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.

Different genetic patterns have been demonstrated for narrowly distributed taxa, many of them linking rarity to evolutionary history. Quite a few species in young genera are endemics and have several populations that present low variability, sometimes attributed to geographical isolation or dispersion processes. Assessing the genetic diversity and structure of such species may be important for protecting them and understanding their diversification history. In this study, we used microsatellite markers and plastid sequences to characterize the levels of genetic variation and population structure of two endemic and restricted species that grow in isolated areas on the margin of the distribution of their respective genera. Plastid and nuclear diversities were very low and weakly structured in their populations. Evolutionary scenarios for both species are compatible with open-field expansions during the Pleistocene interglacial periods and genetic variability supports founder effects to explain diversification. At present, both species are suffering from habitat loss and changes in the environment can lead these species towards extinction.

Evolutionary transitions in flower color often trace back to changes in the flavonoid biosynthetic pathway and its regulators. In angiosperms, this pathway produces a range of red, purple, and blue anthocyanin pigments. Transcription factor (TF) complexes involving members of the MYB, bHLH, and WD40 protein families control the expression of pathway enzymes. Here, we investigate flavonoid pathway evolution in the Petunieae clade of the tomato family (Solanaceae). Using transcriptomic data from 69 species of Petunieae, we estimated a new phylogeny for the clade. For the 65 species with floral transcriptomes, we retrieved transcripts encoding homologs of 18 enzymes and transcription factors to investigate patterns of evolution across genes and lineages. We found that TFs exhibit faster rates of molecular evolution 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. However, expression levels inversely correlated with molecular evolutionary rates, while shifts in floral pigmentation were weakly related to changes affecting coding regions. Nevertheless, shifts in floral pigmentation and presence/absence patterns of MYB transcripts are strongly correlated. Intensely pigmented and patterned species express homologs of all three main MYB anthocyanin activators in petals, while pale or white species express few or none. Our findings reinforce the notion that regulators of the flavonoid pathway 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: Microsatellite markers were developed for Verbenoxylum reitzii (Verbenaceae), a tree endemic to the Brazilian Atlantic Forest, to investigate their usefulness in population genetic studies. The loci were tested for cross-amplification in the related genera Recordia and Duranta.• Methods and Results: Eleven polymorphic microsatellite markers were isolated from an enriched library of V. reitzii and characterized. The primers were tested on 60 individuals from three populations of this species. The number of alleles per locus ranged from two to 11, and the observed and expected heterozygosities varied from 0.0 to 1.0 and from 0.088 to 0.758, respectively. Ten loci successfully amplified in R. boliviana and all failed in D. vestita.• Conclusions: Our results suggest the usefulness of the microsatellite loci developed here to access genetic variability for phylogeographic and population genetic studies in V. reitzii, which are important for the conservation of this rare species.

Calibrachoa pygmaea is a unique species of Calibrachoa, especially concerning its flower morphology and the environment where it occurs. The species is self-incompatible and is narrowly distributed in wet and flooded fields of the Pampas region. We characterize the genetic diversity of the species based on traditional plastid markers and newly developed nuclear microsatellites to identify drivers that guide its evolution. Our results identified markers that are informative and useful for studying the population structure of C. pygmaea, as well as that of other species of Calibrachoa. Both marker sets were congruent in developing conclusions regarding the evolutionary scenario of C. pygmaea, and revealed that the genetic variability and population structure of the species could be explained by common allele fixation or shared ancestral polymorphism, while its diversification can be attributed mainly to the species' dispersal ability and certain ecological features.