Orbicules in flowering plants: a phylogenetic perspective on their form and function.
Orbicules are readily observable by scanning electron microscopy (SEM) in mature anthers as a layer of tiny particles lining the inner locule wall, in close contact with the pollen grains (El-Ghazaly, 1999; Huysmans et al., 2000; Galati, 2003). Orbicules (syn. Ubisch bodies, (1) con-peito grains) are a-cellular structures of sporopollenin that might occur on the inner tangential and radial walls of tapetal cells. Usually they are smaller than 1 [micro]m, but orbicules with a diameter up to 15 [micro]m are reported in Quararibea (Malvaceae; Nilsson & Robyns, 1974). They originate as lipid droplets (pro-orbicules) within the cytoplasm of tapetal cells, most likely from the rough endoplasmic reticulum (ER; Echlin & Godwin, 1968; Risueno et al., 1969; El-Ghazaly & Jensen, 1986). After exocytosis the pro-orbicules nest on the tapetal plasmalemma and get a sporopollenin coat synchronously with the developing pollen exine (Christensen et al., 1972). The orbicule surface ornamentation often resembles that of the pollen sexine (Rowley et al., 1959; Hesse, 1986; Huysmans et al., 2000), touching the prime challenge in palynology, viz. the source of control over the specific ornamentation of the pollen exine. This resemblance in ornamentation indicates that a similar patterned biosynthesis of sporopollenin is possible on an a-cellular sporophytic structure (pro-orbicule) as on a cellular gametophytic structure (microspore).
The existence of orbicules is known since 1865 when Rosanoff published his observations on anthers of Fabaceae species where he noticed small granules on the inner locule wall that were resistant to concentrated sulphuric acid (Rosanoff, 1865). Both von Ubisch (1927) and von Kosmath (1927) have published lists of species with and without orbicules and considered orbicules to be restricted to taxa with a 'secretory' tapetum type. Ubisch and Kosmath are considered as pioneers in orbicule research, but they were most likely inspired by preceding papers by Chatin (1870; angiosperms), Mascre (1922; Boraginaceae), Schnarf (1923; Lilium), and Krjatchenko (1925; Lilium). The latter suggested an intratapetal origin for orbicules, possibly in the mitochondria.
Since the early days of orbicule-research, a positive correlation was hypothesised between the presence of orbicules and a parietal tapetum type (von Ubisch, 1927; von Kosmath, 1927), although several species were identified with parietal tapetal cells but lacking orbicules (Huysmans et al., 1998). For a long time only one exception to this hypothesis was known, viz. Gentiana acaulis that has anthers with orbicules and an amoeboid tapetum type (Lombardo & Carraro, 1976). Parietal tapeta are the dominant type in land plants and occur in the extant 'basal' angiosperm groups and in most fossil taxa. It is considered as the plesiomorphic condition in angiosperms (Furness & Rudall, 2001a). Parietal tapetum cells keep their individuality and position lining the locules throughout their entire life cycle. In literature, this tapetum type is also referred to as 'secretory' or 'glandular'. In amoeboid tapetal cells, on the contrary, cell walls degenerate prior to fusion of the protoplasts into a plasmodium that invades the locule and assures close contact with the developing microspores. This tapetum type is also known as 'plasmodial'. Next to these two main tapetum types, a few intermediates are described illustrating the complex metabolism of this ephemeral tissue (Pacini et al., 1985). In an invasive tapetum type for instance the cell walls degenerate and the individual protoplasts, without formation of a plasmodium, intrude the locules often in a cyclic pattern during microspore development. The latter type is recorded in a few families throughout angiosperm phylogeny (e.g. Furness & Rudall, 1998, 2001a; Furness, 2008a).
Since mature orbicules consist of sporopollenin they are well represented in the fossil record. One of the few papers devoted to fossil orbicules and tapetal membranes is the ultrastructural work on fossil Pennsylvanian (Carboniferous, 286-320 mya) pollen grains of the Schopfipollenites-type (Taylor, 1976). A more recent study on Craigia (Malvaceae, Miocene) focused on orbicules and pollenkitt (Zetter et al., 2002). Most data on orbicules in the fossil record are from Cretaceous flowering plants, e.g. the excellent preserved flowers of Teixeiraea lusitanica (Ranunculales affinities) with abundant doughnut-shaped orbicules (von Balthazar et al, 2005).
Only a decade ago, it was demonstrated that orbicules occur in all higher order taxonomic units of the angiosperms: from the most early diverged groups (ANITA grade) up to and including the most derived clades of the core-eudicots (see review by Huysmans et al., 1998, updated in Huysmans et al., 2000).
To date our knowledge is insufficient to provide plausible answers to why many plants produce orbicules and why they are absent in several evolutionary successful lineages, even when they are characterized by a parietal tapetum type (e.g. entire family Orchidaceae with >22.000 sp.). Many different functions were hypothesized to explain the occurrence of orbicules (for a review see Huysmans et al., 1998), but none of these is yet satisfactorily proven by experiments or available data. The question whether orbicules have an active function in the anther locules (e.g. in pollen release) or merely represent a by-product of the tapetum, as a reminiscence of the phylogenetically shared origin of tapetum and microsporophytic tissue, remains open.
There is a growing body of literature on the possible allergenic properties of orbicules (e.g. Vinckier & Smets, 2001a, b). Several immunocytological studies provided evidence of localisation of allergens on the orbicular wall (Suarez-Cervera et al., 2003; Canini et al., 2004; Jato et al., 2010), but negative results were reported for birch (Schappi et al., 1997; Vinckier et al., 2006). However, the question whether orbicules are dispersed stogether with the pollen grains remains open. Dinis et al. (2007) provided evidence for two grass species showing that the orbicule density of both dehisced and undehisced anthers did not differ significantly.
The study of orbicules could be particularly rewarding since they offer a window on several biological issues on the borderline between gametophyte and sporophyte, such as the ratio of sporophytic and gametophytic genetic control in the development of the sporoderm, the function and chemical composition of lipidic fractions of tapetal origin in flowering plants (Piffanelli et al., 1998), the patterned sporopollenin polymerisation in the anther mediated by 'white lines' and a glycocalyx, and the possible contribution of in vivo self-assembly of sporopollenin in the development of the sporoderm (Gabarayeva & Hemsley, 2006).
Kress (1986: 342) stated that <<the original or true function of any character may only be apparent or correctly interpreted in light of its phylogenetic history>>. In this line of thought we updated the distribution data of orbicules in flowering plants by thoroughly screening the literature since the first review on the topic (Huysmans et al., 1998). Complementary original observations from certain model plants and selected families were added. The current study aims at (1) providing a summary of all data available on orbicule presence/absence in the flowering plants; (2) identifying patterns in the distribution data by mapping them on a recent angiosperm classification; (3) discussing correlations with tapetum types, pollination syndromes and other traits. (4) Finally, the potential of orbicules as a model system for in vivo research on patterned sporopollenin polymerization (including genetic control by gametophyte/sporophyte and self-assembly processes) is demonstrated.
Materials and Methods
In general, mature orbicules are readily visible on the tapetal membrane (remnants of tapetal cells after programmed cell death) and are chemically inert to any preparation method because of their sporopollenin composition. Useful source material therefore also includes herbarium specimens and pickled flowers. Light microscopy is, due to their small size range, less appropriate for orbicule observations (but see Bhandari & Kishori, 1971). Field emission scanning electron microscopy (FE-SEM) is desirable because it provides high-resolution images with negligible electrical charging of the samples at low accelerating voltage.
Dried flowers or anthers were rehydrated for at least 1 h in a wetting agent such as Agepon or Photo Flo (1:200 in distilled water) prior to dehydration by a graded ethanol series (50%-70%-95%-100%), preferably inside the CPD container(s) to avoid distortion. Pickled flowers were dehydrated completely by continuation of the graded ethanol series from concentration of stock fluid. Living flowers were fixed overnight in FAA (90 ml ethanol 50%+5 ml acetic acid glacial+5 ml formaldehyde) prior to dehydration. Critical point drying (CPD) involves two washes in 100% acetone prior to CPD in acetone as intermediary fluid.
Dried anthers were fixed to aluminium stubs with double adhesive carbon tape. If necessary excess pollen grains were gently removed using a cactus spine to clear part of the locule wall. Stubs were sputter coated with platinum (FE-SEM) or gold (SEM). Orbicules were observed using a FE-SEM (Leo supra 55 VP, Zeiss, Jena, Germany) or a SEM (JEOL JSM 6360, Jeol Ltd, Tokyo, Japan) at an accelerating voltage of 5 kV and a working distance of 10 mm.
We have thoroughly screened the scientific literature from January 1997 until December 2012 aiming to provide a state of the art of the distribution of orbicules in angiosperms. Additional papers were found by screening reference lists in consulted works and the palynology reprint collection of the Laboratory of Plant Systematics (KU Leuven, Belgium). Unpublished validated data from lab members and associated collaborators were added as well. Although we aimed to be exhaustive, the resulting dataset might be incomplete because orbicule distribution data has been published in very diverse research fields. Since negative observations require awareness and directed attention, those are most likely underrepresented in many groups.
The data are presented in a phylogenetic order (Appendix S1) following the angiosperm classification by Stevens (2001 onwards). The main arguments for this choice are (1) the accessibility to the entire scientific community by being a free online resource, (2) the fast inclusion of the most recent evolutionary insights in a continuously updated classification, and (3) the presence of genus lists for each family that allows reproducible genus allocation to family level.
For phylogenetic mapping of orbicule distribution data, we used both the APG III topology at order level (APG, 2009) and an updated dahlgrenogram reflecting the relationships between orders in flowering plants (Barthlott, Borsch & Worberg, pers. com., updated by Worberg). The bubble diagram represents a virtual cross section of 'the tree of life' of angiosperms. Distances between bubbles reflect 'evolutionary distances' between orders or families; bubble size is in relative proportion to species number. A dahlgrenogram does not depict phytogeny in the sense of a branching evolutionary history from inferred hypothesized ancestors. But in the tradition of Rolf Dahlgren (1932-1987), we consider bubble diagrams as powerful tools to depict the distribution of any character in extensive taxa such as angiosperms on a single A4 sheet. Mapping data on both representations was done manually, based on the data in Appendix S1.
Morphology and Localisation of Orbicules
When orbicules are present, they are usually abundant and cover the tapetal remnants on the inner locule surface (Fig. 1a, f). In angiosperms they are seldomly observed adhering to the pollen grains. Size, shape and ornamentation of the orbicular wall may vary between species, e.g. irregular orbicules with a central perforation (Fig. 1h) vs. spherical microechinate orbicules (Fig. 1i). Often the ornamentation of the pollen sexine is similar to that of the orbicules (compare Fig. 1b-c, d-e). Echinate pollen in particular is often reflected in spiny orbicules (Fig. 1i). Orbicuies may be more or less embedded in the tapetal remnants (Fig. 1g). When orbicules are absent, the locule wall is smooth and endothecium thickenings may be pronounced (Fig. 1j).
Distribution of Orbicuies in Angiosperms
Appendix S1 summarizes the orbicule data at species level with orbicule absence/ presence and, if available, the occuring tapetum type. The respective references are added. The data from Fluysmans et al. (1998, 2000), covering the period 1865-1996, are included as well, in order to deliver a review as exhaustive as possible. For these entries only cross-reference to Fluysmans et al. (1998) or (2000) is provided.
In Table 1 orbicule distribution data are given at family level for each order recognized the in angiosperms sensu APG in (2009). Figure 2 presents a color-coded cladogram at order level. The resulting pattern indicates that orbicules are indeed present all over the topology. Of all orders that have been investigated for orbicules, only five remain without any positive observations: Asterales, Cannellales, Commelinales, Vitales, and Zingiberales. In total 17 orders (Acorales, Arecales, Buxales, Celastrales, Ceratophyllales, Crossosomatales, Dilleniales, Escalloniales, Garryales, Gunnerales, Huerteales, Paracryphiales, Petrosaviales, Picramniales, Santalales, Trochodendrales, Zygophyllales) remain blank spots on the angiosperm orbicule-map because no data on orbicules is available (Fig. 2). In the present study we were able to increase the angiosperm dataset for orbicule presence/ absence from 88 to 149 families, an increase of 15 to 36% of the total number of families since Fluysmans et al. (1998); (Fig. 3).
All large (informal) groups in angiosperms such as magnoliids, monocots, basal eudicots, rosids and asterids show a patchy image with both positive and negative observations. At family level, however, less variation is encountered: only 24 families (16% of all families studied) show both presence and absence of orbicules between their representatives, viz. Annonaceae, Apocynaceae, Aquifoliaceae, Berberidaceae, Cactaceae, Convolvulaceae, Dioscoreaceae, Euphorbiaceae, Fabaceae, Gentianaceae, Lamiaceae, Linaceae, Loasaceae, Malvaceae, Melianthaceae, Monimiaceae, Nartheciaceae, Oleaceae, Passifloraceae, Plantaginaceae, Polygonaceae, Rubiaceae, Salicaceae and Tetrameristaceae (Table 1). Within these groups, orbicule distribution data generally are consistent at generic level. Only 8 genera deviate from this pattern: Aletris (Nartheciaceae), Coptosapelta (Rubiaceae), Dioscorea (Dioscoreaceae), Gentiana (Gentianaceae), Ilex (Aquifoliaceae), Ipomoea (Convolvulaceae), Monodora (Annonaceae), and Passiflora (Passifloraceae).
In order to visualise the phylogenetic signal inherent in the distribution data at order level compiled in Table 1, we used the bubble diagram by Barthlott, Borsch and Worberg (pers. com., updated by Worberg, Fig. 4). The total absence of orbicules in Orchidaceae (here depicted separately from Asparagales) is conspicuous. However, (negative) data are extremely rare in this family and therefore orchids represent a target group for further studies.
Orbicules: Valuable New Systematic Character?
Orbicule production by tapetal cells in a secretory metabolic phase can be interpreted as a primitive, possibly neotenic feature in flowering plants since orbicules occur also in bryophytes, pteridophytes and gymnosperms. However, data in these groups are highly fragmented. Orbicules are omnipresent in Gnetales: Ephedra (El-Ghazaly & Rowley, 1997; Doores et al., 2007), Gnetum (Camiel, 1966), and Welwitschia (Zavada & Gabarayeva, 1991). Cryptomeria japonica, a coniferous species in Taxodiaceae and a major cause of pollinosis in Japan, produces orbicules (e.g. Hosoo et al., 2005). Audran (1981) described orbicule development in Ceratozamia (Cycadaceae) and Rowley and Walles (1987) in Pinus. Blackmore et al. (2000) reported orbicules in 13 genera of pteridophytes, but see Alarid et al. (2005) for conflicting data on Isoetes. The majority of pteridophytes are shown to have an amoeboid tapetum (Parkinson & Pacini, 1995), however, many homosporous ferns have globular bodies in their sporangial locules that were considered to be homologous to orbicules in spermatophytes (Lugardon, 1981). In biyophytes and lycopods, on the contrary, a parietal tapetum is omnipresent (Pacini et al., 1985) and similar structures to orbicules are recorded, but their homology remains unresolved due to lack of data.
As such the overall systematic value of orbicules in flowering plants is restricted for not being an apomorphic feature. However, mere orbicule presence/absence data reveal an interesting pattern in angiosperms (Appendix SI, Fig. 2). In total 149 out of 416 families (36%) are represented in Appendix SI with orbicule data for at least one species. Both orbicule presence and absence is recorded from the earliest diverging flowering plants up to most recent diversified asterid clades. All large (informal) groups in angiosperms such as magnoliids, monocots, basal eudicots, rosids and asterids show a patchy image with both positive and negative observations. At family level, however, less variation is encountered: only 24 families (16% of families studied) show both absence and presence of orbicules between their representatives (see Results). The majority of families is thus surprisingly constant for orbicule data, providing a predictive value that surely has potential for systematically oriented research questions. In Brassicaceae, for instance, orbicules were never recorded; also in Arabidopsis thaliana they are absent, despite its parietal tapetum (Murgia et al., 1991). However, Staiger et al. (1994), in their immunocytochemical study on Sinapis alba, provided TEM pictures of <<globules resembling pro-Ubisch bodies which appeared at tetrad stage>> labelled with two tapetum specific proteins. According to the authors, the pro-orbicules contain sporopollenin precursors but receive no polymerized sporopollenin wall later in development (Staiger et al., 1994). Lamiaceae is a well-studied family generally lacking orbicules on the inner locule wall. All positive observations in Appendix SI represent former Chloanthaceae species (Ray & El-Ghazaly, 1987), a family that was merged with Verbenaceae of which many taxa were recently accomodated in Lamiaceae (Harley et al., 2004). On a lower level of classification, we see that orbicule distribution data are generally consistent at generic level. The observation of both presence and absence in a single genus is very rare and is only found in 8 genera (see Results). In Rubiaceae for example, the most thoroughly studied angiosperm family for orbicules, intrageneric variation was observed and described in only one (Coptosapelta) out of 163 genera investigated (Verellen et al., 2004; Verstraete et al., 2011). A discussion on this variation can be found in Huysmans et al. (2010) for Monodora (Annonaceae) and in Verstraete (2009) for the other genera. These deviant genera are interesting groups for further study into orbicule distribution and tapetal characteristics.
Recent observations in Annonaceae showed that orbicules are much more common in the family than previously perceived (Huysmans et al., 2010). Moreover, an unequivocal phylogenetic pattern appeared by plotting the available orbicule distribution data on the most recent family phylogeny. In Anaxagorea, basal clade and sister to all other Annonaceae, Ambavioideae and the 'short branch clade', orbicules were recorded. In the most derived Tong branch clade' orbicules are consistently absent (Monodora crispata being the single exception). A recent study in Rubiaceae that investigated the phylogenetic signal of orbicules also demonstrated an evolutionary trend towards the absence of orbicules (Verstraete et al, 2011). The same trend emerges at angiosperm level based on our collective data: orbicules are common in the ANITA-grade and 85% of the monocots studied produce orbicules, with Orchidaceae, Commelinales and Zingiberales as notable exceptions. Within eudicots the asterids are most densely sampled with 61% orbicule presence. Asteraceae and the majority of Lamiaceae lack orbicules. These observations indicate firstly that orbicules are found all over the topology of flowering plants and, secondly, that an evolutionary trend exists towards orbicule absence in more derived clades.
Orbicules come in a wide array of different sizes, shapes and densities (Fig. 1). Do any of these features provide additional potential systematic characters? Orbicule characters are systematically analysed in several taxa of Gentianales such as Gentianaceae (Vinckier & Smets, 2003), Apocynaceae s.l. (Vinckier & Smets, 2002c), Loganiaceae s.l. (Vinckier & Smets, 2002a), and Rubiaceae (Huysmans et al., 1997; Vinckier et al., 2000; Verstraete et al., 2011). Overall conclusion for Gentianales was that orbicules are a common feature and that morphological characteristics might be useful at tribal level (Vinckier & Smets, 2002b).
The key issue here is the character stability at species level. Very few studies have paid attention to the intraspecific variability of orbicule characters; mostly only a single specimen per species is investigated. Verellen et al. (2004) observed five specimens of Coptosapelta tomentosa (Rubiaceae) and found three different orbicule types in different specimens and one specimen without orbicules. Orbicules are absent in most Lamiaceae and for some species such as Heterolamium debile, Melissa officinalis, Prunella vulgaris, Stachydeoma graveolens and Thymbra spicata, this negative observation was confirmed in more than two specimens (Moon et al., 2008b). A detailed developmental study at ultrastructural level of Anaxagorea brevipes (Annonaceae) showed that the parietal tapetum reluctantly invades the locule at the early tetrad stage and secretes both orbicules and other globular lipoidal concretions (Gabarayeva, 1995). The variety of size and chemical composition of these tapetal cytoplasmic globular inclusions reflects the variety of the tapetal functions according to Gabarayeva (1995).
Correlation Orbicules-Tapetum Type
It is impossible to separate orbicules and the tissue where they originate if it comes to explain their form and function. At present, identification keys are available to distinguish between tapetum types (e.g. Pacini, 1997). The various types, however, can be reduced to two basic types that were already recognised by Goebel (1901): (1) parietal (syn. secretory or glandular) tapetum and (2) amoeboid (syn. plasmodial or intrusive) tapetum. An intennediate type, (3) invasive tapetum, is sometimes mentioned. In this third type, the cell walls degenerate and the individual protoplasts, without formation of a plasmodium, intmde the locules often in a cyclic pattern during microspore development. The latter type is recorded in a few families throughout angiosperm phylogeny (e.g. Furness & Rudall, 1998, 2001a; Furness, 2008a). Ultrastructural research of tapetal cells during their entire development, an element of major importance, revealed the highly dynamic nature and amazing morphological and cytological variation of this specialized nutritious tissue (e.g. Rowley et al., 1992; Rowley, 1993). For the great majority of plants no detailed information on tapetum type is available. When Davis (1966) compiled embryological data for the angiosperms, information on the tapetum type (amoeboid or parietal) was available for only 231 families. Pacini et al. (1985: table 2) updated her results and stressed that, astonishingly, the tapetum type was not investigated in almost half of the angiosperm families.
The positive correlation between orbicules and a parietal tapetum type is hypothesized since almost a century. Our present results confirm this general pattern statistically (Table 2). However, we also detected several additional species with a non-parietal tapetum type and orbicules (Table 3). Tradescantia virginiana (Commelinaceae), for example, has an amoeboid tapetum that produces tapetal derived granules but with less obvious sporopollenin accretion on their surface and smaller in size than average orbicules (Tiwari & Gunning, 1986c). The granules were considered analogous to the tapetal pro-orbicules of parietal tapeta by these authors. A highly interesting observation concerns Sauromatum venosum (Araceae), which has inaperturate pollen grains with an endexine and spines, both polysaccharidic in nature (Weber et al., 1998). Remarkably, orbicule-like structures occur, also polysaccharidic in composition (tested with PAS-reaction for detection of neutral polysaccharides). Both PAS-positive spines and orbicule-like structures in Sauromatum originate from the amoeboid tapetum and are formed synchronously (during first pollen mitosis) with an identical pattern formation (Weber et al., 1998). In Asteraceae different tapetum types occur with several intermediate forms (reviewed by Pacini, 1996), while orbicules appear to be consistently absent throughout the entire family and also in sister clades Calyceraceae and Goodeniaceae (Appendix SI). Therefore Asteraceae might provide an interesting case for increasing our understanding of the relationship between tapetum metabolism and orbicule production. An ontogenetic study of pollen and anthers of Cabomba caroliniana (Cabombaceae; Taylor et al., 2008) indicated the presence of an amoeboid tapetum. The degree of migration of tapetal cells into the locules, however, was variable between anthers and the secretory function was conserved. The latter is expressed in the presence of orbicules. These observations highlight the character plasticity present in basal angiosperms and support the conclusion put forward by Furness and Rudall (2001a) that characters present in these taxa potentially represent evolutionary experimentation in early angiosperm lineages.
Finally, it should be noted that the presence of a parietal tapetum is not the single determinative proxy to find orbicules. Of the 242 species that are indicated in Appendix SI as having a parietal tapetum, 31 species (=12.8%) lack orbicules (Table 2). This rough count does not include Orchidaceae, with over 22.000 species, which are believed to be characterized by a parietal tapetum (Pacini, 2009). Orbicule data are extremely scarce for orchids, possibly because negative observations require directed attention. Doritis is actually the only orchid genus where orbicules were reported, but we seriously doubt this interpretation judging their figures 8 and 12 (Wolter et al., 1988).
Correlation Orbicules-Pollination Syndrome
In their analytical key for tapetum types Pacini et al. (1985) correlated the occurrence of orbicules with the presence of pollenkitt, that is with the pollination syndrome. Pacini (1997) suggested that orbicules are absent in species with a strictly entomophilous pollination in which pollenkitt is present, and that they only occur in anemophilous species (without pollenkitt) and entomophilous angiosperms with a non-specific pollination syndrome: <<Only few taxa with a parietal tapetum with a strong entomophilous syndrome, such as the pumpkin (Cucurbita pepo) and orchids lack Ubisch bodies entirely>> (Pacini & Franchi, 1993: 5). Our new data do not contradict this statement. Other examples of taxa that have a parietal tapetum but lack orbicules are found in Brassicaceae and Balsaminaceae. These two families, together with Orchidaceae, are therefore very suitable for future studies on the correlation between orbicules and pollination syndrome.
Orbicules have not been observed in taxa with viscin threads, elastoviscin, massulae or compact pollinia (Pacini, 1997). On the other hand, they are present in Acacia species that develop polyads (Kenrick & Knox, 1979; G. Prenner, pers. com.).
Orbicules as a Model System for Sporopollenin Polymerisation
Orbicules provide an interesting model to study sporopollenin biosynthesis since they are a-cellular structures, independent of cytoplasmic control, contrary to the pollen exine (Clement & Audran, 1993). Very little experimental work has been done concerning the factors controlling orbicule formation. The pioneering work on Canna (Tiwari & Gunning, 1986a) and Tradescantia (Tiwari & Gunning, 1986b, c, d), both with amoeboid tapetum lacking orbicules, deserves special mention. The authors used colchicine treatments to investigate the role of cortical microtubules in the developing invasive tapetum. In Tradescantia the treatments prevented cell fusion. In both species investigated there was a disordered deposition of sporopollenin on all available extracellular lipidic surfaces and also on the outside of tapetal plasma membranes. The effect of colchicine provided evidence that amoeboid tapeta do participate in the synthesis and secretion of sporopollenin even though this activity is only manifest after experimental disturbance. Tiwari and Gunning (1986d) suggested that the availability of lipidic surfaces and extracellular space imposes physical constraints on the amount of sporopollenin deposited at any particular site.
Rowley et al. (1959: 537) accurately pointed out already half a century ago that <<... their [orbicules] morphology, composition, and position outside of the pollen wall seem to touch upon the prime problem of pollen morphology; i.e., the source of control over the specific ornamentation of the exine>>. The question as to whether the wall around the haploid microspore, and especially its ornamentation or patterning, is controlled by the microspore itself or by the diploid, sporophytic tapetum has long challenged angiosperm palynologists (reviewed by Blackmore et al., 2007) and has not yet been fully resolved. Pro-orbicules, after exocytosis and generally during tetrad stage, nest on the tapetal plasmalemma, and it is on this lipidic surface that they receive their sporopollenin coat. Moreover, the mode of development seems to be similar between exine and orbicular wall (Christensen et al., 1972). Evidence is growing that orbicule wall development also involves a glycocalyx-template and white line centred lamellae were observed in sub-mature orbicules of Rondeletia odorata (Rubiaceae; S. Huysmans, unpubl. data) reflecting the process of endexine development in the same species (El-Ghazaly et al., 2001).
The study of taxa with exineless pollen might yield interesting information concerning the potential of sporopollenin production by the tapetum. Pollen grains possessing a much-reduced exine and elaborated intine (omniaperturate and/or exineless) are known to occur in 54 families of angiosperms and are nearly ubiquitous in Zingiberales (Kress, 1986). For this study we have observed Etlingera (Zingiberaceae) with exineless pollen grains and without orbicules. In the large genus Xylopia (Annonaceae) at least four species have sporopollenin-lacking pollen (Tsou & Johnson, 2003) and no orbicules. It is noteworthy that in two species of Xylopia in which the tapetum starts to degenerate before meiosis, the pollen does not develop a typical exine wall (Tsou & Johnson, 2003).
Origin of Orbicules and Tapetal Lipid Metabolism
Insight in the complex lipid metabolism of tapetal cells appears to be crucial in our understanding of the distribution and function of orbicules in angiosperms. Pro-orbicules are indeed simple lipid vesicles originating from the endoplasmic reticulum in the tapetal cytoplasm (see Staehelin, 1997 for a review of ER functional domains in plant cells), while the sporopollenin wall of mature orbicules is considered as a complex mixed polymer of acyl lipid precursors and phenyl-propanoids (Scott, 1994). Piffanelli et al. (1998) reviewed the biogenesis and function of four lipidic structures associated with male gametophytes; exine and pollenkitt as extracellular lipidic structures, and storage oil bodies and a dense membrane network as intracellular lipidic structures. The first two are mostly controlled by the sporophytic genome, while the other two are primarily regulated by the gametophytic genome. Piffanelli et al. (1998) mentioned Ubisch bodies as one of the lipidic bodies produced by the anther in Brassicaceae (their Fig. 1a), which is in conflict with our present results since there are no reports of orbicules in Brassicaceae (but see Staiger et al., 1994). Tapetal cell degradation appears to involve apoptosis-like programmed cell death, which indicates a controlled process (Parish & Li, 2010). Therefore we prefer the term tapetum maturation above degeneration for this process since the long persistence of the tapetal mitochondria indicate the necessity for an energy supply, confirming that it not simply concerns necrosis (Papini et al., 1999). Wu et al. (1997) described a novel class of lipid containing organelles in the tapetum of Brassica that they termed tapetosomes (but see Dunbar, 1973). Whether the initiation or chemical composition of tapetosomes or elaioplasts (Piffanelli & Murphy, 1998) is related to pro-orbicules is unknown.
Evo-Devo and Orbicule Marker Genes
Evolutionary developmental approaches have greatly expanded our understanding of the genetic pathways that are involved in pollen wall development and the rate of gametophytic and sporophytic factors in the patterned polymerisation of sporopollenin on a lipidic interface, i.e. the plasmalemma of the microspores (reviewed by Wilson & Zhang, 2009). Evo-devo studies on model plants reveal an increasing number of genes expressed in the tapetum that when silenced (or in natural mutants) cause male-sterility or an arrest of the wild type microspore development (e.g. Goldberg et al., 1993; Kapoor et al., 2002; Yui et al., 2003; Ariizumi et al., 2004; Suzuki et al., 2008; Wu et al, 2008; McNeil & Smith, 2010; Zhang et al, 2010; Li et al., 2011). In rice, two putative orbicule marker genes, Os Raftinl and Os Raftin2, are downregulated in Wax-deficient anther1 (wda1) anthers that are lacking orbicules contrary to the wild-type (Jung et al., 2006). The rice RAFTIN genes are homologs of the wheat RAFTIN1 gene located at orbicules and exines, and supposed to have a guiding role in the proper fixation of sporopollenin polymers in the exine (Wang et al., 2003). Jung et al. (2006) concluded that the downregulation of both orbicule marker genes and the absence of orbicules and cytoplasmic lipid bodies in wda1 anthers imply that the wda1 mutant possibly affects the transfer of sporopollenin from the tapetum to the pollen walls via these organelles. Our results strongly question this transport function attributed to orbicules because firstly not all species with a parietal tapetum produce orbicules (Appendix S1), and secondly to date no enzyme could be characterized that is able to depolymerise sporopollenin. Thom et al. (1998) provided experimental evidence for reaggregation of materials obtained after fractionation of dissolved sporopollenin. To our knowledge there is no evidence available for reaggregation of sporopollenin in vivo. Moreover, orbicules generally remain associated with the tapetal plasmalemma throughout their development and thus active movement of orbicules is restricted to intrusion into the locules of invasive parietal tapetal cells that maintain their individual protoplasts (e.g. in Anaxagorea: Gabarayeva, 1995; Nymphaea: Gabarayeva & El-Ghazaly, 1997 and Rowley et al., 1992; Rondeletia: S. Huysmans, unpubl. data). Cyclic invasion of secretory tapetal cells into the locular space might be a much more common feature than presently considered (Gabarayeva et al., 2009).
Self-Assembly Processes and Patterning
There is growing evidence on the importance of self-assembly processes in pollen wall development. Gabarayeva and Hemsley (2006) summarized the developmental facts during tetrad and microspore stages and suggested mechanisms of molecular interaction to explain the wide range of variation in ornamentation patterns on angiospenn pollen grains. The authors concluded that in the sequence of exine development four main stages can be recognized, each with a different mechanism for wall construction: (1) formation of glycocalyx by self-assembly of micelles, (2) insertion of sporopollenin receptors under control of genome, (3) accumulation of receptor-dependant sporopollenin under control of sporopollenin receptors and (4) accumulation of receptor-independent sporopollenin by self-assembly. Exine pattern determination was originally attributed to callose (e.g. Blackmore & Barnes, 1990), however, genomic control of glycocalyx construction components is the most likely method by which genetic control of initial patterning is exerted (Gabarayeva & Hemsley, 2006). Evidence exists that during the middle tetrad stage sporopollenin can be produced from both tapetum and microspore although at present there is no data to suggest whether sporopollenin from these two sources differs in chemistry. If sporopollenin monomer is to be derived from the microspore, a site of production and a mechanism of transport are required. Droplets of lipid-like material synthesised in the interior of the ER membrane (Staehelin, 1997) are likely candidates for the monomers while the white-line-centred lamellae (see Scott, 1994) may well provide an appropriate transport conduit (Gabarayeva & Hemsley, 2006). During late tetrad stage sporopollenin deposition continues while callose is disintegrated by callase activity, but the source of this sporopollenin has yet to be determined and may be from either the microspore or the tapetum. The disintegration of callose may facilitate the penetration of sporopollenin precursors from the tapetum. In the subsequent free microspore stage sporopollenin is almost certainly derived from the tapetum and the mechanism of accumulation is largely self-assembly. Consequently the ultimate pattern results from autopolymerisation of bulk monomer (Gabarayeva & Hemsley, 2006 and references therein).
If self-assembly processes interfere with the work of the genome in pattern determination of the sporoderm, we could hypothesize parallel mechanisms for the sporopollenin polymerisation and patterning on the pro-orbicule core. The gametophytic genomic component and a callose wall are missing in the orbicule model and yet a patterned accumulation of sporopollenin with similar ornamentation to the pollen wall (Hesse, 1986; Huysmans et al., 1998) is still achieved.
Function(s) of Orbicules
One of the most intriguing open questions about orbicules is their function. Although many hypotheses on functions attributed to orbicules can be found in the literature, none of them is yet satisfactorily proven (reviewed in Huysmans et al., 1998). Two opposing lines of thought can be distinguished: orbicules play an active role or are just a by-product.
One hypothesis is that the tapetum has the vestigial capacity to polymerize sporopollenin because it shares a phylogenetically identical origin with the sporogenous tissue (Hesse, 1986). Orbicules are just mere by-products of the tapetal cell metabolism. This explains the presence of orbicules in unrelated taxa, the similarities in ornamentation between pollen and orbicules of the same species, and also the absence of orbicules in species with an amoeboid tapetum. Amoeboid tapeta may have lost the capacity to polymerize sporopollenin during evolution. The selective pressure for the evolution of absence of orbicules could be the conservation of resources, viz. sporopollenin precursors. However, the classic model of anther development involving three 'germ layers' that give rise to specific cell lineages (LI to epidermis, L2 to endothecium, middle layers, outer tapetum and archesporial cells, and L3 to connective and inner tapetum) and the well accepted dual origin of tapetum in angiosperms, has been challenged. Tsou and Johnson (2003) showed that tapetum differentiation in Annonaceae may instead be induced by chemical signaling from neighbouring sporogenous cells, and that ontogenetic origin has little or no significance for tapetum formation. Tapetum differentiation might be position-dependent rather than cell autonomous (Tsou & Johnson, 2003).
Another hypothesis is that orbicules participate actively in sporodenn formation by representing a transport system of sporopollenin between the tapetum and the developing microspores. This idea is based on observations of connections between orbicules and pollen sexines, mainly in grasses, or observations of a close contact between microspores and orbicules in other species. However, orbicules do not erode nor are they eliminated during microspore development, on the contrary their sporopollenin coat grows synchronously with the pollen exine (see Christensen et al., 1972 for comparative data on Sorghum). Moreover, so far no enzyme has been found to depolymerize sporopollenin. Since orbicules are not universally present, not even in species with a parietal tapetum, it is unlikely that they have such a general and crucial function as exine construction. Another active role for orbicules could be contributing to pollen dispersal. Orbicules could form a hydrophobic locule surface from which pollen can easily detach (Heslop-Harrison, 1968). Or, because exine and orbicules both consist of sporopollenin, they carry the same electrical charge and therefore repel one another (Pacini & Franchi, 1993). Orbicules could even present a reward for pollinators and therefore help in the attraction of visiting animals (Huysmans et al., 2000). But what about plant species that lack orbicules? Their pollen is being dispersed as well, without the presence of a hydrophobic surface or a repelling force. Orbicules are also very common in anemophilous species where offering a floral reward would be superfluous. Until now, there is no evidence of a correlation between orbicules and a particular pollination syndrome.
Conclusions and Future Directions
Orbicules might represent one of the last 'secrets of the anther', however, we demonstrated that they are actually commonly occurring in all higher order taxa of flowering plants and that they have great potential as model system to increase our knowledge in some fundamental issues in palynology and cell biology. Our results identified families and orders that are lacking any orbicule data. Orchidaceae, Arecales, rosids in general with Malpighiales in particular are 'hot' taxa for future morphological studies. Poaceae (rice, wheat, com, ...) and Solanaceae (tomato, tabacco,...) are plausible candidates for directed evo-devo approaches. Many new developments and data can be expected if functional genetic experiments on model plants, set up to identify the genes and gene products that control sporopollenin polymerisation and patterning, take both microspores and orbicules into account when screening phenotypes. We hope this review might raise awareness of orbicules and inspire a new generation of molecular biologists, palynologists, and systematists alike to explore the potential of orbicules in their own field of research.
Acknowledgments The authors gratefully thank N. Geerts (KU Leuven) and F. Christie (RBG Edinburgh) for technical assistance, A. Wortley and A. Poulsen for hospitality and sampling assistance to the last author at Royal Botanic Gardens Edinburgh, and A. Worberg for drawing an updated and tailor-made bubble diagram of angiosperms. Special thanks are due to all whom kindly shared personal observations of orbicules with special mention of L. Ronse De Craene, F. Gonzalez and G. Prenner, and to all students at the Laboratory of Plant Systematics who contributed to the dense asterid and monocots sampling. S. Blackmore, M. Hesse, and the late J. Rowley are gratefully acknowledged by the last author for stimulating discussions on orbicules. A visit to RBG Edinburgh by the last author was funded by the European Commission's Research Infrastructure Action via the Synthesys Project (GB-TAF-5561). The Research Foundation - Flanders (FWO, G.0268.04, G.0250.05) and the KU Leuven (OT/05/35) financially supported our research. This work is dedicated to Gamal El-Ghazaly (1947-2001) and John Rowley (1926-2010).
Alarid, K. M., R. D. Bray & L. J. Mussulman. 2005. Morphology, ultrastructure and typology of orbicules in Isoetes (Isoetaceae, Lycophyta). Botany 2005: Annual Meeting of the Botanical Society of America in Austin, Texas, USA. [online abstract, www.2005.botanyconference.org/engine/search/index.php?func= detail&aid=267],
Amelia Garcia, M. T., B. G. Galati & A. M. Anton. 2002. Microsporogenesis, microgametogenesis and pollen morphology of Passiflora spp. (Passifloraceae). Botanical Journal of the Linnean Society 139: 383-394.
Anger, E. M. & M. Weber. 2006. Pollen-wall formation in Arum alpinum. Annals of Botany 97: 239-244.
APG. 2009. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161: 105-121.
Ariizumi, T., K. Hatakeyama, K. Hinata, R. Inatsugi, I. Nishida, S. Sato, T. Kato, S. Tabata & K. Toriyama. 2004. Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana. Plant Journal 39: 170-181.
Audran, J.-C. 1981. Pollen and tapetum development in Ceratozamia mexicana (Cycadaceae): sporal origin of the exinic sporopollenin in Cycads. Review of Palaeobotany and Palynology 33: 315-346.
Bhandari, N. N. & R. Kishori. 1971. Ubisch granules on tapetal membranes in anthers; rapid selective staining by spirit blue. Stain Technology 46: 15-17.
Blackmore, S. & S. H. Barnes. 1985. Cosmos pollen ontogeny: a scanning electron microscope study. Protoplasma 126: 91-99.
--&--. 1990. Pollen wall development in angiosperms. Pp 173-192. In: S. Blackmore & R. B. Knox (eds). Microspores: evolution and ontogeny. Academic, London, UK.
--M. Takahashi & K. Uehara. 2000. A preliminary phylogenetic analysis of sporogenesis in Pteridophytes. Pp 109-124. In: M. M. Harley, C. M. Morton, & S. Blackmore (eds). Pollen Spores: Morphology and Biology. Royal Botanic Gardens, Kew, UK.
--, A. H. Wortley, J. J. Skvarla & J. R. Rowley. 2007. Pollen wall development in flowering plants. New Phytologist 174: 483-498.
Buss, P. A. & N. R. Lersten. 1975. Survey of tapetal nuclear number as a taxonomic character in Leguminosae. Botanical Gazette 136: 388-395.
Canini, A., J. Giovinazzi, P. Iacovacci, C. Pini & M. Grilli Caiola. 2004. Localisation of a carbohydrate epitope recognised by human IgE in pollen of Cupressaceae. Journal of Plant Research 117: 147-153.
Carniel, K. 1966. Uber die Komchen-Schicht in den Pollensacken von Gnetum gnemon. Osterreichische Botanische Zeitschrift 113: 368-374.
Chatin, A. 1870. De l'anthere. Recherches sur le developpement, la structure et les fonctions de ses tissus. Bailliere, Paris, France.
Chen, S.-H., J.-P. Liao, M.-Z. Luo & B. K. Kirchoff. 2008. Calcium distribution and function during anther development of Torenia foumieri (Lindemiaceae). Annales Botanici Fennici 45: 195-203.
Chichiricco, G. 1999. Developmental stages of pollen wall and tapetum in some Crocus species. Grana 38: 31-41.
Christensen, J. E., H. T. Jr. Horner & N. R. Lersten. 1972. Pollen wall and tapetal orbicular wall development in Sorghum bicolor (Gramineae). American Journal of Botany 59: 43-58.
Clement, C. & J. C. Audran. 1993. Orbicule wall surface characteristics in Lilium (Liliaceae). An ultrastructural and cytochemical approach. Grana 32: 348-353.
Cortes-B., R. 2003. Systematics and biogeography of Retiniphyllum (Rubiaceae). Ph.D. dissertation. City University of New York, New York, New York, USA.
Davis, G. L. 1966. Systematic embryology of the angiosperms. Wiley, New York, New York, USA.
Dessein, S., H. Ochoterena, P. De Block, F. Lens, E. Robbrecht, P. Schols, E. Smets, S. Vinckier & S. Huysmans. 2005. Palynological characters and their phylogenetic signal in Rubiaceae. Botanical Review 71: 354-414.
D'Hondt, C., P. Schols, S. Huysmans & E. Smets. 2004. Systematic relevance of pollen and orbicule characters in the tribe Hillieae (Rubiaceae). Botanical Journal of the Linnean Society 146: 303-321.
Dinis, A. M., F. Baptista & A. P. Coutinho. 2007. Is the quantity of orbicules released by Dactylis glomerata and Cynosurus echinatus (Poaceae) big enough to play an allergenic role? Grana 46: 140-147.
Doores, A. S., J. M. Osborn & G. El-Ghazaly. 2007. Pollen ontogeny in Ephedra americana (Gnetales). International Journal of Plant Sciences 168: 985-997.
Dunbar, A. 1973. Pollen development in the Eleocharis palustris group (Cyperaceae): 1. Ultrastructure and ontogeny. Botaniska Notiser 126: 197-254.
Echlin, P. & H. Godwin. 1968. The ultrastructure and ontogeny of pollen in Helleborus foetidus L. I. The development of the tapetum and Ubisch bodies. Journal of Cell Science 3: 161-174.
El-Ghazaly, G. 1999. Tapetum and orbicules (Ubisch bodies): development, morphology and role of pollen grains and tapetal orbicules in allergenicity. Pp 157-173. In: M. Cresti, G. Cai, & A. Moscatelli (eds). Fertilization in higher plants. Springer, Berlin, Germany.
--& S. Huysmans. 2001. Re-evaluation of a neglected layer in pollen wall development with comments on its evolution. Grana 40: 3-16.
--,--& E. Smets. 2001. Pollen wall development oIRondeletia odorata (Rubiaceae). American Journal of Botany 88: 14-30.
--& W. A. Jensen. 1986. Studies of the development of wheat (Triticum aesdmm) pollen. I. Formation of the pollen wall and Ubisch bodies. Grana 25: 1-29.
--& S. Nilsson. 1991. Development of tapetum and orbicules of Catharanthus roseus (Apocynaceae). Pp 317-329. In: S. Blackmore & S. H. Barnes (eds). Pollen Spores: Patterns of Diversity. Oxford University Press, Oxford, UK.
--& J. R. Rowley. 1997. Pollen wall of Ephedra foliata. Palynology 21: 7-18.
Fernando, D. D. & D. D. Cass. 1994. Plasmodial tapetum and pollen wall development in Butomus umbellatus (Butomaceae). American Journal of Botany 81: 1592-1600.
Foreman, D. B. 1984. The morphology and phytogeny of the Monimiaceae (sensu lato). Ph.D. dissertation, University of New England, Armindale, Australia.
Furness, C. A. 2008a. A review of the distribution of plasmodial and invasive tapeta in eudicots. International Journal of Plant Sciences 169: 207-223.
--2008b. Successive microsporogenesis in eudicots, with particular reference to Berberidaceae (Ranunculales). Plant Systematics and Evolution 273: 211-223.
--2011. Comparative structure and development of pollen and tapetum in Malpighiales, with a focus on the parietal clade. International Journal of Plant Sciences 172: 980-1011.
--& P. J. Rudall. 1998. The tapetum and systematics in monocotyledons. Botanical Review 64: 201 239.
-- & --. 1999. Microsporogenesis in monocotyledons. Annals of Botany 84: 475-4199.
-- & --. 2001a. The tapetum in basal angiosperms: early diversity. International Journal of Plant Sciences 162: 375-392.
-- & --. 2001b. Pollen and anther characters in monocot systematics. Grana 40: 17-25.
-- & --. 2006. Comparative structure and development of pollen and tapetum in Pandanales. International Journal of Plant Sciences 167: 331-348.
--, -- & A. Eastman. 2002. Contribution of pollen and tapetal characters to the systematics of Triuridaceae. Plant Systematics and Evolution 235: 209-218.
Gabarayeva, N. I. 1995. Pollen wall and tapetum development in Anaxagorea brevipes (Annonaceae): sporoderm substructure, cytoskeleton, sporopollenin precursor particles, and the endexine problem. Review of Palaeobotany and Palynology 85: 123-152.
-- & G. El-Ghazaly. 1997. Sporoderm development in Nymphaea mexicana (Nymphaeaceae). Plant Systematics and Evolution 204: 1-19.
--, V. V. Grigorjeva & J. R. Rowley. 2010a. A new took at sporoderm ontogeny in Persea americana and the hidden side of development. Annals of Botany 105: 939-955.
--, -- & --. 2010b. Sporoderm development in Acer tataricum (Aceraceae): an interpretation. Protoplasma 247: 65-81.
--, --, -- & A. R. Herasley. 2009. Sporoderm development in Trevesia burckii (Araliaceae). II. Post-tetrad period: further evidence for the participation of self-assembly processes. Review of Palaeobotany and Palynology 156: 233-247.
-- & A. R. Hemsley. 2006. Merging concepts: the role of self-assembly in the development of pollen wall structure. Review of Palaeobotany and Palynology 138: 121-139.
Galati, B. G. 2003. Ubisch bodies in angiosperms. Advances in Plant Reproductive Biology 2: 1-20.
--, F. Monacci, M. M. Gotelli & S. Rosenfeldt. 2007. Pollen, tapetum and orbicule development in Modiolastrum malvifolium (Malvaceae). Annals of Botany 99: 755-763.
-- & S. Rosenfeldt. 1998. The pollen development in Ceiba insignis (Kunth) Gibbs & Semir ex Chorisia speciosa St.Hil. (Bombacaceae). Phytomorphology 48: 121-129.
-- & L. I. Strittmatter. 1999a. Correlation between pollen development and Ubisch bodies ontogeny in Jacaranda mimosifolia (Bignoniaceae). Beitrage zur Biologie der Pflanzen 71: 249-260.
-- & --. 1999b. Microsporogenesis and microgametogenesis in Jacaranda mimosifolia (Bignoniaceae). Phytomorphology 49: 67-74.
--, G. Zarlavsky, S. Rosenfeldt & M. M. Gotelli. 2012. Pollen ontogeny in Magnolia liliflora Desr. Plant Systematics and Evolution 298: 527-534.
Geeraerts, A., J. A. M. Raeymaekers, S. Vinckier, A. Pletsers, E. Smets & S. Huysmans. 2009. Systematic palynology in Ebenaceae with focus on Ebenoideae: morphological diversity and character evolution. Review of Palaeobotany and Palynology 153: 336-353.
Goebel, K. 1901. Organographie der Pflanzen, ed. 1st. Gustav Fischer Verlag, Jena, Germany.
Goldberg, R. B., T. P. Beals & P. M. Sanders. 1993. Anther development: basic principles and practical applications. Plant Cell 5: 1217-1229.
Gonzalez, F. 1999. A phylogenetic analysis of the Aristolochioideae (Aristolochiaceae). Ph.D. dissertation, The City University of New York, New York, New York, USA.
--, P. J. Rudall & C. A. Furness. 2001. Microsporogenesis and systematics of Aristolochiaceae. Botanical Journal of the Linnean Society 137: 221-242.
Gotelli, M., B. G. Galati & D. Medan. 2012. Pollen, tapetum, and orbicule development in Colletia paradoxa and Discaria americana (Rhamnaceae). The Scientific World Journal 2012: ID 948469.
Gupta, S. C. & K. Nanda. 1972. Occurrence and histochemistry of the anther tapetal membrane. Grana 12: 99-104.
Halbritter, H. & M. Hesse. 2005. Specific ornamentation of orbicular walls and pollen grains, as exemplified by Acanthaceae. Grana 44: 308-312.
--, -- & M. Weber. 2012. The unique design of pollen tetrads in Dionaea and Drosera. Grana 51: 148-157.
Hansson, T. & G. El-Ghazaly. 2000. Development and cytochemistry of pollen and tapetum in Mitriostigma axillare (Rubiaceae). Grana 39: 65-89.
Harley, R. M., S. Atkins, A. L. Budantsev, P. D. Cantino, B. J. Conn, R. Grayer, M. M. Harley, R. de Kok, T. Krestovskaja, R. Morales, A. J. Paton, O. Ryding & T. Upson. 2004. Labiatae. Pp 167-275. In: J. W. Kadereit (ed). The Families and Genera of Vascular Plants, vol. 7, Flowering plants: Dicotyledons (Lamiales except Acanthaceae including Avicenniaceae). Springer, Berlin, Germany.
Hermann, P. M. & B. F. Palser. 2000. Stamen development in the Ericaceae. I. Anther wall, microsporogenesis, inversion and appendages. American Journal of Botany 87: 934-957.
Heslop-Harrison, J. 1968. Tapetal origin of pollen-coat substances in Lilium. New Phytologist 67: 779-786.
Hesse, M. 1986. Orbicules and the ektexine are homologous sporopollenine concretions in Spermatophyta. Plant Systematics and Evolution 153: 37-48.
-- 1999. Electron-translucent angular areas in developing tapetum cell walls and pollen grains of Tilia platyphyllos. Protoplasma 207: 169-173.
-- 2001. Pollen characters of Amborella trichopoda (Amborellaceae): a reinvestigation. International Journal of Plant Sciences 162: 201-208.
--, H. flalbritter, R. Zetter, M. Weber, R. Buchner, A. Frosch-Radivo & S. Ulrich. 2009. Pollen terminology. An illustrated handbook. Springer, Vienna, Austria.
Hosoo, Y., E. Yoshii, K. Negishi & H. Taira. 2005. A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica. Sexual Plant Reproduction 18: 81-89.
Huysmans, S., S. Dessein, E. Smets & E. Robbrecht. 2003. Pollen morphology of NW European representatives confirms monophyly of Rubieae (Rubiaceae). Review of Palaeobotany and Palynology 127: 219-240.
--, G. El-Ghazaly, S. Nilsson & E. Smets. 1997. Systematic value of tapetal orbicules: a preliminary survey of the Cinchonoideae (Rubiaceae). Canadian Journal of Botany 75: 815-826.
--, -- & E. Smets. 1998. Orbicules in angiosperms. Morphology, function, distribution, and relation with tapetum types. Botanical Review 64: 240-272.
--, -- & --. 2000. Orbicules: still a well hidden secret of the anther. Pp 201-212. In: B. Nordenstam, G. El-Ghazaly, & M. Kassas (eds). Plant Systematics for the 21st Century. Portland Press, London, UK.
--, E. Robbrecht, P. Delprete & E. Smets. 1999. Pollen morphological support for the Catesbaeeae Chiococceae-Emstema-complex (Rubiaceae). Grana 38: 325--338.
--, B. Verstraete, E. Smets & L. W. Chatrou. 2010. Distribution of orbicules in Annonaceae mirrors evolutionary trend in angiosperms. Plant Ecology and Evolution 143: 199-211.
Inamuddin, M., B. Were & M. Saquib. 2009. A contribution to the embryology of Rhynchelytrum repens (Willd.) C.E. Hubbard. Scientific World 7: 37-40.
Jacobs, B., K. Geuten, N. Pyck, S. Huysmans, S. Jansen & E. Smets. 2011. Unraveling the phytogeny of Heptacodium and Zabelia (Caprifoliaceae): an interdisciplinary approach. Systematic Botany 36: 231-252.
Jager-Zurn, I., R. N. Novelo & C. T. Philbrick. 2006. Microspore development in Podostemaceae-Podostemoideae, with implications on the characterization of the subfamilies. Plant Systematics and Evolution 256: 209-216.
Janssens, S., S. Dessein & E. Smets. 2011. Portrayal of Impatiens nzabiana (Balsaminaceae): a morphological, molecular and biogeographic study of a new Gabonese species. Systematic Botany 36: 440-448.
--, E. Knox, S. Dessein & E. Smets. 2009. Impatiens msisimwanensis (Balsaminaceae): Description, pollen morphology and phylogenetic position of a new East African species. South African Journal of Botany 75: 104-109.
--, F. Lens, S. Dressier, K. Geuten, E. Smets & S. Vinckier. 2005. Palynological variation in balsaminoid Ericales. II. Balsaminaceae, Tetrameristaceae, Pellicieraceae and general conclusions. Annals of Botany 96: 1061-1073.
--, Y. Song Wilson, Y.-M. Yuan, A. Nagels, E. F. Smets & S. Huysmans. 2012. A total evidence approach using palynological characters to infer the complex evolutionary history of the Asian Impatiens (Balsaminaceae). Taxon 61: 355-367.
Jato, V., F. J. Rodriguez-Rajo, Z. Gonzalez-Parrado, B. Elvira-Rendueles, S. Moreno-Grau, A. Vega-Maray, D. Fernandez-Gonzalez, J. A. Asturias & M. Suarez-Cervera. 2010. Detection of airborne Par j 1 and Par j 2 allergens in relation to Urticaceae pollen counts in different bioclimatic areas. Annals of Allergy, Asthma & Immunology 105: 50-56.
Jung, K.-H., M.-J. Han, D.-Y. Lee, Y.-S. Lee, L. Schreiber, R. Franke, A. Faust, A. Yephremov, H. Saedler, Y.-W. Kim, I. Hwang & G. An. 2006. Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development. Plant Cell 18: 3015-3032.
Johri, B. M. & S. P. Bhatnagar. 1955. A contribution to the morphology and life history of Aristolochia. Phytomorphology 3: 123-137.
Kapoor, S., A. Kobayashi & H. Takatsuji. 2002. Silencing of the tapetum-specific Zinc finger gene TAZ1 causes premature degeneration of tapetum and pollen abortion in Petunia. Plant Cell 14: 2353-2367.
Kenrick, J. & R. B. Knox. 1979. Pollen development and cytochemistry in some Australian species of Acacia. Australian Journal of Botany 27: 413-127.
von Kosmath, L. 1927. Studien uber das Antherentapetum. Osterreichische Botanische Zeitschrift 76: 235-241.
Kress, W. J. 1986. Exineless pollen structure and pollination systems of tropical Heliconia (Heliconiaceae). Pp 329-345. In: S. Blackmore & I. K. Ferguson (eds). Pollen and Spores: Form and Function. Academic, London, UK.
Kreunen, S. S. & J. M. Osborn. 1999. Pollen and anther development in Nelumbo (Nelumbonaceae). American Journal of Botany 86: 1662-1676.
Krjatchenko, M. D. 1925. De l'activite des chondriosomes pendant le developpement des grains de pollen et des cellules nourricieres du pollen dans Lilium croceum Chaix. Revue Generate de Botanique 37: 193-211.
Lens, F., S. Dressler, S. Vinckier, S. Janssens, S. Dessein, L. Van Evelghem & E. Smets. 2005. Palynological variation in balsaminoid Ericales. I. Marcgraviaceae. Annals of Botany 96: 1047-1060.
Le Thomas, A., M. Suarez-Cervera & P. Goldblatt. 2001. Ontogeny of the exine in pollen of Aristea (Iridaceae). Grana 40: 35-44.
Li, T. & H. Cao. 1986. Microsporogensis and development of mate gametophyte of Camellia chrysantha (Hu) Tuyama. Journal of Beijing Forestry University.
Li, H., Z. Yuan, G. Vizcay-Barrena, C. Yang, W. Liang, J. Zong, Z. A. Wilson & D. Zhang. 2011. PERSISTENT TAPETAL CELLI encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiology 156: 615-630.
Liang, X., Y. Zhang, S. Liu & B. Yu. 1994. Studies on the infrastructure of the development of anther tapetum in Sesame (Sesamum indicum L.). Journal of Agricultural Biotechnology.
Lombardo, G. & L. Carraro. 1976. Tapetal ultrastructural changes during pollen development. III. Studies on Gentiana acaulis. Caryologia 29: 345-349.
Lugardon, B. 1981. Les globules des Filicinees, homologues des corps d'Ubisch des Spermatophytes. Pollen Spores 23: 93-124.
Mascre, M. 1922. Sur l'etamine des Boraginees. Comptes Rendus de l'Academie des Sciences de Paris 175: 987-988.
McNeil, K. J. & A. G. Smith. 2010. A glycine-rich protein that facilitates exine formation during tomato pollen development. Planta 231: 793-808.
Merckx, V., P. Schols, K. Geuten, S. Huysmans & E. Smets. 2008. Phylogenetic relationships in Nartheciaceae (Dioscoreales) with focus on pollen and orbicule morphology. Belgian Journal of Botany 141: 64-77.
--, --, H. Maas-van de Kamer, P. Maas, S. Huysmans & E. Smets. 2006. Phylogeny and evolution of Burmanniaceae (Dioscoreales) based on nuclear and mitochondrial data. American Journal of Botany 93: 1684-1698.
Moon, H.-K., S. Vinckier, E. Smets & S. Huysmans. 2008a. Comparative pollen morphology and ultrastructure of Mentheae subtribe Nepetinae (Lamiaceae). Review of Palaeobotany and Palynology 149: 174-186.
--, --, -- & --. 2008b. Palynological evolutionary trends within the tribe Mentheae with special emphasis on subtribe Menthinae (Nepetoideae: Lamiaceae). Plant Systematics and Evolution 275: 93-108.
--, --, J. B. Walker, E. Smets & S. Huysmans. 2008c. A search for phylogenetically informative pollen characters in the subtribe Salviinae (Mentheae: Lamiaceae). International Journal of Plant Sciences 169: 455-471.
Mu, X., F. Wang & W. Wang. 1988. Development and histochemical observations of tapetum and peritapetal membrane in anther of Pulsatilla chinensis. Acta Botanica Sinica 30: 6-13.
Muasya, A. M., P. M. Musili & A. Vrijdaghs. 2010. Kyllinga mbitheana (Cyperaceae)-Description, floral ontogeny and pollen micromorphology of a new species from Kenya. Journal of East African Natural History 99: 65-75.
Murgia, M., M. Charzynska, M. Rougier & M. Cresti. 1991. Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features. Sexual Plant Reproduction 4: 28-35.
Murphy, D. J. 2006. The extracellular pollen coat in members of the Brassicaceae: composition, biosynthesis, and functions in pollination. Protoplasma 228: 31-39.
Nagels, A., A. M. Muasya, S. Huysmans, A. Vrijdaghs, E. Smets & S. Vinckier. 2009. Palynological diversity and major evolutionary trends in Cyperaceae. Plant Systematics and Evolution 277: 117-142.
Nilsson, S. & A. Robyns. 1974. Pollen morphology and taxonomy of the genus Quararibea s.l. (Bombacaceae). Bulletin du Jardin Botanique National de Belgique 44: 77-99.
Pacini, E. 1996. Tapetum types in the Compositae: forms and function. Pp 21-28. In: D. J. N. Hind & H. J. Beentje (eds). Proceedings of the International Compositae Conference in Kew, 1994, vol. 1. Kew Publishing, Kew, UK.
--1997. Tapetum character states: analytical keys for tapetum types and activities. Canadian Journal of Botany 75: 1448-1459.
--2009. Orchids pollen dispersal units and reproductive consequences. Pp 185-218. In: T. Kull, J. Arditti, & S. M. Wong (eds). Orchid Biology: reviews and perspectives. Springer, Berlin, Germany.
--, G. G. Franchi & M. Hesse. 1985. The tapetum: its form, function and possible phylogeny in Embryophyta. Plant Systematics and Evolution 149: 155-185.
-- & --. 1993. Role of the tapetum in pollen and spore dispersal. Plant Systematics and Evolution Supplement 7: 1-11.
-- & B. E. Juniper. 1979. The ultrastructure of pollen-grain development in the olive (Olea europaea). II. Secretion by the tapetal cells. New Phytologist 83: 165-174.
-- & --. 1983. The ultrastructure of the formation and development of the amoeboid tapetum in Arum italicum Miller. Protoplasma 117: 116-129.
Papini, A., S. Mosti & L. Brighigna. 1999. Programmed-cell-death events during tapetum development of angiosperms. Protoplasma 207: 213-221.
Parish, R. W. & S. F. Li. 2010. Death of a tapetum: A programme of developmental altruism. Plant Science 178: 73-89.
Parkinson, B. M. & E. Pacini. 1995. A comparison of tapetal structure and function in pteridophytes and angiosperms. Plant Systematics and Evolution 198: 55-88.
Parulekar, N. K. 1970. Annonaceae. Bulletin of Indian National Academy of Sciences 41: 38-41.
Passarelli, L. M., S. B. Girarde & N. M. Tur. 2002. Palynology of South American Podostemaceae. Grana 41: 10-15.
Periasamy, K. & B. G. Swamy. 1959. Studies in the Annonaceae I: microsporogenesis in Cananga odorata and Miliusa wightiana. Phytomorphology 9: 251-263.
Piffanelli, P. & D. J. Murphy. 1998. Novel organelles and targeting mechanisms in the anther tapetum. Trends in Plant Science 3: 250-253.
--, J. H. E. Ross & D. J. Murphy. 1998. Biogenesis and function of the lipidic structures of pollen grains. Sexual Plant Reproduction 11: 65-80.
Prakash, N., D. B. Foreman & S. J. Griffith. 1984. Gametogenesis in Galbulimima belgraveana (Himantandraceae). Australian Journal of Botany 32: 605-612.
Radice, S., M. Ontivero, E. Giordani & E. Bellini. 2008. Anatomical differences on development of fertile and sterile pollen grains of Prunus salicina Lindl. Plant Systematics and Evolution 273: 63-69.
Raghavan, V. 1988. Anther and pollen development in rice (Oryza sativa). American Journal of Botany 75: 183-196.
Ray, B. & G. El-Ghazaly. 1987. Morphology and taxonomic application of orbicules (Ubisch bodies) in Chloanthaceae. Pollen Spores 29: 151-166.
Risueno, M. C., G. Gimenez-Martin, J. F. Lopez-Saez & M. I. R. Garcia. 1969. Origin and development of sporopollenine bodies. Protoplasma 67: 361-374.
Ronse De Craene, L. P. 2002. Floral development and anatomy of Pentadiplandra (Pentadiplandraceae): a key genus in the identification of floral morphological trends in the core Brassicales. Canadian Journal of Botany 80: 443-159.
--2005. Floral developmental evidence for the systematic position of Bads (Bataceae). American Journal of Botany 92: 752-760.
--, T. A. Yang, P. Schols & E. Smets. 2002. Flower anatomy and systematics of Bretschneidera (Bretschneideraceae). Botanical Journal of the Linnean Society 139: 29-45.
-- & A. G. Miller. 2004. Floral development and anatomy of Dirachma socotrana (Dirachmaceae): a controversial member of the Rosales. Plant Systematics and Evolution 249: 111-127.
-- & L. Wanntorp. 2008. Morphology and evolution of the flower of Meliosma (Sabiaceae): implications for pollination biology. Plant Systematics and Evolution 271: 79-91.
Rosanoff, S. 1865. Zur Kenntnis des Baues und der Entwicklungsgeschichte des Pollens der Mimoseae. Jahrbuch fur wissenschaftliche Botanik 4: 441-450.
Rosenfeldt, S. & B. G. Galati. 2005. Ubisch bodies and pollen ontogeny in Oxalis articulata Savigny. Biocell 29: 271-278.
-- & --. 2008. Orbicules diversity in Oxalis species from the province of Buenos Aires (Argentina). Biocell 32: 41-47.
-- & --. 2012. Embryological studies of Oxalis debilis Kunth. Plant Systematics and Evolution 298: 1567-1573.
Rowley, J. R. 1962.Nonhomogeneous sporopollenin in microcspores of Poa annua L. Grana Palynologica 3: 3-19.
--1993. Cycles of hyperactivity in tapetal cells. Plant Systematics and Evolution Supplement 7: 23-37.
-- & N. I. Gabarayeva. 2004. Microspore development in Quercus robur (Fagaceae). Review of Palaeobotany and Palynology 132: 115-132.
--, -- & B. Walles. 1992. Cyclic invasion of tapetal cells into loculi during microspore development in Nymphaea colorata (Nymphaceae). American Journal of Botany 79: 801-808.
--, K. Muhlethaler & A. Frey-Wyssling. 1959. A route for the transfer of materials through the pollen grain wall. Journal of Biophysical and Biochemical Cytology 6: 537-538.
-- & B. Walles. 1987. Origin and structure of Ubisch bodies in Pinus sylvestris. Acta Societatis Botanicorum Poloniae 56: 215-227.
Rudall, P. J., E. M. Engleman, L. Hanson & M. W. Chase. 1998. Embryology, cytology and systematics of Hemiphylacus, Asparagus and Anemarrhena (Asparagales). Plant Systematics and Evolution 211: 181-199.
Sajo, M. G., C. A. Furness, C. J. Prychid & P. J. Rudall. 2005. Microsporogenesis and anther development in Bromeliaceae. Grana 44: 65-74.
Santos, R. P. & J. F. A. Mariath. 1999. Ultrastructure of the orbicules (Ubisch bodies) in Ilex paraguariensis St.Hil. (Aquifoliaceae). Acta Microscopica 8 Supplement C: 773-774.
Schappi, G. F., P. E. Taylor, I. A. Staff, C. Suphioglu & R. B. Knox. 1997. Source of Bet v 1 loaded inhalable particles from birch revealed. Sexual Plant Reproduction 10: 315-323.
Schnarf, K. 1923. Kleine Beitrage zur Entwicklungsgeschichte der Angiospermen. IV. Uber das Verhalten des Antherentapetums einiger Pflanzen. Osterreichische Botanische Zeitschrift 72: 242-245.
Schols, P., C. A. Furness, V. Merckx, P. Wilkin & E. Smets. 2005a. Comparative pollen development in Dioscoreales. International Journal of Plant Sciences 166: 909-924.
--, --, P. Wilkin, S. Huysmans & E. Smets. 2001. Morphology of pollen and orbicules in some Dioscorea species and its systematic implications. Botanical Journal of the Linnean Society 136: 295-311.
--, --, --, E. Smets, V. Cielen & S. Huysmans. 2003. Pollen morphology of Dioscorea (Dioscoreaceae) and its relation to systematics. Botanical Journal of the Linnean Society 143: 375-390.
--, P. Wilkin, C. A. Furness, S. Huysmans & E. Smets. 2005b. Pollen evolution in yams (Dioscorea: Dioscoreaceae). Systematic Botany 30: 750-758.
Scott, R. J. 1994. Pollen exine - the sporopollenin enigma and the physics of pattern. Pp 49-81. In: R. J. Scott & M. A. Stead (eds). Molecular and Cellular Aspects of Plant Reproduction. Cambridge University Press, Cambridge, UK.
Staehelin, L. A. 1997. The plant ER: a dynamic organelle composed of a large number of discrete functional domains. Plant Journal 11: 1151-1165.
Staiger, D., S. Kappeler, M. Muller & K. Apel. 1994. The proteins encoded by two tapetum-specific transcripts, Satap35 and Satap44, from Sinapis alba L. are localized in the exine cell wall layer of developing microspores. Planta 192: 221-231.
Stevens, P. F. 2001 onwards. Angiosperm Phylogeny Website. Version 12, July 2012 [and more or less continuously updated since].
Stone, D. E., S. C. Sellers & W. J. Kress. 1979. Ontogeny of exineless pollen in Heliconia, a banana relative. Annals of the Missouri Botanical Garden 66: 701-730.
Strittmatter, L. & B. G. Galati. 2000. Embryological study in Oziroe acaulis (Hyacinthaceae). Phytomorphology 50: 161-171.
-- & --. 2001. Pollen development in Myosolis azorica and M. laxa (Boraginaceae). Phytomorphology 54: 1-9.
--, -- & F. Monacci. 2000. Ubisch bodies in the peritapetal membrane of Abutilon pictum Gill (Malvaceae). Beitrage zur Biologie der Pflanzen 71: 393-402.
Su, Y. C. F. & R. M. K. Saunders. 2003. Pollen structure, tetrad cohesion and pollen-connecting threads in Pseuduvaria (Annonaceae). Botanical Journal of the Linnean Society 143: 69-78.
Suarez-Cervera, M., Y. Takahashi, A. Vega-Maray & J. A. Seoane-Camba. 2003. Immunocytochemical localization of Cry j 1, the major allergen of Cryplomeria japonica (Taxodiaceae) in Cupressus arizonica and Cupressus sempervirens (Cupressaceae) pollen grains. Sexual Plant Reproduction 16: 9-15.
Suzuki, T., K. Masaoka, M. Nishi, K. Nakamura & S. Ishiguro. 2008. Identification of kaonashi mutants showing abnormal pollen exine structure in Arabidopsis thaliana. Plant Cell Physiology 49: 1465-1477.
Taylor, M. L., B. L. Gutman, N. A. Melrose, A. M. Ingraham, J. A. Schwartz & J. M. Osborn. 2008. Pollen and anther ontogeny in Cabomba caroliniana (Cabombaceae, Nymphaeales). American Journal of Botany 95: 399-413.
--, P. J. Hudson, J. M. Rigg, N. Julie, J. Schwartz Green, T. C. Thiemann & J. M. Osborn. 2012. Tapetum structure and ontogeny in Victoria (Nymphaeaceae). Grana 51: 107-118.
-- & J. M. Osborn. 2006. Pollen ontogeny in Brasenia (Cabombaceae, Nymphaeales). American Journal of Botany 93: 344-356.
Taylor, T. N. 1976. The ultrastructure of Schopfipollenites: orbicules and tapetal membranes. American Journal of Botany 63: 857-862.
Teppner, H. & E. Stabentheiner. 2007. Anther opening, polyad presentation, pollenkitt and pollen adhesive in four Calliandra species (Mimosaceae-Ingeae). Phyton 47: 291-320.
Thom, I., M. Grote, J. Abraham-Peskir & R. Wiermann. 1998. Electron and X-ray microscopic analyses of reaggregated materials obtained after fractionation of dissolved sporopollenin. Protoplasma 204: 13-21.
Tiwari, S. C. & B. E. S. Gunning. 1986a. Development of tapetum and microspores in Canna L.: an example of an invasive but non-syncytial tapetum. Annals of Botany 57: 557-563.
-- & --. 1986b. Cytoskeleton, cell surface and the development of invasive plasmodial tapetum in Tradescantia virginiana L. Protoplasma 133: 89-99.
-- & --. 1986c. An ultrastructural, cytochemical and immunofluorescence study of postmeiotic development of plasmodial tapetum in Tradescantia virginiana L. and its relevance to the pathway of sporopollenin secretion. Protoplasma 133: 100-114.
-- & --. 1986d. Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum of Tradescantia virginiana L. Protoplasma 133: 115-128.
Tsou, C.-H. & Y.-L. Fu. 2007. Octad pollen formation in Cymbopetalum (Annonaceae): the binding mechanism. Plant Systematics and Evolution 263: 13-23.
-- & D. M. Johnson. 2003. Comparative development of aseptate and septate anthers of Annonaceae. American Journal of Botany 90: 832-848.
von Ubisch, G. 1927. Zur Entwicklungsgeschichte der Antheren. Planta 3: 490-495.
Van der Ham, R. W. J. M. 1991. Pollen morphology of the Stemonaceae. Blumea 36: 127-159.
--, Y.-M. Zimmermann, S. Nilsson & A. Igersheim. 2001. Pollen morphology and phytogeny of the Alyxieae (Apocynaceae). Grana 40: 169-191.
Verellen, J., S. Dessein, S. G. Razafimandimbison, E. Smets & S. Huysmans. 2007. Pollen morphology of the tribes Naucleeae and Hymenodictyeae (Rubiaceae-Cinchonoideae) and its phylogenetic significance. Botanical Journal of the Linnean Society 153: 329-341.
--, E. Smets & S. Huysmans. 2004. The remarkable genus Coptosapelta (Rubiaceae): pollen and orbicule morphology and systematic implications. Journal of Plant Research 117: 57-68.
Verstraete, B. 2009. Orbicules in angiospermen: distributie en relatie met tapetum. Case-studies in Annonaceae, Rubiaceae en modelplanten. MSc Thesis, KU Leuven, Belgium. [Dutch]
--, I. Groeninckx, E. Smets & S. Huysmans. 2011. Phylogenetic signal of orbicules at family level: Rubiaceae as case study. Taxon 60: 742-757.
Vinckier, S., S. Huysmans & E. Smets. 2000. Morphology and ultrastructure of orbicules in the subfamily Ixoroideae (Rubiaceae). Review of Palaeobotany and Palynology 108: 151-174.
--, S. Janssens, S. Huysmans, A. Vandevenne & E. Smets. 2012. Pollen ontogeny linked to tapetal cell maturation in Impatiens parviflora (Balsaminaceae). Grana 52: 10-24.
-- & E. Smets. 2001a. A survey of the presence and morphology of orbicules in European allergenic angiosperms. Background information for allergen research. Canadian Journal of Botany 79: 757-766.
-- & --. 2001b. The potential role of orbicules as a vector of allergens. Allergy 56: 1129-1136.
-- & --. 2002a. Morphology, ultrastructure and typology of orbicules in Loganiaceae s.l. and related genera, in relation to systematics. Review of Palaeobotany and Palynology 119: 161-189.
-- & --. 2002b. Systematic importance of orbicule diversity in Gentianales. Grana 41: 158-182.
-- & --. 2002c. Morphological and ultrastructural diversity of orbicules in relation to evolutionary tendencies in Apocynaceae s.l. Annals of Botany 90: 647-662.
-- & --. 2003. Morphological and ultrastructural diversity of orbicules in Gentianaceae. Annals of Botany 92: 657-672.
-- & --. 2005. A histological study of microsporogenesis in Tarenna gracilipes (Rubiaceae). Grana 44: 30-44.
--, P. Cadot, M. Grote, J. L. Ceuppens & E. Smets. 2006. Orbicules do not significantly contribute to the allergenic micro-aerosol emitted from birch trees. Allergy 61: 1243-1244.
--, -- & E. Smets. 2005. The manifold characters of orbicules: structural diversity, systematic significance, and vectors for allergens. Grana 44: 300-307.
von Balthazar, M., K. Raunsgaard Pedersen & E. M. Friis. 2005. Teixeiraea lusitanica, a new fossil flower from the early Cretaceous of Portugal with affinities to Ranunculales. Plant Systematics and Evolution 255: 55-75.
Wang, A. M., Q. Xia, W. S. Xie, R. Datla & G. Selvaraj. 2003. The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development. Proceedings of the National Academy of Sciences, USA 100: 14487-14492.
Weber, M., H. Halbritter & M. Hesse. 1998. The spiny pollen wall in Sauromatum (Araceae)--With special reference to the endexine. International Journal of Plant Science 159: 744-749.
Wilson, Z. A. & D.-B. Zhang. 2009. From Arabidopsis to rice: pathways in pollen development. Journal of Experimental Biology 60: 1479-1492.
Wolter, M., C. Seuffert & R. Schill. 1988. The ontogeny of pollinia and elastoviscin in the anther of Doritis pulcherrima (Orchidaceae). Nordic Journal of Botany 8: 77-88.
Wu, S. S. H., K. A. Platt, C. Ratnayake, T.-W. Wang, J. T. L. Ting & A. H. C. Huang. 1997. Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum. Proceedings of the National Academy of Sciences, USA 94: 12711-12716.
Wu, S., S. J. B. O'Leary, S. Gleddie, F. Eudes, A. Laroche & L. S. Robert. 2008. A chalcone synthase-like gene is highly expressed in the tapetum of both wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack). Plant Cell Reports 27: 1441-1449.
Yui, R., S. Iketani, T. Mikami & T. Kubo. 2003. Antisense inhibition of mitochondrial pyruvate dehydrogenase Ela subunit in anther tapetum causes male sterility. Plant Journal 34: 57-66.
Zavada, M. S. & N. Gabarayeva. 1991. Comparative pollen wall development of Welwitschia mirabilis and selected primitive angiosperms. Bulletin of the Torrey Botanical Club 118: 292-302.
Zetter, R., M. Weber, M. Hesse & M. Pingen. 2002. Pollen, pollenkitt, and orbicules in Craigia bronnii flower buds (Tilioideae, Malvaceae) from the Miocene of Hambach, Germany. International Journal of Plant Sciences 163: 1067-1071.
Zhang, D., W. Liang, C. Yin, J. Zong, F. Gu & D. Zhang. 2010. OsC6, encoding a lipid transfer protein, is required for postmeiotic anther development in rice. Plant Physiology 154: 149-162.
Zhang, X.-H., Y. Ren & X.-H. Tian. 2012. Microsporogenesis and megasporogenesis in Sinofranchetia (Lardizabalaceae). Flora 207: 197-202.
Brecht Verstraete [1,2,5] * Hye-Kyoung Moon  * Erik Smets [1,3] * Suzy Huysmans [1,4]
 Laboratory of Plant Systematics, KU Leuven, Kasteelpark Arenberg 31, PO Box 2435, 3001 Leuven, Belgium
 Botanic Garden Meise, Domein van Bouchout, 1850 Meise, Belgium
 Naturalis Biodiversity Center, Leiden University, PO Box 9517, 2300 RA Leiden, The Netherlands
 Faculty of Science, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
 Author for Correspondence; e-mail: firstname.lastname@example.org
Published online: 22 May 2014
(1) Rowley (1962) coined the term to acknowledge Gerta von Ubisch (1882-1965), a German biologist who did pioneering work on orbicules in the 1920's. She was, however, not the first scientist to describe orbicules; to our knowledge Rosanoff (1865) was the first to discover orbicules.
Electronic supplementary material The online version of this article (doi:10.1007/s12229-014-9135-1) contains supplementary material, which is available to authorized users.
Table 1 Orbicule distribution data for flowering plants summarized at order level (sensu APG III) based on data in Appendix S1 Total Order # fam. # fam. + # fam. +/- Amborellales 1 1 Nymphaeales 3 2 Austrobaileyales 3 2 Chloranthales 1 1 Magnoliids Cannellales 2 Piperales 5 3 Laurales 7 2 1 Magnoliales 6 3 1 Monocots Acorales 1 Alismatales 13 2 Petrosaviales 1 Dioscoreales 5 2 Pandanales 5 2 Liliales 10 4 Asparagales (incl. Orchidaceae) 14 6 Commelinids Arecales 1 Commelinales 5 Poales 16 5 Zingiberales 8 Possible sister of eudicots Ceratophyllales 1 Eudicots Ranunculales 7 2 1 Proteales 3 2 Sabiaceae 1 Trochodendrales 1 Buxales 2 Core eudicots Gunnerales 2 Dilleniales 1 Saxifragales 14 3 Rosids Vitales 1 Fabids Zygophyllales 2 Celastrales 2 Oxalidales 7 1 Malpighiales 36 3 4 Cucurbitales 7 1 Fabales 4 1 1 Fagales 7 2 Rosales 9 4 Malvids Geraniales 3 I 1 Myrtales 9 1 Crossosomatales 7 Picramniales 1 Huerteales 3 Brassicales 17 3 Malvales 10 1 1 Sapindales 9 2 Berberidopsidales 2 1 Santalales 7 Caryophyllales 34 6 2 Asterids Cornales 6 4 1 Ericales 22 5 1 Lamiids Garryales 2 Gentianales 5 2 3 Lamiales 23 7 3 Solanales 5 1 1 Campanulids Aquifoliales 5 1 Asterales 11 Escalloniales 1 Bmniales 2 1 Paracryphiales 1 Dipsacales 2 1 Apiales 7 1 Unplaced families 8 1 Total 416 90 24 # fam. Order # fam.- no data Amborellales Nymphaeales 1 Austrobaileyales 1 Chloranthales Magnoliids Cannellales 1 1 Piperales 2 Laurales 1 3 Magnoliales 1 1 Monocots Acorales 1 Alismatales 1 10 Petrosaviales 1 Dioscoreales 1 2 Pandanales 3 Liliales 6 Asparagales (incl. Orchidaceae) 1 7 Commelinids Arecales 1 Commelinales 2 3 Poales 11 Zingiberales 3 5 Possible sister of eudicots Ceratophyllales 1 Eudicots Ranunculales 1 3 Proteales 1 Sabiaceae 1 Trochodendrales 1 Buxales 2 Core eudicots Gunnerales 2 Dilleniales 1 Saxifragales 11 Rosids Vitales 1 Fabids Zygophyllales 2 Celastrales 2 Oxalidales 6 Malpighiales 29 Cucurbitales 1 5 Fabales 2 Fagales 5 Rosales 1 4 Malvids Geraniales 1 Myrtales 1 7 Crossosomatales 7 Picramniales 1 Huerteales 3 Brassicales 5 9 Malvales 8 Sapindales 7 Berberidopsidales 1 Santalales 7 Caryophyllales 1 25 Asterids Cornales 1 Ericales 1 15 Lamiids Garryales 2 Gentianales Lamiales 2 11 Solanales 1 2 Campanulids Aquifoliales 4 Asterales 3 8 Escalloniales 1 Bmniales 1 Paracryphiales 1 Dipsacales 1 Apiales 6 Unplaced families 7 Total 35 267 # fam. number of families, + orbicules present,--orbicules absent, +/- orbicules absent or present Table 2 Summary of the numbers of species with information on tapetum type and presence/absence of orbicules (based on data in Appendix S1). Only taxa where the observations are unambiguous are taken into account Orbicules Orbicules Total present absent Parietal tapetum 211 (87.2%) 31 (12.8%) 242 Amoeboid tapetum 7 (20%) 28 (80%) 35 Invasive tapetum 4 (36.4%) 7 (63.6%) 11 Total 222 66 288 Table 3 Species with an amoeboid or invasive tapetum type that are reported to produce orbicules Family Species Reference Amaranthaceae Beta vulgaris Fluysmans et al., 1998; Furness, 2008a Annonaceae Anaxagorea brevipes (a) Gabarayeva, 1995 Apocynaceae Vinca rosea (a) El-Ghazaly & Nilsson, 1991 Asteraceae Cosmos bipinnatus Blackmore & Barnes, 1985 Bromeliaceae Aechmea dichlamydea var. Sajo et al., 2005 trinitensis (a) Butomaceae Butomus umbellatus (b) Fernando & Cass, 1994 Cabombaceae Cabomba caroliniana Taylor et al., 2008 Commelinaceae Tradescantia virginiana Tiwari & Gunning, 1986c (b) Fabaceae Acacia conferta Kenrick & Knox, 1979 Acacia iteaphylla " Acacia subalata " Fagaceae Qnercus robur Rowley & Gabarayeva, 2004 Gentianaceae Canscora decussate (b) Vinckier & Smets, 2003 Gentiana acaulis Lombardo & Carraro, 1976 Swertia perennis (b) Vinckier & Smets, 2003 Lauraceae Persea palustris (b) Furness & Rudall, 2001a Malvaceae A but i Ion pic turn Strittmatter et al., 2000 Modiolastrum malvifolium Galati et al., 2007 Nymphaeaceae Nymphaea colorata (a) Rowley et al., 1992 Nymphaea mexicana (a) Gabarayeva & El-Ghazaly, 1997 (a) Tapetum type is specified as cyclic invasive (b) Species show very small sporopollenin granules, homology with orbicules is not clear
|Printer friendly Cite/link Email Feedback|
|Author:||Verstraete, Brecht; Moon, Hye-Kyoung; Smets, Erik; Huysmans, Suzy|
|Publication:||The Botanical Review|
|Date:||Jun 1, 2014|
|Previous Article:||The fossil record of the Solanaceae revisited and revised--the fossil record of Rhamnaceae enhanced.|
|Next Article:||The nuts and bolts of doing the Flora of the Bahama Archipelago: how Don Correll worked.|