Floral KNOX expression supports parallel evolution of nectar spurs in snapdragon relatives (Antirrhineae Plantaginaceae)

Nectar spurs are tubular outgrowths of perianth tissue-a key innovation that has evolved many times independently in angiosperms. These novel floral structures are tightly linked to pollination and pollinator shifts may explain their role in increasing diversification rates. In the tribe Antirrhineae (Plantaginaceae), nectar spurs on the ventral petal have convergently evolved four times, in the Kickxia clade, Cymbalaria, Chaenorhinum, and Linaria. Here we explore the molecular mechanism underpinning this convergence, by investigating two Class I KNOX genes (HIRZ and INA) across Antirrhineae. Such KNOX genes are canonically expressed in the shoot apical meristem (SAM). HIRZ and INA were originally identified in mutants of Antirrhinum majus (snapdragons) whereby ectopic floral expression led to mutated spur-bearing petals; subsequently HIRZ/INA were shown to have floral expression in the naturally spur-bearing relative Linaria vulgaris (toadflax). Orthologues of HIRZ and INA were isolated here from Kickxia elatine, Cymbalaria muralis, and Chaenorhinum origanifolium, and confirmed through phylogenetic and gene structure analyses. Similar expression patterns were found in all three species, with moderate-high expression of both HIRZ and INA in floral buds and mature flowers, consistent high expression in apices (i.e., the SAM), and low expression in leaves. Higher levels of expression were found in floral buds compared to leaves (on average 75-86-fold higher) for INA and HIRZ, respectively. Together these data strongly suggest that the four independent evolutions of the petal spur in Antirrhineae are a result of parallel evolution, through the independent recruitment of similar underlying molecular mechanisms.