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PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. https://hdl.handle.net/2066/224834 Please be advised that this information was generated on 2021-10-06 and may be subject to change. Pramanik et al. EvoDevo (2020) 11:16 https://doi.org/10.1186/s13227-020-00160-z EvoDevo RESEARCH Open Access Evolution and development of three highly specialized foral structures of bee-pollinated Phalaenopsis species Dewi Pramanik1,2,3 , Nemi Dorst4, Niels Meesters5, Marlies Spaans4, Erik Smets1,2,6 , Monique Welten1 and Barbara Gravendeel1,2,7* Abstract Background: Variation in shape and size of many foral organs is related to pollinators. Evolution of such organs is driven by duplication and modifcation 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 foral structures of orchids involved in pollination. Early stage and mature tissues were collected from fowers of the bee- pollinated Phalaenopsis equestris and Phalaenopsis pulcherrima, two species that difer in foral 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-stamino- dial 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- of 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 diferential 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 foral structures are very diferently shaped and sized. Our study provides a frst glimpse of the evolutionary developmental mechanisms driving adaptation of Phalaenopsis fowers to diferent pollinators by providing combined micro-mor- phological and molecular evidence for a possible sepaloid–petaloid–staminodial origin of the orchid mentum. Keywords: Callus, Mentum, Stelidia, RT-PCR, Transcriptomics Background One of the key innovations in the evolution of fowering plants is the transfer of pollen by pollinators. Tis adap- *Correspondence: [email protected] tation enabled plants to reproduce much more efciently 1 Naturalis Biodiversity Center, Endless Forms Group, Darwinweg 2, 2333 and played a major role in the diversifcation of the fow- CR Leiden, The Netherlands ering plants [1]. In more basal fowering plant lineages, Full list of author information is available at the end of the article such as Magnoliales, pollinators carry pollen all over © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pramanik et al. EvoDevo (2020) 11:16 Page 2 of 20 their body, resulting in a low pollination success. In more foral organs and structures in diferent foral whorls play derived plant lineages such as orchids, very precise place- an important role in this process. ment of pollen on very specifc body parts of pollinators Orchid fowers consist of fve whorls. Te two outer evolved, that ensured reproductive isolation and species whorls contain three sepals and three petals, of which diversifcation. the median petal is transformed into a labellum. Tere Apart from very precise pollen placement in orchids, are two staminal whorls. Te outer whorl consists of two deceptive pollination also evolved, in which pollinators staminodes and one functional stamen. Te inner whorl are cheated when they visit fowers that ofer no reward. is assumed to contain three staminodes but this is a long Deceptive pollination promotes cross-fertilization by debated issue. In the center of the fower there is a ffth reducing the visitation period of pollinators and discour- whorl that contains a gynoecium that is formed by three aging returns to a single fower or inforescence, in this fused carpels and at least one stamen. Te median petal way preventing self-pollination. Seven mechanisms of of orchid fowers, the labellum, is often enlarged and deceptive pollination of orchids have been described so ornamented with a wart-like structure, the callus. Dur- far: generalized food deception, Batesian foral mimicry, ing landing on the orchid fower, the callus on the label- brood-site imitation, shelter imitation, pseudo-antago- lum provides a holdfast for the insect to grasp with its nism, rendezvous attraction and sexual deception [2]. Te front legs [21] (Fig. 1a). Te reproductive organs (style, generalized food deception syndrome [3] is characterized stigma, stamen) have become fused and incorporated by a model mimicking general foral signals of reward- in a gynostemium [22, 23]. To prevent the insect from ing plants such as a similar inforescence shape, foral walking of the labellum, wing-shaped structures on color, scent, nectar guides, spurs and pollen-like papillae both sides of the gynostemium, so-called stelidia [24], [4, 5]. Tese signals attract recently emerged, immigrant are vital as they keep the insect trapped and also posi- or exploratory pollinators [2]. Te Batesian mimicry tion the head or other body parts of the animal correctly syndrome copies one particular rewarding model plant in front of the reproductive organs (Fig. 1b). Lastly, the species growing nearby [6–8]. In the case of brood-site so-called mentum, an outgrowth formed by the bases imitation, orchids deceive insects by mimicking oviposi- of the gynostemium and labellum and the lateral sides tion substrates such as rotting fruit, dung, or fungi [9]. of the lateral sepals [25] can act as a hinge, slamming In the case of shelter imitation, fowers are shaped in the an insect that walks over the labellum against the upper form of a tube [10] to provide shelter or a warm up place part of the gynostemium, where the anther and stigma for insects in need of thermoregulation [11, 12]. Te are positioned, to ensure that removal and/or deposi- pseudo-antagonism syndrome relates to foraging behav- tion of the pollinia takes place at a very precise position ior of kleptoparasitic pollinators by employing small fo- on the pollinator and orchid fower (Fig. 1c). Te size and ral hairs easily vibrating in a small breeze that are thought shape of these three highly specialized foral structures to mimic insect prey captured in spider webs [13, 14]. Te became adapted to the bodies of specifc pollinators dur- frst step in evolution towards pseudocopulation might ing orchid evolution. Tis resulted in diversifcation of be related with the rendezvous syndrome [15]. In the lat- the shape and size of these structures. Such a diversifca- ter syndrome, fowers emit signals that attract pollina- tion can for example be found in diferent species of the tors of both sexes such as aggregation pheromones [16]. orchid genus Phalaenopsis. Examples of a large callus can In the frst syndrome, fowers mimic the shape and scent be found in Phalaenopsis amabilis (Fig. 2a), P. celebensis of female insects; this elicits male insect sexual behavior (Fig. 2c), and P. equestris (Fig. 2d), pronounced stelidia with the mimicking fower [17–19]. From the seven strat- are present in P. bellina (Fig. 2b), P. celebensis (Fig. 2c) egies described above, the most common one in orchids and P. pulcherrima (Fig. 2f), whereas a large mentum is is general food deception. Around one-third of all pol- present in P. pulcherrima (Figs. 1d and 2f). lination cases in orchids are categorized as general food deceptive [20]. Deceptive fowers look, feel and smell like Models of foral organ development in orchids fowers of nearby rewarding plants and only after visiting Multiple molecular studies show that the foral develop- a cheating fower several times, inexperienced pollina- ment of orchids involves interaction of diferent MADS tors learn to distinguish a cheater from the model. Tis box and other genes that duplicated early during orchid is sufcient to transfer pollen from one orchid to another evolution [26–31].