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Stereomicrographs of fresh flowers (A-H) and cleared flowers stained with safranin (I-K). A-E, Ophrys bombyliflora. A, Longitudinally bisected flower, showing central labellum lobe ending with an apical appendix. B, Enlarged lateral view of the appendix. C, Bottom view of the flower, showing the distal end of central and lateral labellum lobes; the apical appendix and the convex glabrous margins are well seen. D, E, Details of appendix, before (D) and after (E) immersion in neutral red; the outline of the appendix cavity stains red. F-H, Ophrys tenthredinifera, after immersion in neutral red. F, Abaxial surface of labellum, showing the red-stained band near the margin. G, H, Close-up of red-stained appendix in front view (G) and in lateral view (H). I, J, Labellum vasculature of O. tenthredinifera. I, Abaxial surface, showing central vein branched toward labellum margin. J, Venation of appendix. K, Vein-branched appendix tip of O. bombyliflora. In B and D, an arrow indicates the appendix tip with tuft of trichomes, and an asterisk indicates the appendix concavity. ad p adaxial surface of labellum; ap p appendix; cl p central labellum lobe; gm p glabrous margin; gy p gynostemium; ll p lateral labellum lobe; pr p protuberance; sc p stigmatic cavity. Scale bars p 3 mm (A, C, I), 0.5 mm (B), 1 mm (D, E, G, H, J, K), or 6 mm (F).
Source publication
Premise of research. The insect-like flowers of the Ophrys orchids are adapted to sexually deceptive
pollination through pseudocopulation, providing chemical, visual, and tactile stimuli for male insects. Although
the chemical composition of the odor bouquet of several species has long been identified, the precise site of
fragrance production in th...
Contexts in source publication
Context 1
... electron microscope observations revealed a great diversity of epidermal cell types on the adaxial surface of the labellum in both species. Conversely, the abaxial surface is entirely characterized by typical smooth pavement cells, i.e., elongated tabular or lenticular epidermal cells ( fig. A2F, avail- able in the online edition of the International Journal of Plant Sciences), which in O. tenthredinifera become larger and dome shaped toward the margin, especially in the apical region of ...
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... arrangement of the cuticular striations on their cell walls (figs. 4C, 4H, A2C), which varies gradually toward the central area of the speculum, where short, narrow trichomes occur ( fig. 4I). In some flowers of O. tenthredinifera, the glabrous lateral areas of the speculum were found to be larger and composed of small papillae and flat cells (fig. ...
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... part presents elon- gated, flat, polygonal cells with smooth or finely ridged cuti- cles, similar to the pavement cells of the abaxial surface ( fig. A1C). Contorted trichomes with parallel striations lengthwise and a reticulate cuticular pattern on their bases ( fig. 4G) di- verge at the apex of the conical bulge, where they become shorter ( fig. A2A, ...
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... reflexed apical appendix in O. tenthredinifera ( fig. 4J). By contrast, O. bombyliflora presents a shallow longitudinal central groove in the velutinous apical indumentum, and trichomes are found obliquely oriented toward that groove ( fig. 3G). Both species possess a hirsute indumentum of long, contorted, unicellular trichomes near the margins ( fig. A2I). While in O. bombyli- flora these trichomes border only the apical appendix ( fig. 3J), in O. tenthredinifera they form a complete submarginal band throughout the labellum, surrounding the narrow to moderate glabrous margin consisting of dome-shaped papillae with a smooth surface ( fig. 4K). Conversely, the glabrous margin of the ...
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... d (A. Francisco, personal observation). Diluted neutral red intensely stained specific areas of freshly opened flowers but failed to stain late flower buds just before anthesis. The apical appendix and the entire glabrous margins of freshly opened flowers of O. tenthredinifera stained deeply red after less than 3 h of immersion in neutral red ( fig. 2F-2H). Moreover, in most flow- ers, a narrow red-stained band 3-4 mm in width was seen on the abaxial surface of the apical region of the labellum and was especially intense in the first hours of anthesis ( fig. 2F). Although with weaker intensity, neutral red also stained the appendix in freshly opened flowers of O. bombyliflora ( fig. 2D, ...
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... glabrous margins of freshly opened flowers of O. tenthredinifera stained deeply red after less than 3 h of immersion in neutral red ( fig. 2F-2H). Moreover, in most flow- ers, a narrow red-stained band 3-4 mm in width was seen on the abaxial surface of the apical region of the labellum and was especially intense in the first hours of anthesis ( fig. 2F). Although with weaker intensity, neutral red also stained the appendix in freshly opened flowers of O. bombyliflora ( fig. 2D, ...
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... red ( fig. 2F-2H). Moreover, in most flow- ers, a narrow red-stained band 3-4 mm in width was seen on the abaxial surface of the apical region of the labellum and was especially intense in the first hours of anthesis ( fig. 2F). Although with weaker intensity, neutral red also stained the appendix in freshly opened flowers of O. bombyliflora ( fig. 2D, ...
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... red: the apical appendix in O. bombyliflora and O. tenthredinifera and the glabrous margins of the apical labellum in the latter species. Further- more, cleared flowers of these two species revealed that the labellum is supplied by a central vein composed of several vascular bundles ending at the appendix tip, where they branch intensely ( fig. 2I-2K). The central vein is also divided into several secondary veins supplying the margins of the labellum, where further branching occurs, which is especially clear in O. tenthredinifera ( fig. ...
Context 9
... revealed that the labellum is supplied by a central vein composed of several vascular bundles ending at the appendix tip, where they branch intensely ( fig. 2I-2K). The central vein is also divided into several secondary veins supplying the margins of the labellum, where further branching occurs, which is especially clear in O. tenthredinifera ( fig. ...
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Citations
... Some suggest that in this species, the labellum only displays a poorly defined, glabrous basal field due to its flat epidermal cells [64,67]. Yet, these cells closely resemble those typically found not in the labellum but in the contiguous floor of the stigmatic cavity of other Ophrys species [52,59], casting doubt on their homology. Accurately interpreting the floral structure of O. speculum requires a detailed micromorphological comparison with other Ophrys species to establish homology between floral parts. ...
... Building on our previous studies of the labellum micromorphology and anatomy within the clade composed of the groups of O. bombyliflora, O. tenthredinifera, O. speculum, and section Pseudophrys [52,54], the present study focuses on the two subspecies of O. speculum that occur in the western part of its geographic range: O. speculum Link subsp. lusitanica O.Danesch & E.Danesch and O. speculum Link subsp. ...
... The controversy regarding the presence of a basal field in O. speculum arises from varying interpretations of the flower's structure by different researchers [52,59,64,67]. This issue can only be resolved through a detailed micromorphological comparison of the labellum and stigmatic cavity between O. speculum and other Ophrys species, aiming to establish homology between the floral areas. ...
Pollination by sexual deception specifically attracts male insects, through the floral scent and particular morphological features of the flower that serve as visual and tactile stimuli. The unique bond between the Ophrys speculum orchid and the male Dasyscolia ciliata wasp primarily stems from a few distinctive semiochemicals that mimic the female wasp’s sex pheromone, although the floral scent comprises a variety of compounds. An osmophore producing highly volatile compounds has been documented in four close relatives of O. speculum and is now being also investigated in this species. Given the existing debates regarding the structure of the labellum and stigmatic cavity in O. speculum, this study details their micromorphology. Additionally, comparisons of O. speculum flowers and female D. ciliata wasps under stereomicroscopy and scanning electron microscopy are conducted to seek new evidence of visual and tactile mimicry. The findings confirm that (i) an osmophore is present at the apical margin of the labellum in O. speculum flowers; (ii) the labellum features a distinct basal field homologous to those found in other Ophrys species; and (iii) the basal labellum region closely mimics the female wasp’s thorax and wings. The implications of these novel floral features are discussed within an evolutionary context.
... Setting aside very few exceptions, including the comparative SEM images of gynostemia illustrated here (Figure 4), detailed morphological studies of Ophrys have focused almost exclusively on describing the labellum. This emphasis is understandable, given its often rugged three-dimensional topology [22,87]; the complexity of its surface textures and markings [97]; and its equally complex internal anatomy [12][13][14]98]. Even genome size differs among cells within different regions of the labellum, presumably dictating contrasting levels of glandular activity [14]. ...
Simple Summary: Our frequently deployed approach to optimally circumscribing species requires large-scale field sampling within and between populations for large numbers of morphometric characters, followed by multivariate ordinations to objectively seek discontinuities (or, failing that, zones of limited overlap) among sets of populations considered to represent species. Corresponding boundaries are sought in DNA-based outputs, either phylogenies or preferably ordinations based on population genetic data. Herein, we analyse within a molecular phylogenetic framework detailed morphometric data for the charismatic bee orchids (Ophrys), seeking a 'mesospecies' species concept that might provide a compromise between the nine 'macrospecies' recognised primarily through DNA barcoding and the several hundred 'microspecies' recognised primarily through perceived pollinator specificity. Our analyses failed to find robust groupings that could be regarded as credible mesospecies, instead implying that each macrospecies constitutes a morphological continuum. This problematic result encouraged us to reappraise both our morphometric approach and the relative merits of the contrasting macrospecies and microspecies concepts, and to reiterate the key role played by genetics in species circumscription. Abstract: Despite (or perhaps because of) intensive multidisciplinary research, opinions on the optimal number of species recognised within the Eurasian orchid genus Ophrys range from nine to at least 400. The lower figure of nine macrospecies is based primarily on seeking small but reliable discontinuities in DNA 'barcode' regions, an approach subsequently reinforced and finessed via high-throughput sequencing studies. The upper figure of ca. 400 microspecies reflects the morphological authoritarianism of traditional taxonomy combined with belief in extreme pollinator specificity caused by reliance on pollination through pseudo-copulation, enacted by bees and wasps. Groupings of microspecies that are less inclusive than macrospecies are termed mesospecies. Herein, we present multivariate morphometric analyses based on 51 characters scored for 457 individual plants that together span the full morphological and molecular diversity within the genus Ophrys, encompassing 113 named microspecies that collectively represent all 29 mesospecies and all nine macrospecies. We critique our preferred morphometric approach of accumulating heterogeneous data and analysing them primarily using principal coordinates, noting that our conclusions would have been strengthened by even greater sampling and the inclusion of data describing pseudo-pheromone cocktails. Morphological variation within Ophrys proved to be exceptionally multidimensional, lacking strong directional trends. Multivariate clustering of plants according to prior taxonomy was typically weak, irrespective of whether it was assessed at the level of macrospecies, mesospecies or microspecies; considerable morphological overlap was evident even between subsets of the molecularly differentiable macrospecies. Characters supporting genuine taxonomic distinctions were often sufficiently subtle that they were masked by greater and more positively correlated variation that reflected strong contrasts in flower size, tepal colour or, less often, plant size. Individual macrospecies appear to represent morphological continua, within which taxonomic divisions are likely to prove arbitrary if based exclusively on morphological criteria and adequately sampled across their geographic range. It remains unclear how much of the mosaic of subtle character variation among the microspecies reflects genetic versus epigenetic or non-genetic influences and what proportion of any contrasts observed in gene frequencies can be attributed to the adaptive microevolution that is widely considered to dictate speciation in the genus. Moreover, supplementing Biology 2023, 12, 136. https://doi.org/10.3390/biology12010136 https://www.mdpi.com/journal/biology Biology 2023, 12, 136 2 of 52 weak morphological criteria with extrinsic criteria, typically by imposing constraints on geographic location and/or supposed pollinator preference, assumes rather than demonstrates the presence of even the weakest of species boundaries. Overall, it is clear that entities in Ophrys below the level of macrospecies have insufficiently structured variation, either phenotypic or genotypic, to be resolved into discrete, self-circumscribing ("natural") entities that can legitimately be equated with species as delimited within other less specialised plant genera. Our search for a non-arbitrary (meso)species concept competent to circumscribe an intermediate number of species has so far proven unsuccessful.
... Osmophores, elaiophores and nectaries are found in members of the Orchidaceae and are important elements for maintaining the plant-pollinator interaction (Melo, Borba & Paiva, 2010;Aguiar et al., 2012;Pansarin et al., 2012;Nunes et al., 2013;Neubig et al., 2015;Franken, Pansarin & Pansarin, 2016;Kettler, Solís & Ferrucci, 2019). However, in flowers of species of the subtribe Stanhopeinae (Cymbidieae: Epidendroideae), only osmophores located on the adaxial surface of the sepals, petals or labellum have been reported (Curry et al., 1991;Pansarin, Castro & Sazima, 2009;Pansarin & Pansarin, 2011;Antón, Kamińska & Stpiczyńska, 2012;Davies & Stpiczyńska, 2012;Francisco & Ascensão, 2013;Adachi, Machado & Guimarães, 2015;Davies & Stpiczyńska, 2017;Casique et al., 2018). These osmophores are comprised of papillose or non-papillose epidermal secretory cells, characterized as unicellular secretory trichomes (Curry et al., 1991;Stpiczyńska, 1993;Ascensão et al., 2005;Melo et al., 2010). ...
... Free fatty acids are the most common components found in floral oils of the elaiophores (Vogel, 1974;Reis et al., 2006;Possobom & Machado, 2017). The intense staining of the floral tissue with reagents such as neutral red help in osmophore identification (Vogel, 1990;Pansarin & Pansarin, 2011;Francisco & Ascensão, 2013). The stains Sudan black, Sudan III and Sudan IV are useful for the detection of triacylglycerides and total lipids bound to proteins (Effemert et al., 2006). ...
The members of the Stanhopeinae (Orchidaceae) bear osmophores, which are related to pollination since they exude volatile lipids that attract euglossine bees. Gongora spp. are pollinated exclusively by euglossine bees. In view of the structural diversity found in the flowers of this genus and the lack of data on other foraging insects that visit these flowers, we elucidated aspects of the anatomy of floral secreting structures in the Stanhopeinae group, focusing on structures in Gongora jauariensis, Gongora pleiochroma and Gongora minax, species from the Amazon Rainforest. Secretory structures were analysed using light, scanning and transmission electron microscopy, and histochemical and phytochemical procedures. Osmophores, extrafloral nectaries and elaiophores were observed. The three species bear a structured nectary on the bract and osmophores on both the sepals and labellum hypochile. In G. minax, the labellum (hypochile) shows chemical and structural characteristics of elaiophore and osmophores, suggesting that it has both functions. We report interactions between foraging ants and nectaries of Gongora spp. for the first time. Interactions with ants attracted by the nectaries on the bracts are beneficial for orchids, as the ants help to protect their reproductive structures.
... This appealing hypothesis contrasts with the longevity of flowers in Ophrys, even we acknowledge the paucity of published data on this critical biological parameter. Francisco & Ascensão (2013) mention periods of anthesis of 6-8 days and 9-12 days for Ophrys bombyliflora and O. tenthdedinifera, respectively, whereas Neiland & Wilcox (1995) observed that unpollinated flowers of O. tenthredenifera and O. arachnitiformis remained intact in the field for three weeks or more. As each Ophrys inflorescence carries several flowers that open successively, albeit with a slight overlap between subsequent flowers, a given Ophrys individual with a mean of four flowers should present at least one attractive flower during ca. ...
Adaptive radiations occur mostly in response to environmental variation through the evolution of key eco-morphological innovations that allow emerging species to occupy new ecological niches. However, rapid phenotypic evolution and the evolution of key novelties are likely to also occur when a couple or few species are engaged into narrow ecological interactions. To demonstrate coevolution is a difficult task; only elusive evidences confirm that coevolution is a driver of speciation and diversification. Here we propose that the adaptive radiation of the Mediterranean orchid genus Ophrys, which gave rise to ca. 350 species since the apparition of the genus is due to the particular co-evolutionary dynamics between these plants and their pollinators. We suggest that the pollination by sexual swindle used by Ophrys orchids is the main driver of this coevolution. Flowers of each Ophrys species mimic sexually receptive females of one particular insect species, mainly bees. Male bees are attracted by pseudo-pheromones emitted by Ophrys flowers that are similar to the sexual pheromones of their females. Males lured by the flower shape, color and hairiness attempt to copulate with the flower, which glues pollen on their bodies. Pollen is eventually transferred to the stigma of another flower of the same Ophrys species during similar copulation attempts. Three observations led us to propose the scenario of an asymmetric co-evolutionary relationship between Ophrys and their pollinators. Firstly, there is a strong intra-specific competition among Ophrys individuals for the attraction of their species-specific pollinators, which is due to the high learning and memorization abilities of bees that record the pheromone signatures of kin or of previously courted partner to avoid (further) copulation attempts. Mnemonic pollinators induce thus a strong selective pressure for variation in the pseudo-pheromones emitted by individual flowers, which will potentially generate shifts in pollinator species, and hence Ophrys speciation. These pollinator shifts are adaptive for new Ophrys species because they may benefit from a competitor-free space. Secondly, such shifts in pollinator species are due to the random crossing of peaks in the olfactory landscape of the pollinator guild that is syntopic to each particular Ophrys population. This selective process on individual, random variation in pseudo-pheromone bouquets is followed by directional selection on flower phenotypes that will reinforce the attraction of the new pollinator. Thirdly, pollinators use the pseudo-pheromones emitted by Ophrys to locate suitable habitats from a Preprints (www.preprints.org) | NOT PEER-REVIEWED |
... (Wiemer et al. 2009), the lipid-secreting, honey bee-pollinated Grobya amherstia Lindl. (Pansarin et al. 2009), and sexuallydeceptive, male Eucera bee-pollinated Ophrys species (Francisco & Ascensão 2013). In some cases, compounds secreted by osmophores may serve as both an attractant and a reward (Eltz et al. 1999(Eltz et al. , 2005Tan et al. 2002). ...
Nervilia plicata is a morphologically variable terrestrial orchid with a wide geographic range in tropical Asia. Several forms have previously been recognised as distinct taxa due primarily to differences in the size, outline and colour of perianth parts. As a first step towards understanding the links between floral polymorphism, pollination ecology and genetic differentiation within the N. plicata complex, we sought to ascertain whether distinct colour morphs in 18 populations in Thailand and South China were correlated with discrete differentiation in macro- or micromorphological characters. Perianth morphology was studied in the field and under stereomicroscopy, and labellum anatomy was examined using light microscopy and scanning electron microscopy. Phenetic analyses comprising Principal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) of 16 characters revealed four clusters representing distinct floral morphs — pink morph with small, entire labellum; purple morph with large, undulate labellum; white and yellow morph with large, entire labellum; and white and yellow morph with small, entire labellum — among which the size, shape, density and periclinal wall surface of dome-shaped papillae in the secretory zone of the labellar adaxial epidermis varied discontinuously. Our results provide evidence of micromorphological differentiation among N. plicata populations in parts of tropical Asia. Pollinator observations and analyses of genetic diversity are required to establish whether this variation is of any ecological, evolutionary or taxonomic significance.
... Orchids have a widely diverse and complex combination of rewards, visual and olfactory appealing and tactile stimuli, all involved in the attraction of animal visitors (Knudsen et al. 2006;Fay 2010;Vereecken et al. 2011;Davies et al. 2013). Reports of investigations about the floral glands diversity of Orchidaceae have emphasized the presence of osmophores, nectaries, and elaiophores on the labellum (Davies et al. 2005(Davies et al. , 2014Davies 2006, 2008;Stpiczynska et al. 2007Stpiczynska et al. , 2015Pansarin et al. 2009;Pacek et al. 2012;Francisco and Ascensão 2013;Nunes et al. 2013;Borba et al. 2014;Pansarin and Pansarin 2014;Kowalkowska et al. 2015). Other related structures that have been reported are nectaries on the column (Stpiczynska et al. 2003), resin trichomes on tepals , as well as colleters on carpels, sepals and floral/involucral bracts (Cardoso-Gustavson et al. 2014). ...
The structure and composition of the labellar secretory trichomes of Rhetinantha cerifera (Barb. Rodr.) M.A.Blanco were analyzed by scanning electron and light microscopy, as well as histochemistry and chemistry. Histological analyses revealed that the chlorophyllated and dry callus at the basal region of labellum is non-secretory. The white sticky secretion is produced by unicellular secretory trichomes (not papillae), occupying the V-shaped ridge at the apical and median regions of the labellum, and the central portion of the labellar adaxial base, behind the callus. Chemical analyses of dichloromethane extracts of the secretion detected several long carbon chain constituents common in plant waxes (n-alkanes, carboxylic acids, alcohols, esters) and phytosteroids, predominantly cycloartenol derivatives. Histochemical tests showed that the secretion contains terpenoids (oleoresin), free fatty acids, phenolic compounds (including flavonoids), and polysaccharides (mainly mucilage); the results were negative for alkaloids. The secretory unicellular trichomes can concomitantly activate different metabolic pathways, and the exudate should be characterized as “heterogeneous mixtures,” consisting of lipophilic and hydrophilic compounds. Therefore, the labellar secretion is chemically more complex than plant waxes composition. The specific epithet cerifera is thus misleading, and previously reported interpretations regarding the secretion are equivocated. Based on the present results and those from the literature, it is suggested that R. cerifera and R. notylioglossa (Rchb.f.) M.A.Blanco are taxonomical entities that should be merged into a single species, as has been suggested in Flora Brasil 2020 under construction.
... 19 Similarly, Mant et al. 18 demonstrated that a monoterpene such as linalool acts in O. exaltata as a longrange attractant for Colletes cunicularius, whereas saturated and unsaturated hydrocarbons induce short-range mating behavior. As also argued by Francisco and Ascensao, 41 considering the different volatility of the compounds emitted by Ophrys species, we could suppose that the floral volatiles of O. panormitana may play two roles: (1) the higher-volatile fraction of the bouquet (dominated by 2-alcohol and 2-ketones) acting as noncopulatory behavior attractants may guide A. nigroaenea males from the distance while (2) the lower-volatile fraction (longchain alkanes and alkenes), acting at shorter-range, reinforces the attraction and stimulates the pseudocopulatory behavior. ...
Sexually deceptive orchid flowers use visual, tactile and olfactory cues of female insects in order to attract males of one or a few closely related species as pollinators. Ophrys L. is the most species-rich genus of sexually deceptive orchids in the Mediterranean Basin. Despite Ophrys pollinated by Andrena male bees use alkanes and mainly alkenes with specific double-bond positions as key signals that trigger pseudocopulatory behavior, some volatile organic compounds (VOCs) with low molecular weight were found as long-range attractants non-eliciting copulatory behavior. Since floral scents in Ophrys have been extensively studied by solvent extractions here we aimed to understand which floral volatiles are found when two different collection methods are used in Ophrys panormitana flowers. By knowing their chemical composition, we could better understand the scent chemistry of this Ophrys species without overlooking VOCs which could also have a function in its pollination biology.
Scent samples collected by dynamic headspace and by solvent extraction were analyzed by gas chromatography/mass spectrometry (GC/MS).
The floral scent of O. panormitana is composed by a bouquet of VOCs with lower and higher molecular weights. The headspace samples contained VOCs with higher volatility (mainly one aliphatic alcohol and two aliphatic ketones) whereas the solvent extracts were composed by VOCs with lower volatility (exclusively long-chain alkanes and alkenes). Overlapping in VOCs between headspace and solvent samples were not found. For the first time Andrena nigroaenea was observed during the pseudocopulation and removing the pollinaria of a flower of O. panormitana.
... Previous detailed micromorphological studies of Ophrys labella using light microscopy, scanning electron microscopy and transmission electron microscopy highlighted a spectacular diversity of cell shapes and sizes, both within and between labella (Bradshaw et al., 2010;Francisco and Ascensão, 2013). These studies showed that the labella of all species examinedeven relatively simple, early-divergent species such as O. insectifera and O. speculum -could readily be divided into at least four micromorphologically distinct and acceptably homogeneous regions of the adaxial epidermis suitable for detailed cytometric and microscopic investigation of endoreplication (Fig. 2), the number of distinct epidermal cell types generally increasing in the more evolutionarily derived species. ...
Background and aims:
Recent tissue-level observations made indirectly via flow cytometry suggest that endoreplication (duplication of the nuclear genome within the nuclear envelope in the absence of subsequent cell division) is widespread within the plant kingdom. Here, we also directly observe ploidy variation among cells within individual petals, relating size of nucleus to cell micromorphology and (more speculatively) to function.
Methods:
We compared the labella (specialized pollinator-attracting petals) of two European orchid genera: Dactylorhiza has a known predisposition to organismal polyploidy, whereas Ophrys exhibits exceptionally complex epidermal patterning that aids pseudocopulatory pollination. Confocal microscopy using multiple staining techniques allowed us to observe directly both the sizes and the internal structures of individual nuclei across each labellum, while flow cytometry was used to test for progressively partial endoreplication.
Key results:
In Dactylorhiza, endoreplication was comparatively infrequent, reached only low levels, and appeared randomly located across the labellum, whereas in Ophrys endoreplication was commonplace, being most frequent in large peripheral trichomes. Endoreplicated nuclei reflected both endomitosis and endocycling, the latter reaching the third round of genome doubling (16C) to generate polytene nuclei. All Ophrys individuals studied exhibited progressively partial endoreplication.
Conclusions:
Comparison of the two genera failed to demonstrate the hypothesized pattern of frequent polyploid speciation in genera showing extensive endoreplication. Endoreplication in Ophrys appears more strongly positively correlated with cell size/complexity than with cell location or secretory role. Epigenetic control of gene overexpression by localized induction of endoreplication within individual plant organs may represent a significant component of a plant's developmental programme, contributing substantially to organ plasticity.
... Streinzer et al., 2010;Vigniolini et al., 2012) and, if successfully misled into alighting on the highly textured, three-dimensional labellum, various tactile cues come into play (e.g. Bradshaw et al., 2010;Francisco and Ascensão, 2013). Pseudo-copulatory pollination has proven sufficiently unusual and intriguing to render the genus Ophrys something of a research industry in recent years, advancing the genus to the point when it can legitimately be described as a model system in reproductive biology studies (compare Kullenberg, 1961;Borg-Karlson, 1990;Paulus and Gack, 1990;Schiestl et al., 1999;Mant et al., 2005;Jersakova et al., 2006;Vereecken, 2009;Ayasse et al., 2010;Schiestl and Johnson, 2013). ...
... The only previous genus-wide morphological cladistic analysis of Ophrys (Devillers and Devillers-Terschuren, 1994), plus a more focused analysis of a major clade within the genus (Francisco et al., 2015), provided a useful foundation for macromorphological characters. The detailed study by Bradshaw et al. (2010), as slightly amended by Francisco and Ascensão (2013), informed our choice of micromorphological characters. We deliberately adopted a more conservative approach to scoring micromorphological characters than did Francisco et al. (2015), suspecting that epidermal features are prone to extensive pleiotropy; in addition, their potentially valuable empirical observations on the size and location of osmophores would need to be extended across the genus in order to qualify for inclusion in the matrix. ...
Background and aims:
Bee orchids (Ophrys) have become the most popular model system for studying reproduction via insect-mediated pseudo-copulation and for exploring the consequent, putatively adaptive, evolutionary radiations. However, despite intensive past research, both the phylogenetic structure and species diversity within the genus remain highly contentious. Here, we integrate next-generation sequencing and morphological cladistic techniques to clarify the phylogeny of the genus.
Methods:
At least two accessions of each of the ten species groups previously circumscribed from large-scale cloned nuclear ribosomal internal transcibed spacer (nrITS) sequencing were subjected to restriction site-associated sequencing (RAD-seq). The resulting matrix of 4159 single nucleotide polymorphisms (SNPs) for 34 accessions was used to construct an unrooted network and a rooted maximum likelihood phylogeny. A parallel morphological cladistic matrix of 43 characters generated both polymorphic and non-polymorphic sets of parsimony trees before being mapped across the RAD-seq topology.
Key results:
RAD-seq data strongly support the monophyly of nine out of ten groups previously circumscribed using nrITS and resolve three major clades; in contrast, supposed microspecies are barely distinguishable. Strong incongruence separated the RAD-seq trees from both the morphological trees and traditional classifications; mapping of the morphological characters across the RAD-seq topology rendered them far more homoplastic.
Conclusions:
The comparatively high level of morphological homoplasy reflects extensive convergence, whereas the derived placement of the fusca group is attributed to paedomorphic simplification. The phenotype of the most recent common ancestor of the extant lineages is inferred, but it post-dates the majority of the character-state changes that typify the genus. RAD-seq may represent the high-water mark of the contribution of molecular phylogenetics to understanding evolution within Ophrys; further progress will require large-scale population-level studies that integrate phenotypic and genotypic data in a cogent conceptual framework.
... Morphologically, O. leochroma shows all the features common for tenthredinifera-type flowers: square to trapezoid form, distinct lateral lobes and a reflexed apical appendix (Fig. 1). In O. tenthredinifera, a relative of O. leochroma, histochemical and cytological analyses have been used to identify the distribution of scent production on the lip (Francisco & Ascensao, 2013). It was thus shown that, as in most Ophrys species, scent is produced in the epidermal cells on the surface of the lip and in the tissue within the apical appendix and along a narrow portion of the margins of the lip (Vogel, 1962;Francisco & Ascensao, 2013). ...
... In O. tenthredinifera, a relative of O. leochroma, histochemical and cytological analyses have been used to identify the distribution of scent production on the lip (Francisco & Ascensao, 2013). It was thus shown that, as in most Ophrys species, scent is produced in the epidermal cells on the surface of the lip and in the tissue within the apical appendix and along a narrow portion of the margins of the lip (Vogel, 1962;Francisco & Ascensao, 2013). The sexual pheromone analogue of O. leochroma was thereby recently shown to comprise a blend of aldehydes, alcohols, fatty acids and hydrocarbons (Weber, 2012;Cuervo et al., 2017). ...
... All studies investigating the function of floral form in sexually deceptive orchids face one major challenge: altering floral form without affecting the emission of the sexual pheromone analogue (De Jager & Peakall, 2015). For instance in Ophrys where the lip is the main scentproducing organ, alterations of the form of the lip could also result in changes in the amount of scent produced (Francisco & Ascensao, 2013). However, in O. leochroma, we did not find any conclusive evidence for scent bias altering the response of males towards the manipulated flowers. ...
Many species of the sexually-deceptive genus Ophrys are characterized by insect-like flowers. Their form has been traditionally considered to play an important role in pollinator attraction and manipulation. Yet the evolution of the floral form remains insufficiently understood. We hypothesize that pollinator-mediated selection is essential for driving floral form evolution in Ophrys, but that form components are being subjected to varying selection pressures depending on their role in mediating interactions with pollinators. By using the Eucera pollinated Ophrys leochroma as a model, our aim has been to assess whether and in what manner pollination effectiveness is altered by experimental manipulation of the flower form. Our results show that the floral form plays an essential and, so far, underestimated role in ensuring effective pollination by mechanically guiding pollinators towards the reproductive structures of the flower. Pollinators are significantly less effective in interacting with flowers having forms altered to resemble those of species pollinated by different hymenopteran genera. Further, those components used by pollinators as gripping points were found to be more effective in ensuring pollinia transfer than those with which pollinators do not directly interact. Thus, mechanically-active and -inactive components appear to be under different selection pressures. As a consequence, mechanically-active components of the flower form could reflect adaptations to the interaction with particular pollinator groups, while inactive components can vary more freely. Disentangling selection patterns between the functionally different components of flower form may provide valuable insights into the mechanisms driving the morphological diversification of sexually-deceptive pollination-systems. This article is protected by copyright. All rights reserved.
