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Anatomy and Morphology |
2 Division of Science, Truman State University, Kirksville, Missouri 63501-4221 USA
Received for publication July 13, 2005. Accepted for publication December 8, 2005.
ABSTRACT
Brasenia is a monotypic genus sporadically distributed throughout the Americas, Asia, Australia, and Africa. It is one of eight genera that comprise the two families of Nymphaeales, or water lilies: Cabombaceae (Brasenia, Cabomba) and Nymphaeaceae (Victoria, Euryale, Nymphaea, Ondinea, Barclaya, Nuphar). Evidence from a range of studies indicates that Nymphaeales are among the most primitive angiosperms. Despite their phylogenetic utility, pollen developmental characters are not well known in Brasenia. This paper is the first to describe the complete pollen developmental sequence in Brasenia schreberi. Anthers at the microspore mother cell, tetrad, free microspore, and mature pollen grain stages were studied using combined scanning electron, transmission electron, and light microscopy. Both tetragonal and decussate tetrads have been identified in Brasenia, indicating successive microsporogenesis. The exine is tectate-columellate. The tetrad stage proceeds rapidly, and the infratectal columellae are the first exine elements to form. Development of the tectum and the foot layer is initiated later during the tetrad stage, with the tectum forming discontinuously. The endexine lamellae form during the free microspore stage, and their development varies in the apertural and non-apertural regions of the pollen wall. Degradation of the secretory tapetum also occurs during the free microspore stage. Unlike other water lilies, Brasenia is wind-pollinated, and several pollen characters appear to be correlated with this pollination syndrome. The adaptive significance of these characters, in contrast to those of the fly-pollinated genus Cabomba, has been considered. Brasenia does not produce pollenkitt nor develop tectal microchannels as does Cabomba. Instead, the discontinuity of the tectum reduces the amount of sporopollenin in the wall, which may allow for more effective wind dispersal. The importance of reassessing palynological characters in light of new ontogenetic data and the phylogenetic implications of this reevaluation are also discussed.
Key Words: anther • Brasenia • Cabombaceae • development • Nymphaeales • ontogeny • pollen • ultrastructure
Brasenia is one of eight genera comprising the two families of Nymphaeales, or water lilies: Cabombaceae (Brasenia, Cabomba) and Nymphaeaceae (Victoria, Euryale, Nymphaea, Ondinea, Barclaya, Nuphar). The intergeneric relationships within Nymphaeales have been elucidated in recent years, supporting the position of Brasenia and Cabomba as divergent sister taxa within the monophyletic family Cabombaceae (Les et al., 1999
; Liu et al., 2005
).
Brasenia is sporadically distributed in freshwater ponds and lakes in temperate and tropical regions of the Americas, eastern Asia, Australia, Africa, and the West Indies. Although no extant populations occur in Europe, Tertiary and Quaternary fossils have been recovered from several European localities (see Williamson and Schneider, 1993
). The sole species of the genus, Brasenia schreberi J.F. Gmelin, is characterized by bright green, floating, peltate leaves and small, purple flowers. The plant produces thick mucilage that covers all of the underwater organs, including the underside of leaves, petioles, stems, and developing floral buds (Osborn and Schneider, 1988
; Williamson and Schneider, 1993
). This mucilage has anti-algal and anti-bacterial properties and may function in alleopathic weed control (Elakovich and Wooten, 1987
).
Brasenia is unique among water lilies in being the only genus that exhibits anemophily, a condition reflected in many of its floral and pollen characters. Anthesis generally occurs from June through September, and the flowers have a 2-day blooming period, with anther dehiscence occurring on the morning of the second day (Osborn and Schneider, 1988
; Osborn et al., 1991
).
There has been an increased focus on Nymphaeales in recent years, as evidence from a broad spectrum of phylogenetic studies indicates that water lilies are among the most primitive of flowering plants. Studies using both molecular and morphological characters have consistently placed Amborella, or Amborella plus Nymphaeales, as the sister group to the remaining angiosperms (e.g., Qiu et al., 1999
; Barkman et al., 2000
; Doyle and Endress, 2000
; Graham and Olmstead, 2000
; Soltis et al., 2000
; Borsch et al., 2003
; Hilu et al., 2003
; Aoki et al., 2004
; see also Soltis and Soltis, 2004
, and references therein).
Investigations of pollen structure and development, and the accompanying changes within the anther, yield an array of characters potentially useful for assessing phylogenetic relationships (Blackmore and Crane, 1988
; Blackmore and Barnes, 1990
; Kreunen and Osborn, 1999
). However, relatively little is known about the pollen of Nymphaeales, including Brasenia. In the majority of studies, light microscopy (LM) was used exclusively to investigate mature pollen grains (e.g., Erdtman, 1954
; Snigerevskaya, 1955
). Scanning electron microscopy (SEM) has also been used to study the mature pollen of several Nymphaealean genera, including Brasenia (e.g., Walker, 1976
; Zhang, 1984
; Osborn and Schneider, 1988
). Fewer studies have employed the use of transmission electron microscopy (TEM) to investigate the mature pollen wall in Brasenia (e.g., Ueno and Kitaguchi, 1961
; Osborn et al., 1991
; Meyer-Melikyan and Diamandopulu, 1996
).
Previous studies of mature pollen of Brasenia have consistently characterized the grains as monosulcate and as having scabrate or indistinct surface ornamentation (e.g., Zhang, 1984
; Osborn et al., 1991
). The exine, however, has been contradictorily characterized as having a granular infratectum by Snigerevskaya (1955)
, as echinate-intectate with whole or partial columellae by Ueno and Kitaguchi (1961)
, and as tectate-columellate by both Osborn et al. (1991)
and Meyer-Melikyan and Diamandopulu (1996)
. Because tectal and infratectal architecture are potentially valuable phylogenetic characters, it is necessary to resolve these conflicting descriptions in order to accurately assess these characters in Brasenia.
These characters, and others, can be better understood in the context of their ontogeny; however, pollen development has not been extensively studied in Nymphaeales, with the exception of Nymphaea (Gabarayeva and Rowley, 1994
; Gabarayeva and El-Ghazaly, 1997
; Gabarayeva et al., 2001
). Pollen ontogeny has never been investigated at the ultrastructural level for five of the eight genera, including Brasenia. One study by Khanna (1965)
did investigate pollen formation in Brasenia using LM and anther smears. This study, however, was limited in its scope and focused on the cytological (chromosomal) aspects of microsporogenesis.
The objective of this paper was to comprehensively document pollen wall ontogeny in Brasenia using combined LM, SEM, and TEM. The details of pollen wall development and the accompanying changes within the anther in Brasenia were then compared to those of the other Nymphaealean genera, primarily Cabomba, in an effort to elucidate the phylogenetic implications of pollen development within the water lilies.
MATERIALS AND METHODS
Plant material
Floral buds of Brasenia schreberi are submerged and located in clusters just below the surface of the water. Seventy-six submerged buds (Fig. 1) were collected at various stages of development in June and July of 2003, 2004, and 2005 from the Atlanta/Long Branch Conservation area near Macon, Missouri, USA (39°52' N, 92°30' W). In addition, five emergent buds (Fig. 2), 10 first-day flowers (Fig. 3), five second-day flowers (Fig. 4), and one third-day flower (Fig. 5) were collected. Five additional first-day flowers were hand-pollinated and set aside for 1–2 h to allow pollen germination to occur.
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Microscopy
For LM and TEM, two to nine anthers from each bud were further dehydrated, then infiltrated and embedded in Spurr's epoxy resin (Electron Microscopy Sciences, Fort Washington, Pennsylvania, USA) following standard protocols. To examine overall pollen and anther histology and identify specimens suitable for ultrastructural studies, the embedded anthers were thick-sectioned with a Leica (Bannokburn, Illinois, USA) Ultracut UCT Ultramicrotome using glass and diamond knives. Thick-sections (850 nm) were collected on glass slides, stained with Richardson's stain (methylene blue and azure II), and imaged with an Olympus (Lake Success, New York, USA) BHS compound microscope. Well-preserved anthers at a range of developmental stages were then thin-sectioned using a diamond knife. Thin-sections (70 nm) were collected and dried on formvar-coated copper slot grids and were stained with uranyl acetate (8 min) and lead citrate (6 min), then imaged with a JEOL (Peabody, Massachusetts, USA) JEM-100SX transmission electron microscope at 80 kV.
For SEM, anthers, buds, and stigmata were further dehydrated in a graded series of ethanol and critical point dried, following standard procedures. Individual anthers were either cut in cross-section using a double-edge razor blade or macerated using a syringe needle, and mounted onto aluminum stubs with double-sided tape. The edges of the tape were affixed to the stub with colloidal graphite. Stubs were then sputter-coated with gold-palladium and imaged with a JEOL JSM-6100 scanning electron microscope at 5 kV.
Sizes of pollen characters were determined by measuring the characters on micrographs of at least five different microspores/pollen grains.
RESULTS
Timing of anther and pollen development is fairly consistent among the anthers within a single floral bud and is nearly synchronous within individual anthers. Each anther contains abundant pollen. The results are divided into sections based on major ontogenetic stage, including the microspore mother cell, tetrad, free microspore, and mature pollen grain stages.
Microspore mother cell stage
Microspore mother cells are present in anthers that range in length from 0.7 to 1.8 mm. Early microspore mother cells are fairly large and appress against one another and against the tapetum (Fig. 6). The nuclei and nucleoli are distinct, and the plasmalemma of each microspore mother cell is surrounded by an electron-dense microspore mother cell coat (Figs. 6, 7). As microspore mother cells develop, they become further separated from one another, and a layer of callose is deposited between the plasmalemma and the microspore mother cell coat. This callose layer is initially thin, but as development proceeds, the callose thickens to reach a maximum thickness of 0.45 µm (Figs. 9–11). The microspore mother cell coat persists outside of the callose layer throughout the microspore mother cell stage (Figs. 9–11).
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Tetrad stage
Tetrads are present in anthers that range from 1.5 to 2.1 mm in length. Each microspore mother cell has undergone meiosis to form a tetrad of microspores. Thick callose surrounds the entire tetrad, while thinner callose septae extend between the individual microspores (Figs. 12, 13). Tetrads occur regularly in both tetragonal and decussate configurations, with both tetrad geometries consistently occurring within the same anther locules (Fig. 12). In early tetrads, the microspore plasmalemma is appressed against the callose (Fig. 15). As development proceeds, the plasmalemma pulls away from the callose, and an electron translucent primexine forms between the callose and the microspore plasmalemma (Fig. 16). The microspore mother cell coat is still present in the early tetrad stage.
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As the tetrad stage continues, the columellae and tectal elements become more robust and more electron dense (Fig. 19). The microspore mother cell coat disintegrates in the middle tetrad stage and the plasmalemma separates slightly from the developing wall (Fig 19). In the late-tetrad stage, a thin foot layer forms in this space at the base of the developing columellae (Fig. 20). The tapetal cells are intact throughout the tetrad stage. They remain tightly appressed, as in the microspore mother cell stage, and contain abundant endoplasmic reticulum (Figs. 12, 14).
Free microspore stage
Free microspores are found in anthers ranging in length from 2.1 to 2.9 mm. This stage initiates when the callose layer surrounding the tetrad breaks apart and the four microspores separate from one another (Fig. 21). During the early-free microspore stage (Figs. 21–27), the tectum, columellae, and foot layer become more electron dense than in the tetrad stage. Throughout the early-free microspore stage, these layers continue to develop and thicken. In the earliest free microspores, the columellae are 50 nm wide and 100 nm tall. As the early-free microspore stage progresses, the width of the columellae increases to 75 nm and the height doubles to 200 nm. Similarly, the tectal elements expand both laterally and in thickness. The overall thickness of the tectum increases almost three-fold throughout the early-free microspore stage. The thickness of the foot layer doubles during the early free microspore stage to 30–40 nm (Figs. 23, 24).
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During the middle-free microspore stage (Figs. 28–33), the thickness of the foot layer increases more than four-fold, reaching a thickness of 140 nm. As the foot layer thickens, the endexine becomes compressed in non-apertural regions until the individual lamellae are difficult to distinguish (Figs. 30, 31). Individual lamellae do remain apparent in regions of the wall adjacent to the aperture, and some extend into the apertural region (Figs. 32, 33). The columellae no longer increase significantly in width and height during the middle-free microspore stage, but the tectum does increase in thickness. New, larger tectal elements develop above the first-formed elements, filling in gaps in the tectum (Figs. 30, 31). However, many discontinuities in the tectum remain, and several of the first-formed, small tectal elements are still visible on the lower side of the tectum. These elements give the inner tectum a slightly granular appearance (Figs. 30, 31). The outermost, secondary tectal elements are also apparent on the surface of the free microspore as developing rodlets. The rodlets are initially small, measuring 0.5 µm in length and 0.2 µm in width, and appear to be randomly organized (Fig. 29).
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In the late-free microspore stage (Figs. 34–39), the plasmalemma pulls away from the developing pollen wall, and the intine begins to develop in this space in the non-apertural wall (Figs. 37, 38). The intine increases in thickness throughout the late-free microspore stage, becoming thickest beneath the aperture (Fig. 36). The endexine lamellae are not distinguishable in the non-apertural regions of the wall (Figs. 37–39). More secondary tectal elements are deposited, but the tectum remains discontinuous, and the primary tectal elements remain visible (Figs. 37–39). The tapetum continues to undergo programmed cell death throughout the late-free microspore stage (Figs. 34, 35), and a small number of orbicules are released from the dissociating tapetal cells (Fig. 35). By the end of the late-free microspore stage, the tapetum is completely disintegrated; however, remnants of the tapetal cells remain attached to the middle layers (Fig. 34). The free microspores become increasingly vacuolated in the late-free microspore stage (Fig. 34).
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This is the first study to comprehensively study pollen and anther ontogeny in Brasenia. Only one published study (Khanna, 1965
) has examined pollen development in Brasenia, and this study was based exclusively on LM. Several key developmental characters are discussed next and summarized in Fig. 47. These characters are interpreted in light of current phylogenetic hypotheses.
Ontogenetic timing
Microspore mother cells become differentiated very early in bud development and anthers more than double in length before meiosis occurs. In contrast, the tetrad stage occurs rapidly. The range of anther sizes that contain tetrads is smaller (1.5–2.1 mm) and overlaps extensively with the contiguous stages. Only anthers ranging from 1.8 to 2.0 mm exclusively contain tetrads. Free microspores are also found within a relatively narrow range of anther sizes (2.1–2.9 mm). During the free microspore stage, the microspore wall undergoes a substantial increase in size. Most of pollen development occurs in submerged buds; however, the latest free microspores occur in emergent buds. The free microspores in emergent buds have a nearly fully developed intine, but the intine is not yet bi-layered. Fully mature pollen grains are found in the nondehiscent, aggregated anthers of first-day flowers and the pre-dehiscent anthers of second-day flowers. Mature pollen is dehisced early in the morning on the second-day of anthesis.
Tetrad geometry and microsporogenesis
Both tetragonal and decussate tetrad configurations were found regularly in anthers of Brasenia, with both types consistently occurring in the same anther locules. This indicates the occurrence of successive microsporogenesis and contradicts Khanna's (1965) report of simultaneous microsporogenesis.
Two major patterns of microsporogenesis, simultaneous and successive, have been recognized and tetrad geometry reflects which pattern occurs (Furness et al., 2002
). In simultaneous microsporogenesis, the first meiotic division is immediately followed by the second meiotic division. Cytokinesis and callose deposition occur after both divisions have occurred. Simultaneous microsporogenesis results in tetrahedral tetrad configurations. In contrast, during successive microsporogenesis, callose is deposited after the first meiotic event, forming a septum, thereby separating the two haploid nuclei. These nuclei then undergo the second meiotic division. Successive microsporogenesis results in a variety of tetrad geometries, including tetragonal and decussate configurations (Furness et al., 2002
).
The pattern of microsporogenesis is considered labile in basal angiosperms; however, the simultaneous type has been considered plesiomorphic in flowering plants (Furness et al., 2002
). This characterization was based, in part, on Khanna's (1965) study of Brasenia that had reported simultaneous microsporogenesis in the genus. Khanna (1965)
described the tetrads of Brasenia as tetrahedral with occasional tetragonal forms. However, in the present study tetrahedral tetrads were never observed, indicating that microsporogenesis is successive in Brasenia.
Recent investigations of Cabomba have indicated that successive microsporogenesis also occurs in that genus (Osborn et al., 2003
). Moreover, Amborella has been described has having successive microsporogenesis (Furness et al., 2002
). The tetrads of Nymphaea (Gabarayeva and Rowley, 1994
) and permanent tetrads of Victoria (Roland, 1965
; Osborn et al., 2004
), however, are found in tetrahedral arrangements, indicating simultaneous microsporogenesis. As successive microsporogenesis is more prevalent in basal angiosperms than previously thought, the phylogenetic status of this character should be reassessed.
Tectum ultrastructure
The exine of Brasenia is relatively thick, with well-defined layers. This study delineates the pattern of ontogeny of these layers and clarifies the ultrastructure of the mature exine. The mature tectum of Brasenia is bi-layered, with an outer homogeneous layer and an inner granular-like layer. The presence and ultrastructure of this inner layer make it somewhat difficult to interpret the structure of the mature exine, especially in oblique section. From the new ontogenetic data, it is clear that the inner layer consists of small, primary tectal elements that are initially deposited in the middle-tetrad stage. The outer, more homogenous layer is composed of secondary tectal elements that are deposited later in the free microspore stage (Fig. 47).
The tectum in Brasenia is laid down discontinuously throughout ontogeny and remains discontinuous in the mature pollen wall. The mature tectum of Cabomba is also discontinuous, although the origin of the discontinuities is ontogenetically different between the genera. In both Cabomba and Brasenia, the tectum initiates as unfused elements. In Cabomba, the tectal elements partially fuse and form a more continuous tectum during the tetrad stage; the discontinuities secondarily arise from the formation of microchannels during the free microspore stage (Osborn et al., 2003
; Gabarayeva et al., 2003
). These microchannels dissect the entire tectum and have been hypothesized to be reservoirs for the copious pollenkitt in Cabomba, which is correlated with fly pollination (Osborn et al., 1991
). Brasenia is wind-pollinated and does not produce pollenkitt, nor are microchannels present in the tectum. In Brasenia, the discontinuities in the tectum occur because neither the primary nor the secondary, sculptural tectal elements become fused during development. Osborn et al. (1991)
have hypothesized that the discontinuous nature of the tectum in Brasenia may reduce the amount of wall material in the tectum. This would reduce the density and the settling speed of the grains, allowing for more effective wind pollination.
Infratectum ultrastructure
The present study indicates that the exine of Brasenia is tectate-columellate, which supports the description by Osborn et al., (1991)
. The new ontogenetic data unequivocally indicate that the granular-like elements found in Brasenia are part of the tectum and not the infratectum. The columellae of Brasenia are the first elements of the exine that form, first appearing in the middle tetrad stage (Fig. 47). The well-defined columellae are similar in structure to those of Cabomba (Osborn et al., 1991
, 2003
; Gabarayeva et al., 2003
), but are much more robust than those of the six genera of Nymphaeaceae.
The inner granular-like layer of the tectum has created some difficulty in accurately describing infratectum ultrastructure in Brasenia.Ueno and Kitaguchi (1961)
characterized mature pollen grains of Brasenia as intectate with partial or whole columellae, while Walker (1976)
, using Ueno and Kitaguchi's (1961) micrographs, interpreted the infratectum as granular, lacking well-defined columellae. Furthermore, the pollen of all eight Nymphaealean genera has historically been described as having a granular infratectum (Ueno and Kitaguchi, 1961
; Ueno, 1962
; Walker, 1974
, 1976
). This character state has been considered plesiomorphic among angiosperms (Doyle et al., 1994
), and a shared state with gymnospermous anthophytes (e.g., Osborn et al., 1991
, 1993
; Osborn and Taylor, 1995
; Osborn, 2000
). This interpretation has changed in recent years, as phylogenetic studies have called into question the validity of the "anthophyte clade" (Bowe et al., 2000
; Chaw et al., 2000
; Donoghue and Doyle, 2000
and references therein). Moreover, a columellar infratectum has more recently been documented in Cabomba and Brasenia (Osborn et al., 1991
, 2003
; Gabarayeva et al., 2003
), as well as in Nymphaea (Rowley et al., 1992
; Gabarayeva and Rowley, 1994
; Gabarayeva and El-Ghazaly, 1997
; Gabarayeva et al., 2001
), and all other genera of Nymphaeaceae (Osborn et al., 2004
). Columellar pollen is also now recognized in other basal angiosperms, including Illiciales, Trimeniaceae, Austrobaileyaceae, and Schisandraceae (see Doyle and Endress, 2000
).
In addition to granular and columellar states, Doyle and Endress (2000)
recognized an "intermediate" state for the infratectum, and they determined this as the ancestral character state in angiosperms. Doyle and Endress (2000)
scored Amborella as intermediate, whereas they scored Nymphaeales as both intermediate (Cabombaceae and Barclaya) and granular (the five other genera of Nymphaeaceae). The current authors disagree with this scoring of Nymphaeales, but recognize that defining an "intermediate" state for the infratectum is a prudent step and indicates that this trait, as well as other palynological characters, is in need of detailed ontogenetic study.
Endexine lamellations
The endexine in Brasenia has not been well described or well documented prior to this study. Thanikaimoni (1985)
interpreted Ueno and Kitaguchi's (1961) micrographs and described the endexine in Brasenia as lamellate. Osborn et al. (1991)
regarded these structures as either a component of the intine or as an artifact, while Walker (1976)
did not mention the presence of an endexine in Brasenia.
In mature pollen, the endexine appears to be lamellate only in the regions of the wall immediately adjacent to the aperture (Fig. 47M). These lamellations are thin and the endexine, as a whole, is not substantially thick. However, from the developmental data it is clear that the endexine is laid down in well-defined plates, both in regions of the wall adjacent to the aperture and in non-apertural regions, initiating during the early-free microspore stage. As the foot layer thickens significantly throughout the early- and middle-free microspore stages, the endexine becomes compressed in the non-apertural wall. By the middle-free microspore stage, the lamellations have become compressed such that they are no longer apparent.
Surface ornamentation
The pollen surface of Brasenia has been described as scabrate, indistinct, or shapeless (Snigerevskaya, 1955
; Walker, 1976
; Osborn et al., 1991
). In the present study, however, small cylindrical elements, or rodlets, were consistently observed. The sculptural rodlets correspond to the secondary tectal elements. At the early-free microspore stage, the surface does appear scabrate, because the tectum is composed only of small, primary elements. The rodlets appear later in development, after the secondary tectal elements begin to be deposited in the middle-free microspore stage. Initially, these rodlets are small and sparsely cover the microspore. As development proceeds, the number and size of the rodlets increase such that the non-apertural surface of the mature pollen grain becomes completely covered by rodlets. Rodlets are not present on the mature apertural wall.
The pollen of only two genera of Nymphaeales have significant surface ornamentation, Nuphar and Cabomba. The surface of mature pollen of Nuphar is characterized by large, vertical spines (Ueno and Kitaguchi, 1961
; Walker, 1976
; Takahashi, 1992
), whereas the mature pollen of Cabomba has parallel rods that run the length of the grain (Walker, 1976
; Osborn et al., 1991
). The apertural region of Cabomba, however, lacks these parallel rods and is instead characterized by smaller rods that are randomly oriented (Osborn et al., 2003
). These apertural rods in Cabomba resemble the rodlets found on the surface of mature Brasenia pollen.
Tapetal ontogeny
The tapetum in Brasenia first becomes distinguishable in the microspore mother cell stage, when the cells are multinucleate and have abundant endoplasmic reticulum. Tapetal cells persist through the tetrad stage and undergo vacuolization in the early-free microspore stage. In the middle-free microspore stage, the tapetal cells begin to disintegrate, and only a remnant of the tapetum remains by the late-free microspore stage. The cells never become detached from the anther wall and, as such, the tapetum in Brasenia is of the secretory type.
The secretory type of tapetum is predominant in land plants and is found in most gymnosperms (Pacini, 1997
; Furness and Rudall, 2001
and references therein). All Nymphaealean genera, as well as Amborella, have been previously described as having a secretory tapetum, which has been considered the plesiomorphic type within angiosperms (Furness and Rudall, 2001
). However, other investigations have indicated the presence of an amoeboid tapetum in Cabomba (Osborn et al., 2003
) and a cyclic-invasive tapetum in Nymphaea (Rowley et al., 1992
).
Orbicules, or Ubisch bodies, are small bodies composed of sporopollenin and are almost always associated with a secretory tapetum. Their function is unclear, although they may serve to transport sporopollenin from the tapetum to the developing microspore (Huysmans et al., 1998
; Furness and Rudall, 2001
) or as an aid in pollen dispersal. This latter function may play an especially important role in wind pollination (Pacini and Franchi, 1993
). Orbicules are present in Brasenia, but even though Brasenia has a secretory tapetum and is wind-pollinated, they occur in small numbers and appear only in the late-free microspore stage.
Conclusion
As a member of Nymphaeales, Brasenia is among the most primitive of flowering plants. Because of their position near the base of the angiosperm tree, water lilies are of considerable interest. Despite their utility in phylogenetic analysis, pollen characters, especially ontogenetic characters, are not well known in Nymphaeales. The present study elucidated salient pollen developmental characters of Brasenia and reexamined the ultrastructure of the mature pollen wall. Several key pollen characters for Brasenia, such as tetrad geometry and infratectum ultrastructure, have been mischaracterized in the literature, and the subsequent scoring of those characters in a range of phylogenetic studies has been impacted. Other characters, such as the presence of endexine lamellae in the non-apertural wall, the granular-like nature of the inner tectum, and the formation of sculptural rodlets, are only revealed upon investigation of the complete developmental sequence. This emphasizes the need for careful and consistent scoring of pollen characters, the importance of including ontogenetic data in phylogenetic analyses, and the re-evaluation of how a range of traditional characters are interpreted in a phylogenetic context.
FOOTNOTES
1 The authors thank E. A. Hooper, A. S. Doores, A. B. Schwendemann, and T. C. Thiemann (Truman State University) for assistance with field collection and the Missouri Department of Conservation for access to plant material. This study was supported in part by the National Science Foundation (IBN-0212521) and Truman State University (Undergraduate Student Research Grant). 
3 Current address: Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, Tennessee 37996 USA
4 Author for correspondence (e-mail: josborn{at}truman.edu
)
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