Diverse natural products are synthesized in plants by specialized metabolic enzymes, which are often lineage-specific and derived from gene duplication followed by functional divergence. However, little is known about the contribution of primary metabolism to the evolution of specialized metabolic pathways. Betalain pigments, uniquely found in the plant order Caryophyllales, are synthesized from the aromatic amino acid l-tyrosine (Tyr) and replaced the otherwise ubiquitous phenylalanine-derived anthocyanins. This study combined biochemical, molecular and phylogenetic analyses, and uncovered coordinated evolution of Tyr and betalain biosynthetic pathways in Caryophyllales. We found that Beta vulgaris, which produces high concentrations of betalains, synthesizes Tyr via plastidic arogenate dehydrogenases (TyrA /ADH) encoded by two ADH genes (BvADHα and BvADHβ). Unlike BvADHβ and other plant ADHs that are strongly inhibited by Tyr, BvADHα exhibited relaxed sensitivity to Tyr. Also, Tyr-insensitive BvADHα orthologs arose during the evolution of betalain pigmentation in the core Caryophyllales and later experienced relaxed selection and gene loss in lineages that reverted from betalain to anthocyanin pigmentation, such as Caryophyllaceae. These results suggest that relaxation of Tyr pathway regulation increased Tyr production and contributed to the evolution of betalain pigmentation, highlighting the significance of upstream primary metabolic regulation for the diversification of specialized plant metabolism.

Gene tree discordance in large genomic data sets can be caused by evolutionary processes such as incomplete lineage sorting and hybridization, as well as model violation, and errors in data processing, orthology inference, and gene tree estimation. Species tree methods that identify and accommodate all sources of conflict are not available, but a combination of multiple approaches can help tease apart alternative sources of conflict. Here, using a phylotranscriptomic analysis in combination with reference genomes, we test a hypothesis of ancient hybridization events within the plant family Amaranthaceae s.l. that was previously supported by morphological, ecological, and Sanger-based molecular data. The data set included seven genomes and 88 transcriptomes, 17 generated for this study. We examined gene-tree discordance using coalescent-based species trees and network inference, gene tree discordance analyses, site pattern tests of introgression, topology tests, synteny analyses, and simulations. We found that a combination of processes might have generated the high levels of gene tree discordance in the backbone of Amaranthaceae s.l. Furthermore, we found evidence that three consecutive short internal branches produce anomalous trees contributing to the discordance. Overall, our results suggest that Amaranthaceae s.l. might be a product of an ancient and rapid lineage diversification, and remains, and probably will remain, unresolved. This work highlights the potential problems of identifiability associated with the sources of gene tree discordance including, in particular, phylogenetic network methods. Our results also demonstrate the importance of thoroughly testing for multiple sources of conflict in phylogenomic analyses, especially in the context of ancient, rapid radiations. We provide several recommendations for exploring conflicting signals in such situations. [Amaranthaceae; gene tree discordance; hybridization; incomplete lineage sorting; phylogenomics; species network; species tree; transcriptomics.]

Summary Within the angiosperm order Caryophyllales, an unusual class of pigments known as betalains can replace the otherwise ubiquitous anthocyanins. In contrast to the phenylalanine‐derived anthocyanins, betalains are tyrosine‐derived pigments which contain the chromophore betalamic acid. The origin of betalain pigments within Caryophyllales and their mutual exclusion with anthocyanin pigments have been the subject of considerable research. In recent years, numerous discoveries, accelerated by ‐omic scale data, phylogenetics and synthetic biology, have shed light on the evolution of the betalain biosynthetic pathway in Caryophyllales. These advances include the elucidation of the biosynthetic steps in the betalain pathway, identification of transcriptional regulators of betalain synthesis, resolution of the phylogenetic history of key genes, and insight into a role for modulation of primary metabolism in betalain synthesis. Here we review how molecular genetics have advanced our understanding of the betalain biosynthetic pathway, and discuss the impact of phylogenetics in revealing its evolutionary history. In light of these insights, we explore our new understanding of the origin of betalains, the mutual exclusion of betalains and anthocyanins, and the homoplastic distribution of betalain pigmentation within Caryophyllales. We conclude with a speculative conceptual model for the stepwise emergence of betalain pigmentation.

Transcriptomes are useful both for species-tree inference and for uncovering evolutionary complexity within lineages. Through analyses of gene-tree conflict and multiple methods of species-tree inference, we demonstrate that phylogenomic data can provide unparalleled insight into the evolutionary history of Caryophyllales. We also discuss a method for overcoming computational challenges associated with homolog clustering in large data sets.

Our data demonstrate the importance of carefully considering gene tree conflict and taxon sampling in phylogenomic analyses. Moreover, they provide a remarkable example of the propensity for paleopolyploidy in angiosperms, with at least seven such events in a clade of less than 2500 species.