Dissecting the relationship between gene function and substitution rates is key to understanding genome-wide patterns of molecular evolution. Biochemical pathways provide powerful systems for investigating this relationship because the functional role of each gene is often well characterized. Here, we investigate the evolution of the flavonoid pigment pathway in the colorful Petunieae clade of the tomato family (Solanaceae). This pathway is broadly conserved in plants, both in terms of its structural elements and its MYB, bHLH and WD40 transcriptional regulators, and its function has been extensively studied, particularly in model species of petunia. We built a phylotranscriptomic dataset for 69 species of Petunieae to infer patterns of molecular evolution across pathway genes and across lineages. We found that transcription factors exhibit faster rates of molecular evolution (dN/dS) than their targets, with the highly specialized MYB genes evolving fastest. Using the largest comparative dataset to date, we recovered little support for the hypothesis that upstream enzymes evolve slower than those occupying more downstream positions, although expression levels do predict molecular evolutionary rates. While shifts in floral pigmentation were only weakly related to changes affecting coding regions, we found a strong relationship with the presence/absence patterns of MYB transcripts. Intensely pigmented species express all three main MYB anthocyanin activators in petals, while pale or white species express few or none. Our findings reinforce the notion that pathway regulators have a dynamic history, involving higher rates of molecular evolution than structural components, along with frequent changes in expression during color transitions.

PREMISE OF THE STUDY:The evolution of novel fruit morphologies has been integral to the success of angiosperms. The inflated fruiting calyx, in which the balloon-like calyx swells to completely surround the fruit, has evolved repeatedly across angiosperms and is postulated to aid in protection and dispersal. We investigated the evolution of this trait in the tomatillos and their allies (Physalideae, Solanaceae). METHODS:The Physalideae phylogeny was estimated using four regions (ITS, LEAFY, trnL-F, waxy) with maximum likelihood (ML) and Bayesian inference. Under the best-fitting ML model of trait evolution, we estimated ancestral states along with the numbers of gains and losses of fruiting calyx accrescence and inflation with Bayesian stochastic mapping. Also, phylogenetic signal in calyx morphology was examined with two metrics (parsimony score and Fritz and Purvis's D).KEY RESULTS: Based on our well-resolved and densely sampled phylogeny, we infer that calyx evolution has proceeded in a stepwise and directional fashion, from non-accrescent to accrescent to inflated. In total, we inferred 24 gains of accrescence, 24 subsequent transitions to a fully inflated calyx, and only two reversals. Despite this lability, fruiting calyx accrescence and inflation showed strong phylogenetic signal. CONCLUSIONS:Our phylogeny greatly improves the resolution of Physalideae and highlights the need for taxonomic work. The comparative analyses reveal that the inflated fruiting calyx has evolved many times and that the trajectory toward this phenotype is generally stepwise and irreversible. These results provide a strong foundation for studying the genetic and developmental mechanisms responsible for the repeated origins of this charismatic fruit trait.