Prezygotic barriers play a major role in the evolution of reproductive isolation, which is a prerequisite for speciation. However, despite considerable progress in identifying genes and mutations responsible for postzygotic isolation, little is known about the genetic and molecular basis underlying prezygotic barriers. The bumblebee-pollinated Mimulus lewisii and the hummingbirdpollinated M. cardinalis represent a classic example of pollinator-mediated prezygotic isolation between two sister species in sympatry. Flower color differences resulting from both carotenoid and anthocyanin pigments contribute to pollinator discrimination between the two species in nature. Through fine-scale genetic mapping, site-directed mutagenesis, and transgenic experiments, we demonstrate that a single-repeat R3 MYB repressor, ROSE INTENSITY1 (ROI1), is the causal gene underlying a major quantitative trait locus (QTL) with the largest effect on anthocyanin concentration and that cis-regulatory change rather than coding DNA mutations cause the allelic difference between M. lewisii and M. cardinalis. Together with the genomic resources and stable transgenic tools developed here, these results suggest that Mimulus is an excellent platform for studying the genetics of pollinator-mediated reproductive isolation and the molecular basis of morphological evolution at the most fundamental level-gene by gene, mutation by mutation. The pollinator-mediated prezygotic isolation between two sister species of monkeyflowers, Mimulus lewisii and M. cardinalis, represents a classic example for studying reproductive isolation and speciation in a natural ecological setting (Hiesey et al. 1971;Bradshaw et al. 1995;Schemske and Bradshaw 1999;Bradshaw and Schemske 2003;Ramsey et al. 2003). The bumblebee-pollinated M. lewisii has pale pink flowers with broad, flat petals and contrasting yellow nectar guides ( Figure 1A). The hummingbird-pollinated M. cardinalis has red flowers with reflexed petals that form a tubular corolla ( Figure 1B). Although the two species are interfertile with hand pollination, they rarely hybridize in wild sympatric populations due to pollinator preference (Hiesey et al. 1971;Schemske and Bradshaw 1999;Bradshaw and Schemske 2003;Ramsey et al. 2003).The pale pink color of M. lewisii flowers results from a low concentration of anthocyanins and the absence of carotenoids (except in the nectar guides) ( Figure 1A). The red color of M. cardinalis flowers is produced by a combination of high concentrations of both pigments. Both anthocyanin and carotenoid concentrations contribute to pollinator discrimination between

Summary A molecular description of the control of floral pigmentation in a multi-species group displaying various flower color patterns is of great interest for understanding the molecular bases of phenotypic diversification and pollinator-mediated speciation. Through transcriptome profiling, mutant analyses, and transgenic experiments, we aim to establish a ‘baseline’ floral anthocyanin regulation model in Mimulus lewisii and to examine the different ways of tinkering with this model in generating the diversity of floral anthocyanin patterns in other Mimulus species. We find one WD40 and one bHLH gene controlling anthocyanin pigmentation in the entire corolla of M. lewisii, and two R2R3-MYB genes, PELAN and NEGAN, controlling anthocyanin production in the petal lobe and nectar guide, respectively. The autoregulation of NEGAN might be a critical property to generate anthocyanin spots. Independent losses of PELAN expression (via different mechanisms) explain two natural yellow-flowered populations of M. cardinalis (typically red-flowered). The NEGAN ortholog is the only anthocyanin-activating MYB expressed in the M. guttatus flowers. The mutant lines and transgenic tools available for M. lewisii will enable gene-by-gene replacement experiments to dissect the genetic and developmental bases of more complex floral color patterns, and to test hypotheses on phenotypic evolution in general.

SummaryCarotenoids are yellow, orange, and red pigments that contribute to the beautiful colors and nutritive value of many flowers and fruits. The structural genes in the highly conserved carotenoid biosynthetic pathway have been well characterized in multiple plant systems, but little is known about the transcription factors that control the expression of these structural genes.By analyzing a chemically induced mutant of Mimulus lewisii through bulk segregant analysis and transgenic experiments, we have identified an R2R3-MYB, Reduced Carotenoid Pigmentation 1 (RCP1), as the first transcription factor that positively regulates carotenoid biosynthesis during flower development.Loss-of-function mutations in RCP1 lead to down-regulation of all carotenoid biosynthetic genes and reduced carotenoid content in M. lewisii flowers, a phenotype recapitulated by RNA interference in the wild-type background. Overexpression of this gene in the rcp1 mutant background restores carotenoid production and, unexpectedly, results in simultaneous decrease of anthocyanin production in some transgenic lines by down-regulating the expression of an activator of anthocyanin biosynthesis.Identification of transcriptional regulators of carotenoid biosynthesis provides the 'toolbox' genes for understanding the molecular basis of flower color diversification in nature and for potential enhancement of carotenoid production in crop plants via genetic engineering.

Flower color patterns have long served as a model for developmental genetics because pigment phenotypes are visually striking, yet generally not required for plant viability, facilitating the genetic analysis of color and pattern mutants. The evolution of novel flower colors and patterns has played a key role in the adaptive radiation of flowering plants via their specialized interactions with different pollinator guilds (e.g., bees, butterflies, birds), motivating the search for allelic differences affecting flower color pattern in closely related plant species with different pollinators. We have identified LIGHT AREAS1 (LAR1), encoding an R2R3-MYB transcription factor, as the causal gene underlying the spatial pattern variation of floral anthocyanin pigmentation between two sister species of monkeyflower: the bumblebee-pollinated Mimulus lewisii and the hummingbirdpollinated Mimulus cardinalis. We demonstrated that LAR1 positively regulates FLAVONOL SYNTHASE (FLS), essentially eliminating anthocyanin biosynthesis in the white region (i.e., light areas) around the corolla throat of M. lewisii flowers by diverting dihydroflavonol into flavonol biosynthesis from the anthocyanin pigment pathway. FLS is preferentially expressed in the light areas of the M. lewisii flower, thus prepatterning the corolla. LAR1 expression in M. cardinalis flowers is much lower than in M. lewisii, explaining the unpatterned phenotype and recessive inheritance of the M. cardinalis allele. Furthermore, our gene-expression analysis and genetic mapping results suggest that cis-regulatory change at the LAR1 gene played a critical role in the evolution of different pigmentation patterns between the two species.flower color pattern | Mimulus | anthocyanins | flavonols | R2R3-MYB M any flowers display interesting color patterns (e.g., spots, stripes, picotees, bull's-eyes) that are precisely programmed during development. Numerous studies have shown that these color patterns are critically important for plant-pollinator interactions (1-8). Among the most captivating examples are deceptive orchids that display floral pigment patterns remarkably similar to female bees or wasps to lure male counterparts for pseudocopulation, thereby achieving pollination (9-11). Despite the obvious aesthetic and ecological significance of these flower color patterns, the molecular mechanisms of pigment pattern formation is not well understood, nor is the genetic basis underlying pattern variation between related species in nature.From a genetic and developmental viewpoint, the most extensively studied flower color pattern is venation. Studies in snapdragon (Antirrhinum majus) and petunia (Petunia hybrida) have revealed a conserved mechanism for the formation of vein-associated anthocyanin pigmentation pattern in petal epidermis. Pigments are only produced in the overlapping expression domains of the R2R3-MYB and bHLH coregulators of anthocyanin biosynthetic genes; the bHLH expression is confined to the petal epidermis and the R2R3-MYB expression is specific to ce...