Abstract 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. Confocal microscopy, Dactylorhiza, flow cytometry, genome duplication, infra-organ ploidy mapping, labellum, Ophrys, orchid, plasticity, polyteny, progressively partial endoreplication INTRODUCTION In recent years, the general perception of the significance of ploidy change in plants has shifted from presumed evolutionary dead-end to an important – possibly even the most important – driver not only of speciation (e.g. Wendel, 2015; Van de Peer et al., 2017) but also of the origin of major lineages such as flowering plants (e.g. Jiao et al., 2011). Today, polyploidy stands accused of being responsible for evolutionary transitions that range from phylogenetically profound rare or unique events, such as the much-debated origin of the angiosperms, to frequent but smaller-scale events, such as the development of morphologically cryptic species complexes (Pillon et al., 2007; Trávníček et al., 2012). Inevitably, studies seeking process-based interpretations of ploidy change in plants have focused on model organisms such as Arabidopsis (Melaragno et al., 1993; Traas et al., 1998; Kato and Lam, 2003; Cookson et al., 2006; Berr and Schubert, 2007; Roeder et al., 2010; Adachi et al., 2011; Massonet et al., 2011; Schubert et al., 2012; Del Pozo and Ramirez-Parra, 2015; Sliwinska et al., 2015), Gossypium (Guan et al., 2014; Snodgrass et al., 2017) and Nicotiana (Renny-Byfield et al., 2011; McCarthy et al., 2016). However, research emphasis has consistently been placed firmly on mechanisms of ploidy change that affect whole organisms and thereafter characterize the entire evolutionary lineage. Here, we focus on genome duplication events that occur in only some cells of the affected organisms and are not directly heritable (though the ability to generate cells with expanded nuclei evidently is heritable). Only recently have the potentially profound consequences of duplication events affecting only specific cells or tissues been recognized, and most of the insights thus far have emerged from the homogenized tissues necessary for flow cytometric analysis. We also have utilized flow cytometry, but in an unusually precise fashion that targeted localized epidermal regions of a single organ. One innovation reported here is that we have combined flow cytometry with confocal microscopy, which permitted mapping of somatic ploidy that was both direct and unusually finely targeted on specific tissues. Nature and terminology of endoreplication Also known as endoreduplication, the term endoreplication describes replication of the nuclear genome occurring within the nuclear envelope in the absence of spindle formation or subsequent cell division, thereby elevating (typically doubling) the DNA content of the affected cell (Nagl, 1978; Galbraith et al., 1991; Breuer et al., 2010, 2014; Edgar et al., 2014). Two contrasting routes to endoreplication are widely recognized (e.g. Gutierrez et al., 2014; Scholes and Paige, 2015) (Fig. 1). In endomitosis, the cell undergoes partial mitosis but fails to execute telophase or cytokinesis, consequently forming lobed or even multiple nuclei within a single cell if mitosis has progressed sufficiently. By contrast, in endocycling the cell avoids mitosis, so that physical connection often persists between duplicated chromatids. Thus, the chromatids can eventually form large, structurally complex polytene chromosomes if the sister chromatids remain tightly configured to form parallel arrays (Huskins, 1947; Pearson, 1974; Zhimulev and Koryakov, 2009; Maluszynska et al., 2013). In either case, in order to unequivocally qualify as endoreplicated, a cell must bypass the M checkpoint in the cell cycle (Fig. 1), a definition that automatically encompasses all cells exceeding 4C (mitotically dividing cells cycle routinely between 2C and 4C DNA amounts). Fig. 1. View largeDownload slide Major phases, checkpoints and potential short-cuts in the standard mitotic cell cycle, illustrating terminology relevant to endoreplication. Fig. 1. View largeDownload slide Major phases, checkpoints and potential short-cuts in the standard mitotic cell cycle, illustrating terminology relevant to endoreplication. The textbook epitome of well-understood polytene chromosomes is zoological rather than botanical – specifically, the salivary gland of the fruit fly Drosophila, wherein multiply duplicated genes greatly increase the transcriptional rate of late-larval ‘glue’, thereby facilitating pendent pupation (e.g. Rodman, 1967; Zhimulev et al., 2012). Since then, zoological interest has expanded rapidly (reviewed by Neiman et al., 2017), reaching as far as biomedicine (e.g. Biesele and Poyner, 1943; Vitrat et al., 1998; Cremer and Cremer, 2001). The mitotic cell cycle is traditionally divided into a series of phases and checkpoints (e.g. Polyn et al., 2015) (Fig. 1); in plants it is determined primarily by antagonism between auxins and cytokinins (notably cyclin-dependent kinase). On average, larger cells cycle more slowly. Genome size sets a minimum duration of cell cycle and a minimum cell size. Thus, both cell size and replication time are causally positively correlated with genome size, but not with each other. In both plants and animals, endoreplication often leads to increases in cell size/complexity and in transcriptional rates/levels, reflecting massed gene duplications (Traas et al., 1998; Sugimoto-Shirasu and Roberts, 2003; Magyar et al., 2012). Visual detection of endoreplication events is less straightforward than might be imagined. Doubling of genome size would from first principles be predicted to double the nuclear volume but to expand the radius by only 26 % (volume = 4/3πr3) – a comparatively subtle increase that can prove difficult to detect in two-dimensional images, particularly since in practice the nuclei often deviate from perfect spheres and carry differentially condensed chromatin. Similar problems exist when measuring cell sizes. Nevertheless, a broadly allometric positive correlation has been demonstrated between genome size and stomatal guard-cell size in the leaves of phylogenetically broad samples of both angiosperms (‘flowering plants’: Beaulieu et al., 2008) and gymnosperms (Lomax et al., 2014). Endoreplication in plant tissues can more easily be detected using flow cytometry, provided sufficient endoreplicated cells are present in the targeted tissue. By applying flow cytometry to a wide range of orchid species, Travnicek et al. (2015) were able to demonstrate the occurrence of the under-researched phenomenon of progressive partial endoreplication (PPE), which remains poorly understood and to date has only been observed in orchids. During most reported cases of PPE, endoreplication affects not the entire nuclear genome but rather the great majority of it, such that the genome undergoes stepwise increases of less than double (Jersakova et al., 2015; Travnicek et al., 2015; Hribova et al., 2016). Brown et al. (2017, their Table 3) subsequently summarized data on PPE in a diverse suite of 75 orchid taxa and reported replicate fractions that ranged from 19 to 105 %, predictably generating a large variance around the mean value for all taxa. All Orchidoideae, most Cypripedioideae and a significant minority of Epidendroideae were affected. Similarly major effects of PPE had earlier been observed in model animals such as Drosophila (e.g. Bosco et al., 2007). Present study Flowers of cultivated azaleas were shown by Schepper et al. (2001) to be fundamentally diploid but nonetheless to contain cell lineages that often switched via endopolyploidy to tetraploidy towards the margin of the flower, which also diverged from the central region of the perianth in both cellular structure and anthocyanin pigmentation. Kudo and Kimura (2002) observed a range of endopolyploid levels from 2C to 32C in contrasting regions of the petals of cabbage, speculating that the larger proximal epidermal cells contribute to rapid petal expansion in the opening bud. Similar investigations have since been performed on orchid flowers – an intensively researched family that therefore has the potential to provide a stronger and more rounded model system for studying the evolutionary significance of ploidy change and genome duplication. However, all of these previous orchid studies (1) treated each organ of the flower as a homogeneous entity and (2) relied upon indirect estimates of nucleus size obtained via flow cytometry of homogenized tissue samples, rather than through direct microscopic observation of individual cells (cf. Mishiba et al., 2001; Lim and Loh, 2003; Yang and Loh, 2004; Barow and Jovtchev, 2007; Chen et al., 2011; Teixeira et al., 2014; Travnicek et al., 2015; Ho et al., 2016; reviewed by Hribova et al., 2016). Such studies are unable to distinguish between the two contrasting modes of endoreplication (endomitosis and endocycling), though they do offer the newly recognized advantage of being able to detect all but the most subtle cases of PPE. Here, we combine the analytical power of flow cytometry to detect PPE and quantify levels of endoreplication with the anatomical precision and cytostructural insights offered by direct microscopical examination, using in particular confocal microscopy to explore endoreplication within the labellum, a specialized insect-attracting petal that is characteristic of orchids (e.g. Rudall and Bateman, 2002; Mondragón-Palomino and Theissen, 2011). The orchid labellum is an exceptionally complex floral structure that typically exhibits many spectacular adaptations at both the macroscopic and the cellular scale (e.g. Box et al., 2008; Bradshaw et al., 2010). It therefore allows simultaneous comparison of an unusually large number of biological features that might influence, or indeed be influenced by, endoreplication. Two model systems of European orchids in subfamily Orchidoideae subtribe Orchidinae were selected by us to contrast strongly in both phenotypic complexity and predisposition to polyploid speciation (Table 1). The genus Dactylorhiza has a diploid complement of 2n = 2x = 40, develops a labellum that is micromorphologically relatively simple, indulges in no known secretion of either nectar or scent, and frequently undergoes polyploid (especially allopolyploid) speciation (e.g. Pillon et al., 2007; Paun et al., 2010; Hedrén et al., 2011). In contrast, the primary subject of this study, bee orchids of the genus Ophrys, have a diploid complement of 2n = 2x = 36 and, on present (limited) evidence, rarely undergo polyploid speciation. However, they reliably possess a labellum that is exceptionally micromorphologically complex (Fig. 2); it exhibits several specialized epidermal cell types that contrast in both appearance and function (e.g. Bradshaw et al., 2010). Table 1. Details of staining and microscopy methods applied to each orchid species studied Species Clade 2n Staining method Microscopy method PI (all DNA, red) Feulgen (all DNA, dark grey) DAPI (A+T-rich DNA, blue) FISH (18S rDNA, green) Confocal Light Fluorescence Dactylorhiza fuchsii A 40 ✓ ✓ Dactylorhiza foliosa A 40 ✓ ✓ Dactylorhiza praetermissa AB 80 ✓ ✓ Ophrys insectifera 1 36 ✓ ✓ ✓ ✓ Ophrys speculum 2 36 ✓ ✓ ✓ ✓ Ophrys tenthredinifera s.l. 2 36 ✓ ✓ Ophrys apifera 3 36 ✓ ✓ ✓ ✓ Ophrys sphegodes 3 36 ✓ ✓ ✓ Species Clade 2n Staining method Microscopy method PI (all DNA, red) Feulgen (all DNA, dark grey) DAPI (A+T-rich DNA, blue) FISH (18S rDNA, green) Confocal Light Fluorescence Dactylorhiza fuchsii A 40 ✓ ✓ Dactylorhiza foliosa A 40 ✓ ✓ Dactylorhiza praetermissa AB 80 ✓ ✓ Ophrys insectifera 1 36 ✓ ✓ ✓ ✓ Ophrys speculum 2 36 ✓ ✓ ✓ ✓ Ophrys tenthredinifera s.l. 2 36 ✓ ✓ Ophrys apifera 3 36 ✓ ✓ ✓ ✓ Ophrys sphegodes 3 36 ✓ ✓ ✓ View Large Table 1. Details of staining and microscopy methods applied to each orchid species studied Species Clade 2n Staining method Microscopy method PI (all DNA, red) Feulgen (all DNA, dark grey) DAPI (A+T-rich DNA, blue) FISH (18S rDNA, green) Confocal Light Fluorescence Dactylorhiza fuchsii A 40 ✓ ✓ Dactylorhiza foliosa A 40 ✓ ✓ Dactylorhiza praetermissa AB 80 ✓ ✓ Ophrys insectifera 1 36 ✓ ✓ ✓ ✓ Ophrys speculum 2 36 ✓ ✓ ✓ ✓ Ophrys tenthredinifera s.l. 2 36 ✓ ✓ Ophrys apifera 3 36 ✓ ✓ ✓ ✓ Ophrys sphegodes 3 36 ✓ ✓ ✓ Species Clade 2n Staining method Microscopy method PI (all DNA, red) Feulgen (all DNA, dark grey) DAPI (A+T-rich DNA, blue) FISH (18S rDNA, green) Confocal Light Fluorescence Dactylorhiza fuchsii A 40 ✓ ✓ Dactylorhiza foliosa A 40 ✓ ✓ Dactylorhiza praetermissa AB 80 ✓ ✓ Ophrys insectifera 1 36 ✓ ✓ ✓ ✓ Ophrys speculum 2 36 ✓ ✓ ✓ ✓ Ophrys tenthredinifera s.l. 2 36 ✓ ✓ Ophrys apifera 3 36 ✓ ✓ ✓ ✓ Ophrys sphegodes 3 36 ✓ ✓ ✓ View Large Fig. 2. View largeDownload slide Scanning electron micrograph of the labellum of Ophrys speculum with enlargements of three micromorphologically variable regions of the adaxial epidermis that were examined for endoreplication during the present study (speculum, appendix, median/mid-lobe and lateral lobe hirsute margins) and one (the stigmatic surface) that was not (see also Bradshaw et al., 2010). As O. speculum lacks an apical appendix, the appendix shown represents that of O. apifera (see also Fig. 9A). Scale bar: (main figure) = 2 mm. Fig. 2. View largeDownload slide Scanning electron micrograph of the labellum of Ophrys speculum with enlargements of three micromorphologically variable regions of the adaxial epidermis that were examined for endoreplication during the present study (speculum, appendix, median/mid-lobe and lateral lobe hirsute margins) and one (the stigmatic surface) that was not (see also Bradshaw et al., 2010). As O. speculum lacks an apical appendix, the appendix shown represents that of O. apifera (see also Fig. 9A). Scale bar: (main figure) = 2 mm. When the labellum of Ophrys is viewed from a functional perspective, contrasting labellar cell types are collectively responsible for seducing naive male insects into participating in this textbook example of sexually deceptive pollination. The labellum first secretes a cocktail of pseudopheromones as an olfactory cue, then offers a complex mosaic of brightly coloured and reflective regions as visual cues, and finally stimulates the now alighted insect with strategically placed and contrasting patches of trichomes – tactile cues that differ between species in size, shape and/or orientation and therefore supposedly mimic the conspecific female insect sufficiently well to encourage pseudocopulation (e.g. Schiestl, 2005; Bateman et al., 2011; Vereecken et al., 2011; Vignolini et al., 2012). In its attempt to mate with the flower, the naive male removes the cohesive pollen masses from the gynostemium – the fused hermaphrodite structure that defines the orchid family, providing the location of meiosis for both pollen and ovule production. The primary goals initially set for the present study were to use somatic ploidy mapping: (1) To assess, via confocal microscopy applied to the labellar petals of multiple representatives of two orchid genera that contrast strongly in cellular complexity, the frequency and location of endoreplicated nuclei, and to estimate the number of endocycles that each has undergone. (2) To determine in epidermal cells whether the presence and size (average and range distribution) of endoreplicated nuclei are associated with, and thus could have helped to determine, one or more of the following three features: size and/or complexity of cells; location of cells within a specific organ, the labellum (especially central versus marginal locations); presumed overall level of transcriptional activity within cells (notably energy-intensive secretions of nectar and/or fragrances). (3) To determine whether PPE occurs in Ophrys labella and whether it can be detected via direct microscopic observation (this goal was added post hoc in the wake of the recognition through flow cytometry of the existence of PPE in orchids by Travnicek et al., 2015). (4) To begin to explore the external morphology and internal structure of endoreplicated (including polytene) nuclei in orchids, permitting the distinction between the products of endomitosis and endocycling. (5) To use somatic ploidy changes observed in the labella as a proxy for endoreplication within the gynostemium – the site of both ovule and pollen formation in the orchid flower, and thus of the unreduced gametes necessary for polyploid speciation in Dactylorhiza. [This goal was effectively undermined after our laboratory study had concluded, when it was shown that the gynostemium is the one organ in an orchid flower that is not prone to endoreplication (Travnicek et al., 2015.)] In retrospect, we are satisfied that we met objectives (1) to (3), made some progress with objective (4), but were naive to believe that we could achieve objective (5). MATERIALS AND METHODS Materials The three species of Dactylorhiza that we selected for detailed study consist of a diploid that is endemic to the Atlantic island of Madeira (D. foliosa), a much more geographically widespread diploid that has acted as the ovule-parent in a series of speciation events driven by allopolyploidy (D. fuchsii), and one of the allotetraploid species (D. praetermissa) that D. fuchsii has generated in combination with the diploid pollen-parent D. incarnata (e.g. Hedrén et al., 2011). The genus Ophrys is exceptionally taxonomically controversial; some traditional morphological and ethological studies recognize more than 350 species (cf. Delforge, 2006; Vereecken et al., 2011; Paulus, 2015; Delforge, 2016), whereas molecular phylogenies suggest ten or fewer species (cf. Devey et al., 2008; Bateman et al., 2011), a pattern maintained even when next-generation sequencing technologies are employed (Fig. 3) (Bateman et al., 2018). We selected five relatively uncontroversial species to represent each of the three subgenera of Ophrys: the monotypic subgenus insectifera plus morphologically and molecularly divergent species pairs from the two remaining subgenera (Table 1, Fig. 3). Fig. 3. View largeDownload slide Backbone phylogeny of the genus Ophrys based on RAD-Seq data, showing the relationships of the nine macrospecies recognized by Bateman et al. (2018), each of which is illustrated to the right. The five macrospecies included in the present study are highlighted in red, and the three main clades here regarded as subgenera are numbered in red. Black numbers below branches indicate all bootstrap support values. Scale: the long axis of each rectangular flower image = 25 mm. Fig. 3. View largeDownload slide Backbone phylogeny of the genus Ophrys based on RAD-Seq data, showing the relationships of the nine macrospecies recognized by Bateman et al. (2018), each of which is illustrated to the right. The five macrospecies included in the present study are highlighted in red, and the three main clades here regarded as subgenera are numbered in red. Black numbers below branches indicate all bootstrap support values. Scale: the long axis of each rectangular flower image = 25 mm. Fresh inflorescences were sampled from either the living collections of the Royal Botanic Gardens, Kew, for Dactylorhiza, and from either natural populations in the UK and the Mediterranean or the private seed-raised horticultural collection of Barry Tattersall for Ophrys. Plant material intended for microscopic examination was placed immediately in fixative (3:1 glacial acetic acid:ethanol) and later refrigerated at 4 °C. Fresh material for analysis via flow cytometry was simply placed in damp tissue. Staining methods Four different staining techniques, each providing different cytological emphases, were employed for contrasting purposes (Table 1). Non-fluorescent Feulgen (Schiff reagent) staining provided the focus for preliminary observations. Of the three remaining, uniformly fluorescent stains employed, propidium iodide (PI) stains DNA uniformly, whereas 4,6-diamidino-2-phenylindole (DAPI) stains predominantly A+T-rich regions. Fluorescent in situ hybridization (FISH) was used to label 18S ribosomal DNA (rDNA) in order to determine whether signals were dispersed or alternatively clustered as an indication of polytene chromosomes. Although fluorescent stains are less convenient than Feulgen stains, they allowed us to pursue both FISH experiments and confocal examination on the same preparation. Cold hydrolysis was employed in the Feulgen procedure to encourage the stain to pass through the cell membrane and reach the nucleus. Inflorescences were immersed in 5 m HCl in test tubes placed in a water bath at 20 °C for 30 min. Samples were then subjected to four 5-min washes in distilled water on a low-speed rotator. Flowers were subsequently stained in Feulgen tubes, enclosed in aluminium foil, and placed in the dark for 30 min. Individual labella were then excised from the stained inflorescence, placed on glass slides, mounted under coverslips and sealed using vulcanizing rubber solution. For PI staining, labella were immediately excised from the fixed inflorescence and passed through four 5-min washes in distilled water on a low-speed rotator before being placed on a glass slide. The PI was diluted 1:1000 in distilled water before application. Staining with DAPI followed a broadly similar protocol but was confined to O. speculum and O. apifera (Table 1). One drop (~25 μL) of photoreactive DAPI, dissolved in Vectashield® to preserve fluorescent reactivity, was added with care in subdued light, and the labellum coverslipped. Any substandard DAPI slides were washed in the non-ionic detergent Tween® before being re-stained with PI. The more complex FISH procedure was adapted from Lim et al. (2006). The protocol requires a three-day preparatory period in total [a ‘standard wash’ is here defined as a 5-min wash at 37 °C in 2× saline sodium citrate buffer (SSC; 3 m NaCl, 0.3 m sodium citrate, pH7)]. Each labellum was quartered, and each quarter was placed in a 1.5-mL microtube. Samples received two 5-min washes in citric buffer (4 mm citric acid plus 6 mm sodium citrate) followed by enzyme digestion in 0.3 % w/v cellulase R10, 0.3 % w/v pectolyase Y23 and 0.3 % w/v Driselase in enzyme buffer for 20 min, and transfer to enzyme buffer for 2 h at room temperature. Samples then received two further 5-min washes before being exposed to 0.12 mg mL−1 RNase in 2× SSC at 37 °C for 60 min. Three further standard washes rendered the labellar fragments fragile, so they were rinsed with care in 0.01 m HCl for 2 min followed by pepsin treatment: 0.1 μg mL−1 pepsin in 0.01 m HCl for 5 min at room temperature. Material was then fixed in 2 % formaldehyde in water at room temperature for 10 min and washed in 2× SSC in preparation for probe hybridization. The petal fragments were then immersed in the hybridization mix overnight. This mix contained the 18S rDNA probe, which had been isolated by PCR amplification of 18S rDNA from Allium cernuum using PCR primers (18S2F 59-CGGAGAATTAGGGTTCGATTC-39 and AB101R 59-ACGAATTCATGGTCCGGTGAAGTGTTCG-39) and labelled with digoxigenin-11-dUTP by nick translation, as described by Renny-Byfield et al. (2012). The hybridization mix contained 4 µg mL−1 labelled 18S rDNA probe in 50 % w/v formamide, 10 % w/v dextran sulphate and 0.1 % w/v sodium dodecyl sulphate in 2× SSC. After overnight incubation, the samples were denatured in a water bath at 70 °C for 3 min, followed by a further phase of hybridization for 24 h at 37 °C. Material was then given three standard washes plus two 5-min, 42 °C washes in a mixture of 20 % formamide in 0.1× SSC. Sites of probe hybridization were then detected by immersing the material in 20 mg mL−1 fluorescein conjugated anti-digoxigenin immunoglobulin G (Roche Biochemicals) in 2× SSC for 4 h. Three further standard washes preceded a wash in 4× SSC/0.2 % Tween at room temperature for 5 min. The surviving tissues were then mounted in subdued light on glass slides in a single drop of DAPI in Vectashield®, together with a single drop of PI as a counterstain. All prepared slides were stored in a refrigerator at 4 °C prior to microscopic examination. Microscopy methods Feulgen-stained slides were observed under standard compound light microscopy and digitally imaged at magnifications ranging from ×10 to ×100, allowing an initial phase of rapid screening. Slides stained in PI and DAPI were then subjected to more detailed examination under a Leica DMRA2 epifluorescence microscope, and images were captured using a Hamamatsu Orca ER camera. There exists a trade-off when measuring genome size through direct microscopic observation rather than automated flow cytometry; a much smaller number of observations is feasible, but in compensation nuclear size can be related to nuclear micromorphology. For each of the three Dactylorhiza species, areas of 100 nuclei were measured from multiple labella using confocal microscopy and Improvision Openlab software. A nuclear volume was calculated from each measured area by assuming that the recorded area represented a circle and the volume a sphere. Cumulative curves of the resulting measures were then constructed in search of comparatively high-angle break points in size distributions that would allow us to assign each nucleus to one of multiple bins in order to construct histograms competent to indicate contrasting ploidy levels (while bearing in mind the possible modifying effects of PPE). Interpretation focused on the proportions of 4C and especially 8C nuclei, which are the smallest nuclei incontrovertibly resulting from endoreplication. The PI-based slides that constituted the backbone of this study, together with the FISH slides of O. sphegodes (Table 1), were in addition transferred to a controlled-environment suite for high-resolution microscopy using a Leica SP5 confocal laser microscope. This approach allowed three-dimensional reconstruction of nuclei, albeit incurring image acquisition times considerably exceeding those necessary for epifluorescence microscopy. In order to detect multiple fluorescence channels simultaneously, successive parallel planes through a specimen were typically captured at intervals of 1 μm, though closer intervals were used when z-stacking successive images collectively representing single nuclei. The resulting sets of high-quality two-dimensional images and stacks were converted into rotatable three-dimensional images (arguably more accurately described as two-and-a-half dimensional images) using Improvision’s Velocity® software. Flow cytometry methods Flow cytometry was introduced into the present study only towards its close. It is in many ways complementary to direct microscopic observation of nuclei; many more nuclei can be measured, arguably with greater accuracy, but at the expense of losing precise knowledge of the spatial relationships of individual nuclei (Supplementary Data). Flow cytometric study was confined to leaves and flowers of two individuals of O. tenthredinifera villosa (a putative microspecies/subspecies confined to the eastern Mediterranean) that were grown from seed wild-collected at two localities on Crete, together with leaf apices of a further two Ophrys species: O. speculum and O.sphegodes incubacea. Analyses were performed separately on two flowers from each plant of O. tenthredinifera, run 2 weeks apart. Each labellum was dissected into six regions for separate analysis according to the dominant micromorphology of the adaxial epidermis: top-left margin, lower-left margin (both dominantly trichomes), appendix (domed), speculum margin, speculum, and stigmatic surface (all typically papillate). It is noteworthy that regrettably, the small excised regions of labellum that were subjected to flow cytometry also encompassed the underlying tissues of the mesophyll and abaxial epidermis, together with any vascular tissues penetrating the mesophyll. Also, given the small areas of tissues excised, counts of nuclei were inevitably smaller than would be ideal. We assessed the nuclear DNA content of each sample by flow cytometry, using interphase nuclei stained with PI and following the one-step procedure described in detail by Doležel et al. (1998). Individual labellum fragments were placed in Petri dishes containing 1 mL of general-purpose isolation buffer (GPB) with 3 % polyvinylpyrrolidone PVP40 (Loureiro et al., 2007) plus leaf tissue of the selected internal standard (Pisum sativum ‘Ctrirad’, 2C = 9.09 pg; Doležel et al., 1998), and each combined sample was diced using a fresh razor blade. Nuclear suspensions were then filtered through a nylon mesh (30 µm pore size) to remove unwanted debris. The filtrate was stained with 1 mg mL−1 PI to a final concentration of 60 µg mL−1. After incubation on ice for 20 min, the relative fluorescence of at least 1000 (typically 5000) particles was recorded using a Partec Cyflow SL3 (Partec, Münster, Germany) flow cytometer fitted with a 100-mW, 532-nm green solid-state laser (Samba, Cobolt, Solna, Sweden). The resulting histograms were analysed with FlowMax software (v2.4, Partec). The individual genomic DNA contents were estimated as 2C values by multiplying the known 2C value of the chosen standard (2C = 1 pg) by the ratio between the mean relative fluorescence intensities of the G1 peak of the Ophrys tissue sample and that of the G1 peak of the standard. Calculation of genome sizes (2C and 4C nuclei, respectively) assumes that 1 pg of unmodified statistical dsDNA represents 978 Mbp (Doležel et al., 1998). We here use 1C to indicate the monoploid genome size, as recommended by Greilhuber et al. (2005). RESULTS AND DISCUSSION Dactylorhiza: contrasting diploidy with allopolyploidy Speciation in European Dactylorhiza occurs dominantly through hybridization plus chromosome doubling (allopolyploidy) between the phylogenetically divergent D. fuchsii and D. incarnata groups (Pillon et al., 2007; Bateman, 2011; Hedrén et al., 2011). Several analytical approaches employed during the last half-century have all indicated multiple independent origins via allopolyploidy of species that are only subtly morphologically distinct, having originated from different yet conspecific parental races that exhibit contrasting habitat preferences (certainly the case in the D. majalis allopolyploid complex). The relevant Dactylorhiza species have a uniformly papillate labellum (Box et al., 2008) and non-secretory spur; operating by food-deceit, they are dominantly pollinated by typically wide spectra of bee species (e.g. Claessens and Kleynen, 2011). We therefore elected to use this genus to test indirectly whether a predisposition to endoreplication might also indicate a predisposition to polyploid speciation. Previous chromosome counts and genetic studies have both demonstrated D. foliosa and D. fuchsii to be diploid (the latter with 2C of 5.94 pg; Aagaard et al., 2005), whereas D. praetermissa is the allotetraploid product of a diploid ovule-parent broadly resembling D. fuchsii and a diploid pollen-parent broadly resembling D. incarnata (e.g. Hedrén et al., 2011). Nonetheless, our analysis of labellar tissues in our three Dactylorhiza study species revealed nuclear size distributions that differed little from expectations of phases G1 or G2 (Fig. 4); we found no evidence of highly reduplicated cells. The distributions of microscopically estimated nucleus size in the three Dactylorhiza species showed less pronounced peaks and troughs than we anticipated (Fig. 5). Nonetheless, the troughs did permit the identification of break points that could be used to delimit bins, and mean values calculated from these bins yielded arithmetically credible estimates of the volumes of 2C, 4C and (in the case of D. praetermissa) <5 % of putative 8C nuclei – presumably the products of small-scale endoreplication. Fig. 4. View largeDownload slide Propidium iodide confocal images of the central region of the labellum of the diploid Dactylorhiza fuchsii (A) and allotetraploid D. praetermissa (B), presented at the same magnification to show the comparatively low variation in the nuclear genome size of the epidermal cells in each species compared with those observed in Ophrys species. Insets show inflorescences of the respective species. Scale bar = 25 μm; long axis of insets = 30 mm. Fig. 4. View largeDownload slide Propidium iodide confocal images of the central region of the labellum of the diploid Dactylorhiza fuchsii (A) and allotetraploid D. praetermissa (B), presented at the same magnification to show the comparatively low variation in the nuclear genome size of the epidermal cells in each species compared with those observed in Ophrys species. Insets show inflorescences of the respective species. Scale bar = 25 μm; long axis of insets = 30 mm. Fig. 5. View largeDownload slide Histogram comparing nuclear volume distributions of the more or less planar labella of the diploid species Dactylorhiza fuchsii (red bars) and D. foliosa (blue bars) with that of the micromorphologically similar but allotetraploid species D. praetermissa (green bars). Arrows indicate the mean size that corresponds with each modal genome size. Fig. 5. View largeDownload slide Histogram comparing nuclear volume distributions of the more or less planar labella of the diploid species Dactylorhiza fuchsii (red bars) and D. foliosa (blue bars) with that of the micromorphologically similar but allotetraploid species D. praetermissa (green bars). Arrows indicate the mean size that corresponds with each modal genome size. In summary, epifluorescence microscopy images of PI-stained labella of the three species of Dactylorhiza studied by us (Fig. 4) revealed uniform nuclear sizes consistent with prior assumptions of their respective ploidy levels (Fig. 5). No clear evidence of endoreplication was found in the two diploid Dactylorhiza species, and only a hint of a low-frequency, low-level endoreplication was detected in the allotetraploid species. Given that the comparative epidermal homogeneity of the labellum is mirrored in the comparative homogeneity of nuclear size, we found no evidence that predisposition to allopolyploid speciation within this genus is positively correlated with predisposition to endoreplication within individual dactylorchid plants. Admittedly, this outcome contrasts with a previous study (Chen et al., 2011) that used flow cytometry to estimate levels of endoreplication in the tropical epidendroid orchid Phalaenopsis aphrodite and found that diploid plants assigned to this species maintained higher overall levels of endoreplication than did corresponding tetraploid plants. Given the comparative homogeneity of patterns of nuclear size variation in Dactylorhiza, we elected to focus our study on the genus Ophrys. Ophrys: presence of partial progressive endoreplication Leitch et al. (2009, their Fig. 1) reported 1C genome size values for 42 species of subfamily Orchidoideae that collectively yielded a mode of 6–7 pg and a mean of 8.4 pg. Four of their 42 data points were derived from unspecified species of Ophrys, which collectively yielded a slightly higher mean of 10.2 pg from a comparatively narrow range of 1C = 9.5–10.8 pg; this figure remains lower than the mean of 18.3 pg/1C calculated by Leitch et al. for aggregated terrestrial species of all orchid subfamilies. Although the two plants analysed by us for flow cytometry and presented in Fig. 6 ostensibly represent the same microspecies (O. villosa, placed molecularly within the macrospecies O. tenthredinifera; Devey et al., 2008), we unexpectedly discovered that the plant that was marginally larger in both vegetative organs and flowers was tetraploid (supposedly an unusual phenomenon within the genus), whereas the somewhat smaller plant yielded the expected diploid result. The labellar results are more readily interpreted when considered in the context of flow cytometry results obtained from leaves of the same two plants (Fig. 6A, D). In particular, the 2C peak that could not be detected with confidence in the labellum data derived from the diploid plant is clearly present, though admittedly modest in size, in the leaf-based histogram. Arithmetic comparison of flow cytometric count peaks over five runs of contrasting regions of the labellum also made clear that these plants routinely show partial replication throughout the labellum; recorded values for the transition from 2C to 4C nuclei demonstrated size increases of 86 ± 3 % rather than the 100 % that would be expected from complete replication of the nuclear DNA. Halving the mean value obtained from the 2C peaks in the flow histogram yields an estimated 1C value of 10.0 ± 0.3 pg – midway within the range previously reported for the genus by Leitch et al. (2009). Fig. 6. View largeDownload slide Fluorescence intensity distributions (a proxy for nuclear genome size) generated by flow cytometry from populations of cells in diploid and tetraploid individuals of Ophrys tenthredinifera villosa. The fluorescence peaks generated by nuclei isolated from leaf tissue of diploid (A) and tetraploid (D) individuals are presented to enable the different endoreplication fluorescence peaks in floral tissues to be correctly interpreted. (B, E) Fluorescence peaks from the trichome-rich lateral lobe of labellum of (B) diploid and (E) tetraploid plants. (C, F) Fluorescent peaks from the labellum appendix in (C) diploid and (F) tetraploid plants. The inverted triangle indicates the expected position of 2C peaks in the lateral lobe and appendix of the labellum; such peaks were absent from the diploid plant. Fig. 6. View largeDownload slide Fluorescence intensity distributions (a proxy for nuclear genome size) generated by flow cytometry from populations of cells in diploid and tetraploid individuals of Ophrys tenthredinifera villosa. The fluorescence peaks generated by nuclei isolated from leaf tissue of diploid (A) and tetraploid (D) individuals are presented to enable the different endoreplication fluorescence peaks in floral tissues to be correctly interpreted. (B, E) Fluorescence peaks from the trichome-rich lateral lobe of labellum of (B) diploid and (E) tetraploid plants. (C, F) Fluorescent peaks from the labellum appendix in (C) diploid and (F) tetraploid plants. The inverted triangle indicates the expected position of 2C peaks in the lateral lobe and appendix of the labellum; such peaks were absent from the diploid plant. Thus, our flow cytometric data suggest that ~14 % of the chromosomal material is lost during endoreplication, offering clear evidence of PPE. Interestingly, 86 % is precisely the fixed fraction reported for PPE in a Lebanese accession attributed to O. fusca by Brown et al. (2017). However, these modest losses of nuclear material are not evident in our small number of nuclear measurements obtained via confocal microscopy. It is therefore possible that whichever elements in the genome fail to replicate during PPE do not affect nuclear diameters as perceived via direct microscopic observation. Further research is urgently required in this area. Ophrys: location and spatial extent of endoreplication 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 examined – even 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. The four regions chosen for study here were (1) the often reflective speculum in the centre of the labellum, (2) the appendix that terminates the labellar apex of most Ophrys species (although this feature was well developed only in O. apifera and O. tenthredinifera among the five species studied here; Table 1, Fig. 3), and the variably papillate–trichomatous margins of (3) the mid-lobe and (4) the lateral lobes, respectively. Future studies could usefully examine other regions, such as the typically papillate stigmatic surface of the gynostemium immediately above the labellar base (Fig. 2), as the stigma should share with the appendix (a putative osmophore) the trait of being secretory and thus presumably constituting a focus of significant gene expression. Preliminary light microscope investigation of Feulgen-stained O. speculum (Fig. 7A) demonstrated an absence of endoreplication in the well-developed speculum located at the centre of the labellum (Fig. 7C) but extensive formation of endoreplicate nuclei in the trichomes (Fig. 7B, D) that form the unusually well-developed hirsute border that extends across both the middle and lateral lobes (Figs 2 and 7A). It also revealed some details of both the diploid and the endoreplicated nuclei, indicating the simultaneous occurrence of at least two kinds of endoreplication: regular nuclei formed through endocycling (Fig. 7F; see also Fig. 9A) and irregular lobed (‘lobulated’) nuclei most likely produced through endomitosis (Fig. 7E, G; see also Fig. 9E). Fig. 7. View largeDownload slide Feulgen light microscopy images of the labellum of Ophrys speculum (A), contrasting the comparative uniformity of nucleus size in the speculum (C) with the greater variability in nucleus size and shape observed along the hirsute margin (B, D). (E–G) Higher-magnification images contrast the likely products of endomitosis (E, G) versus the more spheroidal nuclei thought to result from endocycling (F). The locations on the flowers of the remaining images are shown in (A). Scale bars: (A) = 5 mm; (B) 200 μm; (C, D) 100 μm; (E–G) = 20 μm. Fig. 7. View largeDownload slide Feulgen light microscopy images of the labellum of Ophrys speculum (A), contrasting the comparative uniformity of nucleus size in the speculum (C) with the greater variability in nucleus size and shape observed along the hirsute margin (B, D). (E–G) Higher-magnification images contrast the likely products of endomitosis (E, G) versus the more spheroidal nuclei thought to result from endocycling (F). The locations on the flowers of the remaining images are shown in (A). Scale bars: (A) = 5 mm; (B) 200 μm; (C, D) 100 μm; (E–G) = 20 μm. Ophrysapifera (Fig. 8A) was examined under the confocal microscope using both PI and DAPI staining. Like O. speculum, this species showed no evidence of endoreplication in its comparatively poorly developed speculum (Fig. 8C). Unlike O. speculum, this species possesses a well-developed appendix, but that too showed only modest evidence of endoreplication (Fig. 8B). Had endoreplication been present in the appendix, we would have found it difficult to determine whether its presence reflects the marginal location or physiological activity of the osmophoric appendix, but its comparatively weak expression suggests that neither explanation may apply (but see the flow cytometry evidence for O. sphegodes discussed below). Fig. 8. View largeDownload slide Microscopic images of the labellum of Ophrys apifera (A), contrasting the uniformity of nuclear genome size in the speculum (C) and the apical appendix (B), which is believed to be responsible for much of the pseudopheromone production in the flower, with the much greater variability in size and shape evident along the hirsute margins (D–F). Some of the endoreplicate nuclei occur towards the distal ends of large unicellular trichomes (F) wherever the trichomes are clustered on the labellum. The locations on the flowers of the remaining images are shown in (A). Propidium iodide confocal (C, F) and DAPI confocal (B, D, E). Scale bars: (A) = 2 mm; (B) = 100 μm; (C) = 50 μm; (D) = 500 μm; (E) = 1 mm; (F) = 100 μm. Fig. 8. View largeDownload slide Microscopic images of the labellum of Ophrys apifera (A), contrasting the uniformity of nuclear genome size in the speculum (C) and the apical appendix (B), which is believed to be responsible for much of the pseudopheromone production in the flower, with the much greater variability in size and shape evident along the hirsute margins (D–F). Some of the endoreplicate nuclei occur towards the distal ends of large unicellular trichomes (F) wherever the trichomes are clustered on the labellum. The locations on the flowers of the remaining images are shown in (A). Propidium iodide confocal (C, F) and DAPI confocal (B, D, E). Scale bars: (A) = 2 mm; (B) = 100 μm; (C) = 50 μm; (D) = 500 μm; (E) = 1 mm; (F) = 100 μm. In contrast, the hirsute regions of the labellum margin, rich in unicellular yet highly elongated trichomes, once again proved to be rich in a diverse spectrum of endoreplicate nuclei (Fig. 8D, E). These trichomes were arguably the most complex, and certainly the largest, epidermal cells that we investigated. As well as being larger and more structurally complex than diploid nuclei, the endoreplicate nuclei of O. apifera also exhibited unusual behaviours. Some – particularly those adorning the prominent horn-like lateral lobes – had apparently been squeezed outward until becoming lodged in the narrowly conical apices of the trichomes (Fig. 8D, F), rather than remaining in the much more spacious basal regions of the cells. Similar results were obtained through PI staining of O. insectifera and O. tenthredinifera (results not shown). We sought more quantitative confirmation of these confocal observations in our flow cytometry data. Examples of relative fluorescence histograms for the lateral-lobe trichomes and appendix cells of the diploid and tetraploid plants, respectively, of O. tenthredinifera villosa are given in Fig. 6 (when interpreting this figure, it is important to remember that 2C peaks clearly evident in leaf tissue were far less evident in the corresponding flower tissues). Comparisons between the labella of the two plants analysed, different labella of the same plant and different regions of the same labellum all suggested that broad patterns of nuclear size distributions were reliable but that the quantitative details of those distributions were not, as significant discrepancies were evident at every hierarchical level. Interestingly, contrasting results derived from replicated analyses were most obvious in those labellar regions that are characterized by a papillate epidermis – the stigma (not examined under the confocal microscope) and the speculum. The only confident generalization that can be made here is that the flow cytometry data indicate a greater inclination towards multiple endoreplication events in the appendix than was suggested by confocal examination, levels within the appendix at least matching those evident in the trichomes. This observation encourages us to suspect that the detailed discrepancies reflect the fact that our confocal observations were carefully adjusted to chosen levels in the cellular ‘stratigraphy’ of the labellum, whereas in the case of our flow cytometry samples, size data derived from the adaxial epidermal cells were subordinate in number to those derived from other tissues present in the analysed homogenate. Any such distortion would impact less upon the appendix, a structure that is comparatively small, is composed of comparatively small cells and is more dorsiventrally homogeneous when viewed at a cellular level. Ophrys: genome size and micromorphology of endoreplicated nuclei In terms of levels of endoreplication reached, our flow cytometric data (Fig. 6) indicated that, for the diploid plant of O. tenthredinifera (and irrespective of precise location on the labellum; Fig. 6B, C, E, F), 8C cells were either dominant or were co-dominant with 4C cells; 2C and 16C cells were either infrequent or, less commonly, seemingly absent. Results differed according to precise location on the labellum, obliging us to rely upon the corresponding leaf data for clear evidence of the presence of 2C nuclei (Fig. 6A, D). Similar nucleus size distributions were observed in the tetraploid plant, except that its fundamental ploidy level meant that, strictly, it was 4C cells that were most frequent, whereas clear evidence of a small minority of 16C cells was found only among the trichomes of the lateral labellar lobes (Fig. 6B, E). Similar flow cytometry profiles were obtained from the leaves of the four plants analysed, the only slight deviation being that no clear peak was identified at 16C in the leaves of either O. tenthredinifera (Fig. 6A, D) or O. speculum (not shown). Interestingly, irrespective of the ploidy of the plant, results obtained from leaves matched those obtained from the labellar appendix more closely than those obtained from trichome-rich regions of the labellum (cf. Fig. 6). Closer examination of endoreplicated nuclei through confocal means relied primarily upon PI and FISH preparations of O. sphegodes (Fig. 9). Individual nuclei were selected for more detailed examination and the ploidy level of each was estimated by measuring its nuclear diameter. Still images of nuclei carefully selected by repeatedly pausing movies obtained through z-stacked confocal microscopy (Fig. 9) contrasted nuclei that are diploid (likely to be 2C; Fig. 9A left, B), endoreplicate non-polytene (likely to be 8C; Fig. 9A right) – both clearly possessing single, proportionately sized nucleoli – and endoreplicate polytene (likely to be 16C; Fig. 9D, E). The mean presumed diploid diameter was 21.7 μm. The right-hand nucleus shown in Fig. 9A had a mean diameter of 36.6 μm and that in Fig. 9D of 38 μm, and these are therefore assumed to be 8C, whereas that shown in Fig. 9E averaged 44 μm in diameter and is therefore assumed to be 16C, implying that it had undergone three rounds of endoreplication. No significantly larger nuclei were observed in any Ophrys species, either through confocal microscopy (Figs 7–10) or flow cytometry (Fig. 6). Fig. 9. View largeDownload slide Propidium iodide images of the labellum margin of Ophrys sphegodes (C) selected from within confocal image stacks of reconstructed nuclei contrast nuclei that are diploid at 2C (A left, B), endoreplicate non-polytene at an estimated 8C (A right) (both possessing single, proportionately sized nucleoli) and endoreplicate polytene at an estimated 16C (D, E). (A right) This nucleus is assumed to be endocyclic whereas the remainder of the illustrated endoreplicate nuclei are assumed to be endomitotic. Vertical dimensions of nuclei: (A) left = 21.7 μm, right = 36.6 μm; (B) = 19 μm, (D) = 38 μm, (E) = 44 μm; (C) vertical dimension of image = 25 mm. Fig. 9. View largeDownload slide Propidium iodide images of the labellum margin of Ophrys sphegodes (C) selected from within confocal image stacks of reconstructed nuclei contrast nuclei that are diploid at 2C (A left, B), endoreplicate non-polytene at an estimated 8C (A right) (both possessing single, proportionately sized nucleoli) and endoreplicate polytene at an estimated 16C (D, E). (A right) This nucleus is assumed to be endocyclic whereas the remainder of the illustrated endoreplicate nuclei are assumed to be endomitotic. Vertical dimensions of nuclei: (A) left = 21.7 μm, right = 36.6 μm; (B) = 19 μm, (D) = 38 μm, (E) = 44 μm; (C) vertical dimension of image = 25 mm. Fig. 10. View largeDownload slide FISH confocal images of a fragmented labellum of Ophrys sphegodes, distinguishing diploid (A) from endoreplicate (B, C) nuclei (PI, red fluorescence; FISH, green fluorescence). The diploid nucleus (A) has a single nucleolus containing diffuse 18S rDNA chromatin (FITC, yellow fluorescence) at various intensities, suggesting differential condensation. The most diffuse chromatin is assumed to be the most transcriptionally active. The endoreplicate nuclei (C) are larger, and have a larger nucleolus, with more highly intense 18S rDNA signals, revealing greatly amplified copy numbers of rDNA units resulting from endoreplication. The higher-magnification image (C) shows most clearly 18S rDNA captured at three contrasting condensation states. In (B) and (C), condensed rDNA units surround the nucleolus (red arrow) and envelop diffuse, decondensed (transcriptionally active) rDNA within. Concentrations of rDNA occurring elsewhere in the nucleus (e.g. green arrow) may be inactive and extranucleolar. Scale bars: (A–B) = 20 μm; (C) = 10 μm. Fig. 10. View largeDownload slide FISH confocal images of a fragmented labellum of Ophrys sphegodes, distinguishing diploid (A) from endoreplicate (B, C) nuclei (PI, red fluorescence; FISH, green fluorescence). The diploid nucleus (A) has a single nucleolus containing diffuse 18S rDNA chromatin (FITC, yellow fluorescence) at various intensities, suggesting differential condensation. The most diffuse chromatin is assumed to be the most transcriptionally active. The endoreplicate nuclei (C) are larger, and have a larger nucleolus, with more highly intense 18S rDNA signals, revealing greatly amplified copy numbers of rDNA units resulting from endoreplication. The higher-magnification image (C) shows most clearly 18S rDNA captured at three contrasting condensation states. In (B) and (C), condensed rDNA units surround the nucleolus (red arrow) and envelop diffuse, decondensed (transcriptionally active) rDNA within. Concentrations of rDNA occurring elsewhere in the nucleus (e.g. green arrow) may be inactive and extranucleolar. Scale bars: (A–B) = 20 μm; (C) = 10 μm. Moreover, structural differences are evident between the different categories of nucleus. Diploid nuclei clearly have chromatin concentrated around the periphery (Fig. 10B), whereas the endoreplicate polytene nuclei (Fig. 9D, E) have a much more complex micromorphology that features interchromosomal domains; frayed telomeric regions form a terminal ‘fan’ of chromatids (Fig. 9E, green arrow). There exists a long history of the study of plant polytene chromosomes (Tschermak-Woess, 1956; Barlow, 1974; Gostev and Asker, 1978; Carvalhiera, 2000), but previous reports have been sporadic, have represented few plant families, and have been confined to tissues intimately associated with ovules (Maluszynska et al., 2013). When applied to O. sphegodes, FISH not only readily distinguished diploid from endoreplicate nuclei, but also clearly demonstrated the presence of a single nucleolus around which rDNA transcription was concentrated (Fig. 10). It imaged 18S rDNA at three contrasting condensation states. Condensed rDNA units surrounded the nucleolus, enveloping diffuse, decondensed (i.e. transcriptionally active) rDNA within (red arrows in Fig. 10B, C). By contrast, the rDNA located elsewhere in the nucleus was probably inactive (green arrow in Fig. 10), an overall situation commonly found in plants (Leitch et al., 1992). There was no indication that rDNA was under-replicated and might therefore be a component of the DNA regions that fail to replicate during PPE; it is more likely that PPE reflects failure to replicate some non-functional regions of the nuclear genome. These results make clear that rDNA number has substantially expanded and most likely been completely amplified during endoreplication – an essential prerequisite for mRNA translation to proteins and indicative of higher overall metabolic activity in the endocycled cells. The clear presence of a single nucleolus containing labelled rDNA in both diploid and endoreplicated nuclei suggests that the chromosomes carrying rDNA remain in close proximity, even when clear signs of polyteny are absent. Placing our observations in a broader orchidological context Previous studies of endoreplication in orchids have primarily examined tropical taxa using flow cytometry, with decidedly mixed results. All investigations found evidence of endoreplication in their case-study species, though they differed in which organs of the plant showed the most frequent endoreplication and in what maximum C-value those endoreplicated nuclei could attain (cf. Mishiba et al., 2001; Barow and Meister, 2003; Lim and Loh, 2003; Yang and Loh, 2004; Barow and Jovtchev, 2007; Chen et al., 2011; Teixeira et al., 2014; Travnicek et al., 2015). Collectively, these studies implicate most of the organs that constitute a typical orchid plant: root hair, root, stem, leaf, pedicel, ovary, gynostemium and tepals (i.e. labellum, lateral petals, sepals), though most authors reported that at least some of the organs of their study species appeared to lack endoreplication. The most commonly reported exceptions are the two organs/part-organs that provide the sites of meiosis: ovary (reliably 2C) and pollinium (reliably 1C) (Chen et al., 2011; Travnicek et al., 2015). Reports typically limit observed endoreplication in any particular orchid organ to two or three rounds of duplication, that is, showing a minority of cells attaining either 8C or 16C (though rare 32C nuclei were reported in the intergeneric hybrid ×Doritaenopsis by Mishiba et al., 2001). These results fall well short of the estimated 256C once recorded in the pericarp of Solanum lycopersicum fruits (Yang and Loh, 2004), but are consistent with previous observations on members of several other flowering plant families, such as Fabaceae (Kocova et al., 2016), Brassicaceae, Caryophyllaceae (Agullo-Antón et al., 2013), Cucurbitaceae and Aizoaceae (Travnicek et al., 2015). The most ambitious flow cytometry study on orchids published thus far was conducted by Travnicek et al. (2015), who compared roots, leaves (base and apex), tepals (not differentiated into individual organs, but usually focusing on sepals), ovaries and pollinaria in 48 orchid species: 36 from subfamily Epidendroideae and six from subfamily Orchidoideae (none of them European), plus smaller numbers from the remaining three subfamilies. Clade (i.e. subfamily) membership appeared to be a strong predictor of endoreplicatory behaviour, as it was absent from all tested members of Apostasioideae and Cypripedioideae and from half of the tested members of Epidendroideae. Where endoreplication was observed, it took two forms: ‘classically’ complete (i.e. basic nucleus size doubled) or PPE (basic nucleus size less than doubled; Bory et al., 2008; Trávníček et al., 2012) – a phenomenon thus far observed within the plant kingdom only in a small minority of orchids and one that is presently poorly understood (Travnicek et al., 2015; Hribova et al., 2016). Remarkably, no orchid was observed to exhibit both forms of endoreplication, suggesting plant-wide control of this potentially important mechanistic divergence. According to Travnicek et al. (2015), all tested members of subfamilies Vanilloideae and Orchidoideae showed only partial endoreplication, along with one-sixth of species sampled in subfamily Epidendroideae (mainly tribe Pleurothallideae). The remaining one-third of Epidendroideae reportedly showed no endoreplication of any kind, a conclusion that contrasts with those of all previously published studies of orchids. Lastly, these authors argued that in those orchids that show PPE, tepals can entirely lack G1-phase (i.e. 2C) nuclei. All six Orchidoideae genera examined by Travnicek et al. (2015) showed PPE, though these authors (1) failed to target labella when examining tepals and (2) analysed only non-European species placed phylogenetically outside the limits of subtribe Orchidinae, whereas we analysed only European species confined to subtribe Orchidinae. We can now confirm that, as expected, PPE extends to subtribe Orchidinae in the form of the genus Ophrys, and affects all regions of the labellum (Fig. 6). It appears increasingly likely that the ability to undergo the unusual phenomenon of PPE is clade-specific and hence is phylogenetically constrained. Functional and evolutionary implications of endoreplication Recent insights obtained through genomics have reduced the formerly predominant evolutionary focus on nuclear protein-coding genes, reflecting increasing recognition that potentially all eukaryotes conservatively maintain 15–30 k functional protein-coding genes irrespective of their phenotypic complexity (e.g. Liu et al., 2013). Orchids are no exception – the genome sequence of the classic model orchid Phalaenopsis equestris projected a total of ~29 k genes (Cai et al., 2015) and that of the earliest-divergent orchid Apostasia shenzhenica predicted ~22 k genes (Zhang et al., 2017). Instead, phenotypic complexity shows a far better positive correlation with the amount of non-protein DNA responsible for RNA transcripts and other regulatory elements (Liu et al., 2013). This observation implies a greater evolutionary role for other factors (both endogenous and exogenous) that influence the detailed progress of organismal development – a cycle repeated through time ad nauseam in the relevant phylogenetic lineage. Endogenously, it is likely that mediation of complex epidermal differentiation in plants such as Ophrys is achieved at least partly through influencing production of a minimum of three ‘molecular patterning modules’: (1) the MYB-bHLH-WD40 protein complex; (2) the transmembrane calpain protease DEFECTIVE KERNEL1 (DEK1); and (3) homeodomain leucine zipper (HD-ZIP) class IV transcription factors acting in concert with SIAMESE-related, cyclin-dependent kinase inhibitors. This combination of factors has been shown to be critical to epidermal patterning in several model angiosperms (reviewed by Robinson and Roeder, 2015). Numerous quantitative trait loci have proved capable of modulating successive endocycles to increase resistance to both biotic and abiotic stresses. For example, endoreplication is enhanced by exposure to increased levels of UV-B (Gegas et al., 2014), increased growth temperature (Lee et al., 2007) or more intense herbivory (Scholes and Paige, 2014), in turn enhancing growth rate and yield in crop plants (Breuer et al., 2014). However, the crucial extent to which endoreplication is pre-programmed into ontogeny rather than induced by environmental change remains controversial (Yang and Loh, 2004). The habitat preferences of the orchids in question may be of relevance, given that most tropical orchids are epiphytes and most temperate orchids, though terrestrial geophytes, occupy low-competition soils. In both cases, nutrients and often also water are limited resources, a situation that might confer economic advantage on plants that can increase nuclear size (and thus nuclear products via gene upregulation) without being obliged to expend energy duplicating the remaining components of the cell. Unsurprisingly, the bulk of research into endoreplication in plants has been conducted on that most academically ubiquitous of species, Arabidopsis thaliana (Galbraith et al., 1991, et seq.). Greater frequency and/or number of cycles of endopolyploidy have been shown to increase organ size, not only in Arabidopsis but also in the model epidendroid orchid Phalaenopsis (Ho et al., 2016). Indeed, phenotypic expression of endoreplication is not confined to mere size differences; for example, the complexity of each unicellular but multiply branched trichome that adorns an Arabidopsis leaf is positively correlated with, and potentially dictated by, the number of endoreplications undergone by its nucleus (Folkers et al., 1997; Traas et al., 1998). The range of nuclear sizes found in the hypocotyl and epidermal pavement cells of Arabidopsis leaves precisely matched that observed by us in bee orchid labella (2C–16C), though Arabidopsis trichomes can exceptionally reach 64C (Traas et al., 1998). In contrast, no correlation was observed between degree of endoreplication and length of collet (hypocotyl) root hairs in a later study of six mutant strains of Arabidopsis (Sliwinska et al., 2015), weakening any attempt on our part to draw general conclusions. Traas et al. (1998) argued that, at least in the case of Arabidopsis pavement cells and trichomes, endoreplication represents a response to both internal and external stimuli. More startlingly, they also claimed that successive endoreplication events occurring within the same cell lineage can consistently reflect different combinations of factors. If both of these statements prove upon further evidence to be valid, endoreplication has the potential to constitute a family of complex processes and influences that together are capable of exceptionally rapid and fine-tooled responses during organismal development. There may even be a requirement for a degree of environmental predictivity by the affected cell, as current evidence suggests that endocycles determine the final size and shape of the affected cell but cease early in cell development, before the large central vacuole has formed and certainly before the bulk of cell expansion has occurred. Thus, on the basis of their observations on the model epidendroid orchids Phalaenopsis and Oncidium, Ho et al. (2016; see also Lee et al., 2004) argued that once endoreplication in an orchid flower ceases, so does its ability to further expand through cell division. Traas et al. (1998, p. 500) therefore warned that the correlation between nucleus size and cell size/complexity would most likely be clearest in those plant cell types that are the least physically constrained, such as trichomes and root hairs, noting that ‘in expanding tissues, growth of neighbouring cells must be tightly coordinated in order to avoid local distortions of tissues’. Similar observations were made by Guan et al. (2014) on the trichome-homologue cotton fibres of Gossypium. We speculate that such ‘local distortions of cells’, driven by an appropriate number of endoreplication cycles, would form a ready explanation for the unusual degree of three- dimensionality that characterizes most orchid labella, including the deep invagination that forms the labellar spur in orchids such as Dactylorhiza (Box et al., 2008; Bell et al., 2009) and the overall convexity plus horn-like and/or breast-like ‘evaginations’ that characterize the labellum of most Ophrys species (Bradshaw et al., 2010). Our previous observations suggest that both sets of features emerge late in flower development. We introduced this paper by outlining the multiple adaptations present in Ophrys that are assumed to be required for efficient pseudocopulatory pollination. The labellum first secretes a cocktail of pseudopheromones as an olfactory cue, then offers a complex mosaic of brightly coloured and reflective regions as visual cues, and finally further stimulates the now alighted insect with strategically placed and contrasting patches of trichomes – tactile cues that differ in size, shape and/or orientation and therefore supposedly mimic the conspecific female insect. It seems reasonable to assume that the number of endoreplications undergone by nuclei in the labellum influences the quantities generated of the many biochemicals that they are competent to produce, even if the relationship between genome size and gene products is not precisely arithmetic. If so, it becomes feasible to hypothesize that the epidermal gradation evident in the Ophrys labellum from flat pavement cells through domed and papillate cells to short straight-sided trichomes and longer spiral trichomes could also reflect concentration-dependent biochemical interactions in different epidermal regions. In other words, differential endoreplication could, through dosage effects, dictate differentiation of the panoply of subtly variable visual and tactile cues offered by the bee orchid labellum. In particular, overexpression induced by highly localized endoreplication could substantially shift the concentrations (both relative and absolute) of the biochemicals present in the cocktail of pseudopheromones emitted by Ophrys flowers – changes that are likely to have at least some impact on the spectrum of pollinating insects attracted to the flowers (cf. Gögler et al., 2009; Breitkopf et al., 2013; Sedeek et al., 2014, 2016). Admittedly, demonstrating such an effect would not necessarily resolve current impassioned debates regarding the nature of species and speciation within the genus; opinions already expressed regarding the downstream consequences of such pollinator shifts have ranged from greatly enhanced speciation rate driven by novel pollinator specificities (e.g. Vereecken et al., 2011; Paulus, 2015) to prevention of speciation through enhanced gene flow (e.g. Bateman et al., 2011; Hennecke et al., 2015). Superimposed on continuing uncertainty regarding the nature and significance of endoreplication per se is our almost complete ignorance regarding the nature of progressively partial endoreplication (Fig. 6). Does this phenomenon occur beyond the bounds of the orchid family? Which modestly sized portions of the orchid genome fail to copy during the genome replication process, and why? Hribova et al. (2016, p. 2003) concluded that ‘the mechanism behind PPE is the incomplete replication of nuclear DNA. Together with the precise control of the extent of DNA under-replication, our results indicate that PPE is a highly controlled process accompanying cell and tissue differentiation’. They then ‘hypothesize[d] that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development’ (p. 1996). The results obtained from Ophrys labella during the present study are consistent with, though by no means conclusively demonstrate, the views recently expressed by Hribova et al. Perhaps the most interesting of the many as yet unanswered questions is whether endoreplication does indeed offer selective advantages or is merely the happenstance product of relaxation in developmental control – relaxation occurring towards the end of the ontogenetic trajectories that add the final details to determinate, disposable organs such as petals. But in practice the converse argument may apply. The ability to determine the approximate levels of endoreplication occurring in different specified regions of a single tissue (in this case the epidermis) in a single organ (in this case the labellar petal) could be taken as indicating remarkably subtle and sophisticated (epi)genetic control of development – one that permits the considerable phenotypic plasticity that is evident in so many bona fide orchid species. CONCLUSIONS (1) Endoreplicated nuclei were observed in all five species of Ophrys examined by us, involving both endomitosis and endocycling. In contrast, endoreplicated nuclei were less evident in the corresponding three species of Dactylorhiza. Thus, no link has been demonstrated here between the stronger predisposition to allopolyploid speciation evident in Dactylorhiza and the stronger predisposition to localized endopolyploidy here demonstrated in Ophrys. (2) Endoreplication in the labella of Ophrys species appears to be more strongly positively correlated with cell size/complexity (it especially characterizes trichomes) than with secretory role or marginal location (less evident in the appendix, which is both marginal to the labellum and highly physiologically active, yet yielded nucleus size distributions more consistent with the corresponding leaves). However, this provisional conclusion should be tested further, using even smaller microdissected tissue samples that reliably encompass only a single cell type. (3) Both fluorescence microscopy and flow cytometry revealed three size categories of endoreplicated nuclei in both O. sphegodes and O. tenthredinifera villosa, the polytene nuclei reaching 16C in size (i.e. maximally having undergone three rounds of endoreplication). (4) Progressively partial endoreplication may occur throughout tribe Orchideae within the orchid family, indicating strong phylogenetic control of the underlying mechanism. It is now essential to determine which portion(s) of the orchid genome are escaping replication during PPE events. (5) The possibility therefore exists for epigenetic control of gene overexpression via local induction of endoreplication in particular tissues. If so, endoreplication should be viewed as an important element in the epigenetic palette available to a plant, and a possible explanation of the plastic responses that are being observed with increasing frequency in plants. The evolutionary-developmental significance of endoreplication may thus far have been massively underestimated by the biological community. (6) Combining flow cytometry with confocal microscopy represents a powerful approach to determining the nature and scale of endoreplication within organisms. Direct visualization through confocal microscopy allows observations to be focused precisely within target tissues and to include the nanomorphology of the nucleus, whereas indirect measurement via flow cytometry provides fully quantitative size distributions. In future studies, we would aim to microdissect and then bulk up the organs of interest so that the flow cytometry results can be more rigorously confined to the target tissue. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob. They consist of text relating to measurement of nuclear size, comparing flow cytometry with direct microscopic observation. The text incorporates a table (Table S1) of observed and estimated mean nuclear peaks in the confocal size distributions of nuclei located in the labella of the diploid D. fuchsii and the allotetraploid D. praetermissa analysed in parallel with the diploid D. foliosa. It also incorporates a figure (Fig. S1) showing fluorescence histograms of leaf material in diploid (A, B) and tetraploid (C, D) cytotypes of O. tenthredinifera villosa. ACKNOWLEDGEMENTS We thank Michaela Egertova for guidance on the use of the confocal microscope, Heike Brinkman for additional technical support, and Thomas Cremer and Rachel Walker for sharing insights from their own related confocal research. We are grateful to Catalina Romila for measuring the nuclei of Dactylorhiza fuchsii and D. praetermissa, and Beth Bradshaw for capturing some of the scanning electron micrographs inset into Fig. 2. Barry Tattersall kindly provided material for flow cytometry. 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Annals of Botany – Oxford University Press
Published: Apr 17, 2018
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