2 ± 0.1 mg eq. phloroglucinol/g, P < 0.05). Males fed the Ulva also had a lesser carbohydrate content in the hepatopancreas (P < 0.01) and muscle (P < 0.01). In conclusion, supplementing fresh maturation diets with a small dose of dried Ulva allowed for improvement of reproductive performance and to decrease melanization in spermatophores and the male reproductive tract.

Carotenoids as natural functional pigments.

Carotenoids are tetraterpene pigments that are distributed in photosynthetic bacteria, some species of archaea and fungi, algae, plants, and animals. About 850 naturally occurring carotenoids had been reported up until 2018. Photosynthetic bacteria, fungi, algae, and plants can synthesize carotenoids de novo. Carotenoids are essential pigments in photosynthetic organs along with chlorophylls. Carotenoids also act as photo-protectors, antioxidants, color attractants, and precursors of plant hormones in non-photosynthetic organs of plants. Animals cannot synthesize carotenoids de novo, and so those found in animals are either directly accumulated from food or partly modified through metabolic reactions. So, animal carotenoids show structural diversity. Carotenoids in animals play important roles such precursors of vitamin A, photo-protectors, antioxidants, enhancers of immunity, and contributors to reproduction. In the present review, I describe the structural diversity, function, biosyntheses, and metabolism of natural carotenoids.

Individual and interactive role of Trichoderma viride and arbuscular mycorrhizal fungi on growth and pigment content of onion plants.

The study of interactions between beneficial micro-organisms associated with plant roots is important, because such interactions might either enhance or inhibit the beneficial effects of individual species. The effect of the combined inoculation of arbuscular mycorrhizal (AM) fungi and a biocontrol fungus (Trichoderma viride) on different growth parameters and chlorophyll a,b, carotenoids, total chlorophyll and total pigments of onion (Allium cepa) plants was studied under glasshouse conditions. The results proved that AM fungi and T. viride are compatible with each other and their combined use was effective not only in improving onion growth parameters such as fresh and dry weights, root and shoot lengths and leaf area but also increasing total chlorophyll, carotenoids and total pigments content in onion leaves. Where, inoculation of onion plants by AM fungi and T. viride alone or in combination significantly increased bulb diameters of onion plants 20, 12·5 and 17·5% increase; respectively, when compared with control ones. Also percentage of AM fungal colonization increased greatly with T. viride inoculation. Therefore, AM fungi and T. viride could be a good alternate of chemical fertilizer for improving the growth of onion. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: The need for increasing agricultural productivity and quality has led to an excessive use of chemical fertilizers; creating serious threats to human health and the environment. The use of biofertilizers is an alternative for sustaining high production with low ecological impact. Thus the goal of this study was to propose a biological technique using arbuscular mycorrhizal symbiosis and Trichoderma viride (plant-microbe interaction) for increasing plant growth that represent a great opportunities for recent agricultural practices. This may be an indication displaying the AM and Trichoderma significance for plant progress and growth.

Do glucocorticoids or carotenoids mediate plumage coloration in parrots? An experiment in Platycercus elegans.

Conspicuous coloration can indicate phenotypic quality, and may reflect exposure or vulnerability to stress, or access to essential nutrients such as pigments. Although the production of pigmented colours is well understood, much less is known about how structural colours are affected by physiological state. In this study, we tested whether glucocorticoids (corticosterone) predicted expression of plumage coloration in an Australian parrot, the crimson rosella (Platycercus elegans). Parrots provide an interesting and unique test, as they possess conspicuous coloration produced by distinctive pigments known as psittacofulvins, in addition to structural coloration. We have previously documented that coloration in P. elegans is condition-dependent and responds to dietary manipulation. Here, n = 21 P. elegans underwent a dietary manipulation (including food restriction or carotenoid supplementation) during which they moulted, and the change in reflectance was measured for three structural and three pigmentary plumage patches. Stress-induced corticosterone (10 min after handling) measured at the start of the experiment predicted change in coloration in two pigmentary patches (crown and front). We also found that change in stress-induced corticosterone during the experiment was associated with the change in coloration of the crown and two structural patches (cheek and epaulette). Baseline corticosterone (<3 min after handling) was not associated with any measure of coloration. We found no effects of dietary manipulation on baseline or stress-induced corticosterone, but carotenoid supplementation was associated with an increase in a measure of chronic stress (heterophil/lymphocyte ratio), and the corticosterone response to handling decreased over the course of the study. Our results suggest that corticosterone may be linked to colour expression more broadly than previously recognised, including psittacofulvin and structural coloration in parrots, and they confirm the independence of plumage pigmentation in parrots from carotenoid accumulation. Moreover, our study provides new insight into the stress responses of Psittaciformes, one of the most highly threatened avian orders.

Reproductive trade-offs maintain bract color polymorphism in Scarlet Indian paintbrush (Castilleja coccinea).

Populations of scarlet Indian paintbrush (Castilleja coccinea) in the Midwestern United States exhibit a bract color polymorphism, with each population having predominantly yellow or scarlet bracts. We investigated a possible mechanism for this maintenance of bract color polymorphism in C. coccinea by conducting hand-pollination experiments in two nearby populations, one predominantly yellow and one predominantly scarlet. The hand-pollination treatments were either self-pollination or cross pollination using pollen from within and between populations. Both color morphs were used as pollen donors for the within and between crosses. We found that both color morphs of C. coccinea were self-compatible. When the scarlet morph was the maternal plant it had higher seed set. When pollinators were excluded, the yellow morph outperformed the scarlet morph in fruit set and seed set. The apparent trade-offs between a higher reproductive output in the scarlet morph and a reproductive assurance advantage in the yellow morph may explain the maintenance of the polymorphism in C. coccinea. While many previous studies have provided evidence for pollinator preference playing a role in floral color polymorphism, the results of the current study indicate that reproductive assurance, which would be important for fluctuations in pollinator abundance or colonizing new areas, may act as a selective agent to maintain such polymorphisms.

One for all and all for one: retention of colour-unchanged old flowers increases pollinator attraction in a hermaphroditic plant.

Long-lived flowers increase pollen transfer rates, but these entail high water and carbon maintenance costs. The retention of pollinated and reward-free old flowers enhances pollinator visitation to young receptive flowers by increasing floral display size. This mechanism is associated with acropetal inflorescences or changes in flower colour and openness, but the retention of unchanging solitary flowers remains overlooked. We examined pollination-dependent variation in floral longevity and determined stigmatic receptivity, pollen viability and pollen removal rates among flower ages in Kielmeyera regalis, a Neotropical savanna shrub. We also evaluated the effects of floral display size on pollinator visitation rates. Lastly, we determined whether old flowers are unvisited and exclusively increase pollinator attraction to young flowers through flower removal experiments. Regardless of pollination treatment, flowers lasted fully open with no detectable physical changes for 3 days. Over time, stigmas remained receptive but >95% of pollen was removed. Pollinator visitation significantly increased with floral display size and intermediate percentages (15-30%) of newly opened flowers. Accordingly, the retention of reward-free and unvisited old flowers increased young flower-pollinator interaction. Our results reveal the importance of a prolonged floral longevity in increasing pollinator attraction toward newly opened receptive flowers without changes in flower colour and form. We conclude that the retention of pollinated, reward-free and unvisited colour-unchanged old flowers in K. regalis is a strategy that counteracts the water use costs associated with the maintenance of large flowers with increased mate opportunities in a pollen-limited scenario.

Plumage coloration and social context influence male investment in song.

Animals use multiple signals to attract mates, including elaborate song, brightly coloured ornaments and physical displays. Female birds often prefer both elaborate male song and intense carotenoid-based plumage coloration. This could lead less visually ornamented males to increase song production to maximize their attractiveness to females. We tested this possibility in the highly social and non-territorial house finch (Haemorhous mexicanus), in which females discriminate among males based on both song and on the intensity of red carotenoid-based plumage coloration. We manipulated male plumage coloration through carotenoid supplementation during moult, so that males were either red or yellow. Males were then housed under three social environments: (i) all red birds, (ii) all yellow birds or (iii) a mixture of red/yellow birds. We recorded song after presentation of a female. Red males produced more song than yellow males. But when yellow males were housed with red conspecifics, they produced more song relative to yellow males housed with equally unattractive yellow males. This study provides novel evidence that a male's plumage coloration and the plumage colour of his social competitors influence investment in song.

Molecular adaptations to phosphorus deprivation and comparison with nitrogen deprivation responses in the diatom Phaeodactylum tricornutum.

Phosphorus, an essential element for all living organisms, is a limiting nutrient in many regions of the ocean due to its fast recycling. Changes in phosphate (Pi) availability in aquatic systems affect diatom growth and productivity. We investigated the early adaptive mechanisms in the marine diatom Phaeodactylum tricornutum to P deprivation using a combination of transcriptomics, metabolomics, physiological and biochemical experiments. Our analysis revealed strong induction of gene expression for proteins involved in phosphate acquisition and scavenging, and down-regulation of processes such as photosynthesis, nitrogen assimilation and nucleic acid and ribosome biosynthesis. P deprivation resulted in alterations of carbon allocation through the induction of the pentose phosphate pathway and cytosolic gluconeogenesis, along with repression of the Calvin cycle. Reorganization of cellular lipids was indicated by coordinated induced expression of phospholipases, sulfolipid biosynthesis enzymes and a putative betaine lipid biosynthesis enzyme. A comparative analysis of nitrogen- and phosphorus-deprived P. tricornutum revealed both common and distinct regulation patterns in response to phosphate and nitrate stress. Regulation of central carbon metabolism and amino acid metabolism was similar, whereas unique responses were found in nitrogen assimilation and phosphorus scavenging in nitrogen-deprived and phosphorus-deprived cells, respectively.

Geographic variation in pollen color is associated with temperature stress.

The evolution of flower color, especially petal pigmentation, has received substantial attention. Less understood is the evolutionary ecology of pollen pigmentation, though it varies among and within species and its biochemical properties affect pollen viability. We characterize the distribution of pollen color across 24 populations of the North American herb Campanula americana, and assess the degree to which this variation is genetically based. We identify abiotic factors that covary with pollen color and test whether germination of light and dark pollen is differentially affected by variable temperature and UV. Pollen color varies from white to deep purple in C. americana and is genetically determined. There was a longitudinal cline whereby pollen was darkest in western populations. Accounting for latitudinal variation, western populations experience elevated temperature and UV irradiance. Germination of light-colored pollen was reduced by 60% under high temperature, but dark pollen was unaffected. Exposure to UV reduced germination of light and dark pollen similarly. The cline in pollen color across the range may reflect adaptation to heat stress. This study supports thermal tolerance as a novel function of pollen pigmentation and contributes to growing evidence that abiotic factors can drive floral diversity.

Application of Paracoccus marcusii as a potential feed additive for laying hens.

Carotenoids have been used for many years as an added pigment to enhance egg yolk color. One such carotenoid, astaxanthin, has a strong antioxidant activity and is produced by several microorganisms, including the bacterium Paracoccus marcusii, which has shown promise to be used as a feed additive. Therefore, this study investigated the use of P. marcusii as a possible source of pigmentation in layer hen feed to enhance egg yolk color. Paracoccus marcusii was fed to hens in a sucrose solution (10% m/v). The hens were fed daily and all eggs were collected for analysis. Dilutions of egg contents were plated onto selective media to detect the presence of known food pathogens (E. coli, Listeria, and Salmonella). In the feeding trial, there was no negative effect on hen body weight, egg production, or overall egg quality. There was a significant increase (P < 0.05) in yolk color as well as an increase in whole egg and yolk weight. There were also no known food pathogens detected in any of the egg samples. This study has shown promising results in using this bacterium as an effective feed additive for laying hens.

Sex Difference in Condition Dependence of Carotenoid Gapes in the Eurasian Roller (Coracias garrulus).

In altricial birds, sex differentiation can start early in the ontogeny in the form of color, physiology, and/or growth and may potentially result in sex-specific condition dependence of traits mediating parent-offspring communication. Carotenoids have long been hypothesized to modulate the expression of gape coloration, but their sex-specific role enforcing honesty of gape coloration remains poorly studied. In a within-nest design, we provided carotenoid supplementation to nestlings of the Eurasian roller (Coracias garrulus) and measured the response in circulating carotenoids, coloration of the gape, cutaneous immune responsiveness to phytohemagglutinin, and growth while accounting for the sex of nestlings. Male nestlings supplemented with carotenoids displayed enhanced pigmentation of their gapes and grew faster than control nestlings, but there was no within-individual correlation between gape color and growth in either carotenoid-supplemented or control males. Female nestlings, however, diverted most supplemented carotenoids into growing fast at the expense of reducing their level of circulating carotenoids and displaying less-pigmented gapes. Nestling cutaneous immune response was not affected by carotenoid supplementation in either sex. Our results provide only weak support for the hypothesis that carotenoids enforce the honesty of gape color signals in nestling rollers and demonstrate sex specificity in how nestlings divert a surplus of carotenoids into different physiological functions.

Are pollinators the agents of selection for the extreme large size and dark color in Oncocyclus irises?

Pollinator-mediated selection is a major evolutionary driver of floral traits; yet, such selection has rarely been tested for floral extreme traits. The Oncocyclus irises have exceptionally large, dark-colored flowers, associated with night-sheltering pollination and heat reward by the dark flowers. We quantified phenotypic selection on stem length, floral size and color in two species of iris (Iris atropurpurea and I. haynei), using an experimental approach. We estimated selection gradients for both flowers open to natural pollination and for flowers receiving supplementary hand pollination, assuming that open-pollinated flowers are affected by all factors that could influence fitness, whereas supplementary pollination removes the possible influence of pollinators. We found evidence for pollinator-mediated selection to increase floral size and stem length in I. atropurpurea, but floral color in this species was not under pollinator-mediated selection. In I. haynei, no pollinator-mediated selection on any of the traits was detected. We conclude that the extreme floral size of I. atropurpurea has probably evolved as a result of pollinator behavior. Lack of such evidence for I. haynei and for the dark floral color in both species suggests that other non-pollinator agents are selecting for these prominent traits, or that phenotypic color variation in these irises is neutral.