The road less taken: Dihydroflavonol 4-reductase inactivation and delphinidin anthocyanin loss underpins a natural intraspecific flower colour variation.

Visual cues are of critical importance for the attraction of animal pollinators, however, little is known about the molecular mechanisms underpinning intraspecific floral colour variation. Here, we combined comparative spectral analysis, targeted metabolite profiling, multi-tissue transcriptomics, differential gene expression, sequence analysis and functional analysis to investigate a bee-pollinated orchid species, Glossodia major with common purple- and infrequent white-flowered morphs. We found uncommon and previously unreported delphinidin-based anthocyanins responsible for the conspicuous and pollinator-perceivable colour of the purple morph and three genetic changes underpinning the loss of colour in the white morph - (1) a loss-of-function (LOF; frameshift) mutation affecting dihydroflavonol 4-reductase (DFR1) coding sequence due to a unique 4-bp insertion, (2) specific downregulation of functional DFR1 expression and (3) the unexpected discovery of chimeric Gypsy transposable element (TE)-gene (DFR) transcripts with potential consequences to the genomic stability and post-transcriptional or epigenetic regulation of DFR. This is one of few known cases where regulatory changes and LOF mutation in an anthocyanin structural gene, rather than transcription factors, are important. Furthermore, if TEs prove to be a frequent source of mutation, the interplay between environmental stress-induced TE evolution and pollinator-mediated selection for adaptive colour variation may be an overlooked mechanism maintaining floral colour polymorphism in nature.