Background: Variation in shape and size of many floral organs is related to pollinators. Evolution of such organs is driven by duplication and modification of MADS-box and MYB transcription factors. We applied a combination of micro-morphological (SEM and micro 3D-CT scanning) and molecular techniques (transcriptome and RT-PCR analysis) to understand the evolution and development of the callus, stelidia and mentum, three highly specialized floral structures of orchids involved in pollination. Early stage and mature tissues were collected from flowers of the beepollinated Phalaenopsis equestris and Phalaenopsis pulcherrima, two species that differ in floral morphology: P. equestris has a large callus but short stelidia and no mentum, whereas P. pulcherrima has a small callus, but long stelidia and a pronounced mentum. Results: Our results show the stelidia develop from early primordial stages, whereas the callus and mentum develop later. In combination, the micro 3D-CT scan analysis and gene expression analyses show that the callus is of mixed petaloid-staminodial origin, the stelidia of staminodial origin, and the mentum of mixed sepaloid-petaloid-staminodial origin. SEP clade 1 copies are expressed in the larger callus of P. equestris, whereas AP3 clade 1 and AGL6 clade 1 copies are expressed in the pronounced mentum and long stelidia of P. pulcherrima. AP3 clade 4, PI-, AGL6 clade 2 and PCF clade 1 copies might have a balancing role in callus and gynostemium development. There appears to be a tradeoff between DIV clade 2 expression with SEP clade 1 expression in the callus, on the one hand, and with AP3 clade 1 and AGL6 clade 1 expression in the stelidia and mentum on the other. Conclusions: We detected differential growth and expression of MADS box AP3/PI-like, AGL6-like and SEP-like, and MYB DIV-like gene copies in the callus, stelidia and mentum of two species of Phalaenopsis, of which these floral structures are very differently shaped and sized. Our study provides a first glimpse of the evolutionary developmental mechanisms driving adaptation of Phalaenopsis flowers to different pollinators by providing combined micro-morphological and molecular evidence for a possible sepaloid-petaloid-staminodial origin of the orchid mentum.

Deceptive Ceropegia pitfall flowers are an outstanding example of synorganized morphological complexity. Floral organs functionally synergise to trap fly-pollinators inside the fused corolla. Successful pollination requires precise positioning of flies headfirst into cavities at the gynostegium. These cavities are formed by the corona, a specialized organ of corolline and/or staminal origin. The interplay of floral organs to achieve pollination is well studied but their evolutionary origin is still unclear. We aimed to obtain more insight in the homology of the corona and therefore investigated floral anatomy, ontogeny, vascularization, and differential MADS-box gene expression in Ceropegia sandersonii using X-ray microtomography, Light and Scanning Electronic Microscopy, and RT-PCR. During 10 defined developmental phases, the corona appears in phase 7 at the base of the stamens and was not found to be vascularized. A floral reference transcriptome was generated and 14 MADS-box gene homologs, representing all major MADS-box gene classes, were identified. B- and C-class gene expression was found in mature coronas. Our results indicate staminal origin of the corona, and we propose a first ABCDE-model for floral organ identity in Ceropegia to lay the foundation for a better understanding of the molecular background of pitfall flower evolution in Apocynaceae.

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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.