Evolutionary blocks to anthocyanin accumulation and the loss of an anthocyanin carrier protein in betalain-pigmented Caryophyllales

{square} The order Caryophyllales exhibits complex pigment evolution, with mutual exclusion of anthocyanin and betalain pigments. Given recent evidence for multiple shifts to betalain pigmentation, we re-evaluated potential mechanisms underpinning the exclusion of anthocyanins from betalain-pigmented lineages. {square}We examined the evolution of the flavonoid pathway using transcriptomic and genomic datasets covering 309 species in 31 families. Orthologs and paralogs of known flavonoid synthesis genes were identified by sequence similarity, with gene duplication and gene loss inferred by phylogenetic and syntenic analysis. Relative transcript abundances were assessed to reveal broad-scale gene expression changes between betalain- and anthocyanin-pigmented lineages. {square}Most flavonoid genes are retained and transcribed in betalain-pigmented lineages, and many also show evidence of extensive gene duplication within betalain-pigmented lineages. However, expression of several flavonoid genes is reduced in betalain-pigmented lineages, especially the late-stage genes dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). Notably flavonoid 3',5'-hydroxylase (F3'5'H) homologs have been repeatedly lost in belatain-pigmented lineages, and Anthocyanin9 (AN9) homologs are undetectable in any betalain-pigmented lineages. {square}Down-regulation of ANS and DFR homolog expression (limiting synthesis) and reiterative loss of AN9 homologs (limiting transport), coincident with multiple shifts to betalain pigmentation, are likely crucial the loss of anthocyanins in betalain-pigmented Caryophyllales.