Floral spurs are key innovations associated with elaborate pollination mechanisms that have evolved independently several times across angiosperms. Spur formation can shift the floral symmetry from radial to bilateral, as it is the case in Tropaeolum, the only member of the Brassicales with floral nectar spurs. The genetic mechanisms underlying both spur and bilateral symmetry in the family have not yet been investigated. We studied flower development and morphoanatomy of Tropaeolum longifolium. We also generated a reference transcriptome and isolated all candidate genes involved in adaxial-abaxial differential growth during spur formation. Finally, we evaluated the evolution of the targeted genes across Brassicales and examined their expression in dissected floral parts. Five sepals initiate spirally, followed by five petals alternate to the sepals, five antesepalous stamens, three antepetalous stamens, and three carpels. Intercalary growth at the common base of sepals and petals forms a floral tube. The spur is an outgrowth from the adaxial region of the tube, lined up with the medial sepal. We identified Tropaeolum specific duplications in the TCP3/4L and STM gene lineages, which are critical for spur formation in other taxa. In addition, we found that TM6 (MADS-box), RL2 (RAD-like7), and KN2/6L2 and OSH6L (KNOX1 genes), have been lost in core Brassicales but retained in Tropaeolum. Three genes are pivotal during the extreme adaxial-abaxial asymmetry of the floral tube, namely, TlTCP4L2 restricted to the adaxial side where the spur is formed, and TlTCP12 and TlSTM1 to the abaxial side, lacking a spur.
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