Evolutionary relationships among plants have been inferred primarily using chloroplast data. To date, no study has comprehensively examined the plastome for gene tree conflict. Using a broad sampling of angiosperm plastomes, we characterize gene tree conflict among plastid genes at various time scales and explore correlates to conflict (e.g., evolutionary rate, gene length, molecule type). We uncover notable gene tree conflict against a backdrop of largely uninformative genes. We find alignment length and tree length are strong predictors of concordance, and that nucleotides outperform amino acids. Of the most commonly used markers, matK, greatly outperforms rbcL; however, the rarely used gene rpoC2 is the top-performing gene in every analysis. We find that rpoC2 reconstructs angiosperm phylogeny as well as the entire concatenated set of protein-coding chloroplast genes. Our results suggest that longer genes are superior for phylogeny reconstruction. The alleviation of some conflict through the use of nucleotides suggests that stochastic and systematic error is likely the root of most of the observed conflict, but further research on biological conflict within plastome is warranted given documented cases of heteroplasmic recombination. We suggest that researchers should filter genes for topological concordance when performing downstream comparative analyses on phylogenetic data, even when using chloroplast genomes.

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.

The evolution of L-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that L-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear.To address this, we functionally characterised 23 distinct DODA proteins for L-DOPA 4,5dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of L-DOPA 4,5-dioxygenase activity.We find that low L-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated L-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny.In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated L-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.

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