Differential expressions of anthocyanin synthesis genes underlie flower color divergence in a sympatric Rhododendron sanguineum complex

Background The Rhododendron sanguineum complex is endemic to alpine mountains of northwest Yunnan and southeast Tibet of China. Varieties in this complex exhibit distinct flower colors even at the bud stage. However, the underlying molecular regulations for the flower color variation have not been well characterized. Here, we investigated this via measuring flower reflectance profiles and comparative transcriptome analyses on three coexisting varieties of the R. sanguineum complex, with yellow flush pink, bright crimson, and deep blackish crimson flowers respectively. We compared the expression levels of differentially-expressed-genes (DEGs) of the anthocyanin / flavonoid biosynthesis pathway using RNA-seq and qRT-PCR data. We performed clustering analysis based on transcriptome-derived Single Nucleotide Polymorphisms (SNPs) data, and finally analyzed the promoter architecture of DEGs. Results Reflectance spectra of the three color morphs varied distinctively in the range between 400 and 700 nm, with distinct differences in saturation, brightness, hue, and saturation/hue ratio, an indirect measurement of anthocyanin content. We identified 15,164 orthogroups that were shared among the three varieties. The SNP clustering analysis indicated that the varieties were not monophyletic. A total of 40 paralogous genes encoding 12 enzymes contributed to the flower color polymorphism. These anthocyanin biosynthesis-related genes were associated with synthesis, modification and transportation properties (RsCHS, RsCHI, RsF3H, RsF3′H, RsFLS, RsANS, RsAT, RsOMT, RsGST), as well as genes involved in catabolism and degradation (RsBGLU, RsPER, RsCAD). Variations in sequence and cis-acting elements of these genes might correlate with the anthocyanin accumulation, thus may contribute to the divergence of flower color in the R. sanguineum complex. Conclusions Our results suggested that the varieties are very closely related and flower color variations in the R. sanguineum complex correlate tightly with the differential expression levels of genes involved in the anabolic and catabolic synthesis network of anthocyanin. Our study provides a scenario involving intricate relationships between genetic mechanisms for floral coloration accompanied by gene flow among the varieties that may represent an early case of pollinator-mediated incipient sympatric speciation.

Changes at a Critical Branchpoint in the Anthocyanin Biosynthetic Pathway Underlie the Blue to Orange Flower Color Transition in Lysimachia arvensis

Anthocyanins are the primary pigments contributing to the variety of flower colors among angiosperms and are considered essential for survival and reproduction. Anthocyanins are members of the flavonoids, a broader class of secondary metabolites, of which there are numerous structural genes and regulators thereof. In western European populations of Lysimachia arvensis, there are blue- and orange-petaled individuals. The proportion of blue-flowered plants increases with temperature and daylength yet decreases with precipitation. Here, we performed a transcriptome analysis to characterize the coding sequences of a large group of flavonoid biosynthetic genes, examine their expression and compare our results to flavonoid biochemical analysis for blue and orange petals. Among a set of 140 structural and regulatory genes broadly representing the flavonoid biosynthetic pathway, we found 39 genes with significant differential expression including some that have previously been reported to be involved in similar flower color transitions. In particular, F3′5′H and DFR, two genes at a critical branchpoint in the ABP for determining flower color, showed differential expression. The expression results were complemented by careful examination of the SNPs that differentiate the two color types for these two critical genes. The decreased expression of F3′5′H in orange petals and differential expression of two distinct copies of DFR, which also exhibit amino acid changes in the color-determining substrate specificity region, strongly correlate with the blue to orange transition. Our biochemical analysis was consistent with the transcriptome data indicating that the shift from blue to orange petals is caused by a change from primarily malvidin to largely pelargonidin forms of anthocyanins. Overall, we have identified several flavonoid biosynthetic pathway loci likely involved in the shift in flower color in L. arvensis and even more loci that may represent the complex network of genetic and physiological consequences of this flower color polymorphism.

Why Variation in Flower Color May Help Reproductive Success in the Endangered Australian Orchid Caladenia fulva

Caladenia fulva G.W. Carr (Tawny Spider-orchid) is a terrestrial Australian endangered orchid confined to contiguous reserves in open woodland in Victoria, Australia. Natural recruitment is poor and no confirmed pollinator has been observed in the last 30 years. Polymorphic variation in flower color complicates plans for artificial pollination, seed collection and ex situ propagation for augmentation or re-introduction. DNA sequencing showed that there was no distinction among color variants in the nuclear ribosomal internal transcribed spacer (ITS) region and the chloroplast trnT-trnF and matK regions. Also, authentic specimens of both C. fulva and Caladenia reticulata from the reserves clustered along with these variants, suggesting free interbreeding. Artificial cross-pollination in situ and assessment of seed viability further suggested that no fertility barriers existed among color variants. Natural fruit set was 15% of the population and was proportional to numbers of the different flower colors but varied with orchid patch within the population. Color modeling on spectral data suggested that a hymenopteran pollinator could discriminate visually among color variants. The similarity in fruiting success, however, suggests that flower color polymorphism may avoid pollinator habituation to specific non-rewarding flower colors. The retention of large brightly colored flowers suggests that C. fulva has maintained attractiveness to foraging insects rather than evolving to match a scarce unreliable hymenopteran sexual pollinator. These results suggest that C. fulva should be recognized as encompassing plants with these multiple flower colors, and artificial pollination should use all variants to conserve the biodiversity of the extant population.

Reproductive Assurance Maintains Red-Flowered Plants of Lysimachia arvensis in Mediterranean Populations Despite Inbreeding Depression

Flower color polymorphism, an infrequent but phylogenetically widespread condition in plants, is captivating because it can only be maintained under a few selective regimes but also because it can drive intra-morph assortative mating and promote speciation. Lysimachia arvensis is a polymorphic species with red or blue flowered morphs. In polymorphic populations, which are mostly Mediterranean, pollinators prefer blue-flowered plants to the red ones, and abiotic factors also favors blue-flowered plants. We hypothesize that the red morph is maintained in Mediterranean areas due to its selfing capacity. We assessed inbreeding depression in both color morphs in two Mediterranean populations and genetic diversity was studied via SSR microsatellites in 20 natural populations. Results showed that only 44–47% of selfed progeny of the red plants reached reproduction while about 72–91% of blue morph progeny did it. Between-morph genetic differentiation was high and the red morph had a lower genetic diversity and a higher inbreeding coefficient, mainly in the Mediterranean. Results suggest that selfing maintaining the red morph in Mediterranean areas despite its inbreeding depression. In addition, genetic differentiation between morphs suggests a low gene flow between them, suggesting reproductive isolation.