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.

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Herkogamy, spatial separation between stigma and anthers within a flower, is important in regulating plant‐mating systems. We studied phenotypic variation and heritability of herkogamy traits in Lysimachia arvensis (=Anagallis arvensis) that show both lateral and vertical herkogamy in the same flower, a rare strategy in flowering plants. Both lateral and vertical herkogamy showed continuous variation in 15 natural populations. Lateral herkogamy, measured as the angle between style and stamens, ranged from 5.6 to 66.5°; vertical herkogamy ranged from reverse to approach herkogamy. Herkogamy traits were constant within plants but variable among plants and populations. Flowers with marked lateral herkogamy showed mainly reverse herkogamy, whereas flowers with low lateral herkogamy showed mainly approach herkogamy. Both herkogamy traits showed a high degree of narrow sense heritability (h2 = 0.843 for lateral and h2 = 0.635 for vertical herkogamy). We discuss the possibility that variation in both herkogamy traits among populations of L. arvensis is a consequence of differential selective pressures under different pollination environments.

Background and Aims Herkogamy, or anther–stigma separation, is known to reduce self-pollen deposition, but little is known about the relative efficacy of different modes or conformations of herkogamy. We assessed the effectiveness of vertical versus lateral herkogamy in preventing or promoting self-pollen deposition in the annual herb Lysimachia arvensis, a plant with lineages that differ in flower colour, and in which flowers first display lateral and then vertical herkogamy. Because mating between the two lineages compromises fitness through the production of low-quality hybrid offspring, we tested the prediction that individuals sampled from sites occupied by both lineages should have flowers that promote autonomous self-pollen deposition and self-fertilization as a result of selection to reduce deleterious reproductive interference. Methods We characterized variation in herkogamy within and among 25 pure and mixed populations of L. arvensis in its European range and assessed the effectiveness of lateral versus vertical herkogamy in avoiding self-pollen deposition. Results Lateral herkogamy was more effective than vertical herkogamy in limiting self-pollen deposition. In the case of vertical herkogamy, only approach herkogamy was effective. Lineages showed consistent differences in herkogamy traits. In general, angles were smaller for blue than red flowers in most populations, and blue flowers showed approach herkogamy, while red flowers showed predominantly reverse herkogamy. In sympatry, the red lineage showed a reduction of both herkogamy traits while for the blue lineage only lateral herkogamy was reduced. Conclusions Our results demonstrate that pollen deposition is affected not only by the degree but also the spatial conformation of herkogamy. They also highlight reduced herkogamy as a potential mechanism for promoting reproductive assurance under pollen limitation, as well as for avoiding reproductive interference between genetically divergent lineages.

Summary Genetic divergence between species depends on reproductive isolation (RI) due to traits that reduce interspecific mating (prezygotic isolation) or are due to reduced hybrid fitness (postzygotic isolation). Previous research found that prezygotic barriers tend to be stronger than postzygotic barriers, but most studies are based on the evaluation of F1 hybrid fitness in early life cycle stages. We combined field and experimental data to determine the strength of 17 prezygotic and postzygotic reproductive barriers between two Lysimachia species that often co‐occur and share pollinators. We assessed postzygotic barriers up to F2 hybrids and backcrosses. The two species showed near complete RI due to the cumulative effect of multiple barriers, with an uneven and asymmetric contribution to isolation. In allopatry, prezygotic barriers contributed more to reduce gene flow than postzygotic barriers, but their contributions were more similar in sympatry. The strength of postzygotic RI was up to three times lower for F1 progeny than for F2 or backcrossed progenies, and RI was only complete when late F1 stages and either F2 or backcrosses were accounted for. Our results thus suggest that the relative strength of postzygotic RI may be underestimated when its effects on late stages of the life cycle are disregarded.