Developmental analysis of Ophrys sphegodes labellum helps to unravel the secrets of sexual mimicry

The orchids of the genus Ophrys represent an excellent system to study pollinator-driven speciation. These orchids have a particular pollination strategy that involves the attraction of the males of one specific species of insect, mimicking the pheromone and the appearance of the conspecific females. After landing on the flower, the deceived male starts to perform a “pseudocopulation”, which leads to removal of pollinia. Different species of Ophrys typically attract different species of insects due to the perfection of the decoy. Thus, if two orchids vary enough in their floral traits to attract different species, the genetic flow between them is interrupted, promoting plant differentiation and speciation. However, until now we lacked an understanding of the mechanisms behind the development of the floral traits employed in the mimicry. In this study, we investigate the Ophrys sphegodes labellum, the highly specialized petal responsible for pollinator attraction, in order to uncover the genetic basis of these floral traits. We used a combination of microscopy, micro-computed tomography, and photography to describe the morphology of the labellum during the development. We delineated, for the irst time, seven distinct developmental stages, which will be pivotal for subsequent studies. We also found three main cell types that are localized in three different areas of the labellum. With this knowledge in hand, we created the first expression atlas of labellum stages and tissues by RNA-Seq, so as to be able to uncover the genes involved in pollinator-relevant traits of the labellum. These results will facilitate future detailed evo-devo studies between O. sphegodes and other Ophrys species with different pollinators.