Flower color variation among plant populations might reflect adaptation to local conditions such as the interacting animal community. In the northwest Iberian Peninsula, flower color of Gentiana lutea varies longitudinally among populations, ranging from orange to yellow. We explored whether flower color is locally adapted and the role of pollinators and seed predators as agents of selection by analyzing the influence of flower color on (i) pollinator visitation rate and (ii) escape from seed predation and (iii) by testing whether differences in pollinator communities correlate with flower color variation across populations. Finally, (iv) we investigated whether variation in selective pressures explains flower color variation among 12 G. lutea populations. Flower color influenced pollinator visits and differences in flower color among populations were related to variation in pollinator communities. Selective pressures on flower color vary among populations and explain part of flower color differences among populations of G. lutea. We conclude that flower color in G. lutea is locally adapted and that pollinators play a role in this adaptation.

Animals which interact with plants often cause selective pressures on plant traits. Flower color variation within a species might be shaped by the action of animals feeding on the plant species. Pollinators might exert natural selection on color if flower color is related to their foraging efficiency. For example, some pollinator species might require more time to detect particular colors. If that is the case, flower color might have evolved as a pollination exploitation barrier-ensuring that flowers are more visited by the most efficient pollinators. In addition, non-pollinator agents such as predispersal seed predators may select on flower color, if color indicates food resources (seeds) or if color is related to deterrent compounds. We address selection on flower color in a population of Gentiana lutea where color varies among individuals from yellow to orange. We hypothesize that opposed selection from mutualists (pollinators) and antagonists (predispersal seed predators) maintains flower color variation in this population. By means of path analysis we addressed the role of both interactors in flower color selection. We found that selection acts on flower color, mediated by both pollinators and seed predators. Both agents favored yellow-flowered individuals, thus selection by pollinators and seed predators does not maintain flower color variation in this population.

Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.

Background: Flower colour variation among populations may result from the spatial variation of selective agents. The structure of phenotypic variation informs on the ecological processes related to this variation. Variation in floral traits is mainly attributed to variation in the pollinator fauna, while variation in vegetative traits is usually linked to abiotic factors or herbivores. Aims: We investigated the geographical variation of flower colour (and correlated traits) in Gentiana lutea and the relationship with the variation in abiotic factors. Methods: Phenotypic variation (flower colour, petal length, petal width, stalk length, leaf length, flower number, petal number and number of basal leaves) was assessed in 429 plants of 12 populations located at north-west Iberia. Additionally, we obtained data on the geographical coordinates, elevation, temperature, rainfall and radiation for each population. Results: Populations mostly differed in flower colour, from orange to yellow from west to the east. Abiotic factors were unrelated to variation in either floral or vegetative traits. Conclusions: Phenotypic variation among G. lutea populations does not result from adaptation to environmental factors. Other factors, such as historical events or selective pressure exerted by biotic interactions, might explain the flower colour variation in G. lutea along the Cantabrian Range.

In Gentiana lutea two varieties are described: G. lutea var. aurantiaca with orange corolla colors and G. lutea var. lutea with yellow corolla colors. Both color varieties co-occur in NW Spain, and pollinators select flower color in this species. It is not known whether a hybridization barrier exists between these G. lutea color varieties. We aim to test the compatibility between flower color varieties in G. lutea and its dependence on pollen vectors. Within a sympatric population containing both flower color morphs, we analyzed differences in reproductive success (number, weight, viability and germinability of seeds) depending on fertilization treatments (autogamy and xenogamy within variety and among varieties). We found a 93% reduction in number of seeds and a 37% reduction in seed weight respectively of autogamy treatments compared to xenogamy crossings. Additionally, reproductive success is higher within color varieties than among varieties, due to a 45% seed viability reduction on hybrids from different varieties. Our results show that G. lutea reproductive success is strongly dependent on pollinators and that a partial hybridization barrier exists between G. lutea varieties.