Sub- and neo-functionalization of APETALA3 paralogs have contributed to the evolution of novel floral organ identity in Aquilegia (columbine, Ranunculaceae).

Previous studies of the lower eudicot model Aquilegia have revealed differential expression patterns of two APETALA3 (AP3) paralogs that appear to coincide with the development of a distinct fifth floral organ type, the staminodium. The AqAP3-1 locus quickly becomes limited to the staminodia while AqAP3-2 becomes stamen-specific. We used transient RNAi-based methods to silence each of these loci individually and in combination, followed by detailed studies of the resultant morphologies and the effects on gene expression patterns. Silencing of AqAP3-1 had a strong effect on the staminodia, causing transformation into carpeloid organs, while silencing of AqAP3-2 only affected the stamens, resulting in sterility, stunting or weak transformation towards carpel identity. Much more dramatic phenotypes were obtained in the doubly silenced flowers, where all stamens and staminodia were transformed into carpels. Quantitative reverse-transcription polymerase chain reaction analyses of B gene homolog expression in these flowers are consistent with complex patterns of regulatory feedback among the loci. These findings suggest that the presence of ancient AP3 paralogs in the Ranunculaceae has facilitated the recent evolution of a novel organ identity program in Aquilegia. Specifically, it appears that downregulation of AqAP3-2 in the innermost whorl of stamens was a critical step in the evolution of elaborated sterile organs in this position.