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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 bee-pollinated 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 trade-off 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.

Phalaenopsis is an important ornamental pot plant for the global horticultural market. The inflorescences of Phalaenopsis horticultural hybrids require support from a stick during plant cultivation because of the weight of multiple large flowers. Developing a horticultural hybrid with a sufficiently lignified inflorescence stem that does not require additional support could be a way to reduce the costs of production. This study aimed to (i) determine the orientation and degree of lignification in the inflorescence stem of different species and horticultural hybrids of Phalaenopsis and investigate whether these lignification patterns follow any (ii) topological or (iii) phylogenetic pattern of interest to further explore in genetic precision breeding. Inflorescences of comparable devel- opmental stages of six species and 17 horticultural hybrids of flowering Phalaenopsis orchids were sampled. The orientation of the inflorescence varied from erect, sub-erect, arching, to pendant. The degree of lignification was measured with ImageJ using stained microscopic tissue sections and statistically analyzed. A molecular phylogeny of the species of Phalaenopsis was reconstructed based on plastid and nuclear ribosomal DNA sequences to analyze phylogenetic patterns. Our results show a significant difference in the degree of lignification between the different Phalaenopsis species and hybrids, between peduncle and rachis, and among the six different inflorescence positions analyzed. We found a positive correlation between inflorescence orientation and the proportion of lignified area per total stem area and the proportional thickness of the lignified fiber walls in the peduncle. We conclude that the degree of lignification is heritable, as we observed among our sample size a higher positive correlation between stem lignification variables among closely related taxa compared to more distantly related ones. However, a larger species sampling is needed to further validate our results.