How different is similar – exploring development of Ceropegia sandersonii pitfall flowers to elucidate convergent evolution of floral synorganization

Abstract Background: Lantern plants from the genus Ceropegia (Apocynaceae-Asclepiadoideae) have radially symmetric pitfall flowers that are an outstanding example of functional floral complexity with high synorganization of specialized organs. The evolutionary origin and development of these complex flowers is unclear, and the genetic background of floral organ formation is unknown. Flowers with similar deceptive pollination strategies and floral traits convergently evolved in non-related plant lineages. The partially bilaterally flattened trap flowers of pipevines are functionally similar to Ceropegia pitfall flowers; many orchid taxa evolved complex fully bilaterally flattened flowers with specialized organs to trap pollinators. This study is the first to investigate the genetic background of pitfall flower development in Ceropegia, and to explore (i) convergent evolution of extremely synorganized and complex flowers as well as (ii) the homology of a highly specialized floral organ, the gynostegial corona. Methods: We obtained transcriptomes from C. sandersonii early floral buds and mature sepals, petals, and gynostegia, and analyzed differential expression of selected MADS-box genes in buds and mature floral organs using RT-PCR. In addition, we studied floral ontogeny and vascularization using SEM and 3D X-ray micro-CT scanning. Results: We identified ten phases of floral development from primordia to mature flowers, and for the first time visualized the vascular system of mature Ceropegia pitfall flowers in a 3D-model. We identified 14 MADS-box gene homologs, representing all major MADS-box gene classes, in the floral transcriptomes of Ceropegia. Vascular bundle patterns, as revealed by 3D X-ray micro-CT scanning, in combination with high expression of GLOBOSA and AGAMOUS indicate a staminoid origin of this highly specialized floral organ which starts developing from stage seven onwards. Interestingly, AGAMOUS-LIKE6 was neither expressed in early floral buds nor in any mature floral organ, in line with the radial symmetry of all Ceropegia floral organs. Conclusion: We detected differential expression of MADS-box genes involved in Ceropegia floral organ identity and propose a new ABCDE-model for parachute flowers. We compare this with current models of unrelated plants with similar floral traits but (partially) bilaterally flattened flowers, i.e. Aristolochia fimbriata and Erycina pusilla. With this comparative approach we lay the foundation for understanding the genetic mechanisms driving convergent evolution of highly specialized deceptive trap flowers.