| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Division of Science, Truman State University, Kirksville, Missouri 63501-4221
Received for publication August 31, 1998. Accepted for publication April 22, 1999.
ABSTRACT
The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera and Nelumbo lutea. Historically, the genus has been considered to be closely related to Nymphaeales, however new systematic work has allied Nelumbo with lower eudicots, particularly Platanus. In recent years, studies of pollen development have contributed greatly to the understanding of phylogenetic relationships, but little has been known about these events in Nelumbo. In this paper, pollen and anther development are morphologically described for the first time in N. lutea. A comprehensive ontogenetic sequence is documented, including the sporogenous tissue, microspore mother cell, tetrad, free spore, and mature pollen grain stages. The deposition of a microspore mother cell coat and callose wall, the co-occurrence of both tetrahedral and tetragonal tetrads, the formation of a primexine in tetrads, and primexine persistence into the late free spore stage are shown. The majority of exine development occurs during the free spore stage with the deposition of a tectate-columellate ectexine, a lamellate endexine, and an unusual granular layer below and intermixed with the endexine lamellae. A two-layered intine forms rapidly during the earliest mature pollen stage. Major events of anther development documented include the degradation of a secretory-type tapetum during the free spore stage and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The majority of mature pollen grains are tricolpate, however less common monosulcate and diaperturate grains also develop. Co-occurring aperture types in Nelumbo have been suggested to be an important transition in angiosperm aperture number. However, aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus, which occurs in the early free spore stage. This character, as well as other details of pollen and anther ontogeny in Nelumbo, are compared to those of Nymphaeales and Platanus in an effort to provide additional insight into systematic and phylogenetic relationships. Although Nelumbo is similar to both groups in several characters, the ontogenetic sequence of the genus is different in many ways.
Key Words: anther • development • morphology • Nelumbo • Nelumbonaceae • pollen • ultrastructure
The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera Gaertn. and Nelumbo lutea (Willd.) Pers. Nelumbo nucifera, the Indian or sacred lotus, is found throughout Asia and Australia, whereas N. lutea, the American lotus or water chinquapin, occurs in eastern and southern North America (Williamson and Schneider, 1993
). More recently, Nelumbo lutea has been considered to be a subspecies of N. nucifera (Borsch and Barthlott, 1994
).
Historically, many authors have considered Nelumbo to be closely related to Nymphaeales (water lilies; Moseley, 1958
; Ueno and Kitaguchi, 1961
; Meyer, 1964
; Walker, 1976
; Batygina and Shamrov, 1983
; Ito, 1987
; Cronquist, 1988
). This taxonomic position was based primarily on similarities in floral and vegetative morphology, as well as in habitat (e.g., Cronquist, 1988
). However, there has been disagreement regarding this classification, and several studies have attempted to resolve the phylogenetic relationship of lotuses and water lilies. Past investigations have included studies of floral anatomy (e.g., Moseley and Uhl, 1985
; Ito, 1986
), leaf anatomy and alkaloids (e.g., Goleniewska-Furmanowa, 1970
; Kristen, 1971
; Rao and Banerjee, 1979
; Barthlott et al., 1996
), and seed anatomy (e.g., Collinson, 1980
). One especially important difference between lotuses and water lilies is the triaperturate pollen of Nelumbo, which differs from the monoaperturate pollen grains of most Nymphaeales (Ueno and Kitaguchi, 1961
; Walker, 1976
; Kuprianova, 1979
; Osborn, Taylor, and Schneider, 1991
). The aforementioned suite of characters has supported the placement of Nelumbo in its own family and order, distinct from Nymphaeales (Williamson and Schneider, 1993
, and references therein). Williamson and Schneider (1993)
have suggested that the similarities between Nelumbo and water lilies are attributed either to a shared ancestor or to convergent evolution.
Recent studies have offered support for the convergence hypothesis. Phylogenetic analyses based on chloroplast rbcL gene sequence data have indicated that Nelumbo may only be remotely related to water lilies (Les, Garvin, and Wimpee, 1991
; Chase et al., 1993
; Qui et al., 1998
). Studies of 18S ribosomal DNA sequences have also found Nelumbo and Nymphaea to be only distantly related (Soltis et al., 1997
). Most molecular studies have placed Nelumbo among lower eudicots, although there is no consensus on its exact taxonomic position within this group. Nelumbo has been placed in the Hamamelidae, closely related to Platanus (Chase et al., 1993
; Qui et al., 1998
), as well as among the Ranunculids (Soltis et al., 1997
). Additional phylogenetic analyses, based on both molecular and nonmolecular characters, have also placed Nelumbo among the Hamamelids or Ranunculids (Donoghue and Doyle, 1989
; Nandi, Chase, and Endress, 1998
).
Another line of evidence for assessing phylogenetic relationships among plants comes from studies of pollen development, specifically ontogeny of the pollen wall (e.g., Blackmore and Crane, 1988
; Blackmore and Barnes, 1990
; Gabarayeva, 1991
; Zavada, 1991
). However, no published studies have documented a complete pollen developmental sequence in the genus Nelumbo, and none has addressed any aspect of pollen ontogeny in Nelumbo lutea. One study examined pollen wall development in Nelumbo nucifera using transmission electron microscopy (TEM; Flynn and Rowley, 1971a
), but these authors observed samples only in the tetrad stage specifically with respect to primexine and aperture formation (Flynn and Rowley, 1971a
; see also Rowley, 1975
). Furthermore, only four studies have focused on anther development in Nelumbo. Each of these used light microscopy (LM) exclusively and examined the single taxon N. nucifera (Khanna, 1965
; Gupta and Ahluwalia, 1979
; Batygina, Kravtsova, and Shamrov, 1980
; Batygina and Shamrov, 1983
).
In contrast to the few studies on pollen development, more is known about the morphological aspects of mature pollen grains of Nelumbo. Ueno and Kitaguchi (1961) used TEM to examine ultrastructural features of the mature pollen wall from nine species of Nymphaeaceae, including Nelumbo nucifera. Walker (1976)
studied pollen of Nelumbo lutea using scanning electron microscopy (SEM), and several other studies have used LM and SEM to focus on the morphology of both extant and fossil Nelumbo pollen in a comparative context (e.g., Kuprianova and Tarasevich, 1983
; Skawinska, 1985
; Zetter and Keri, 1989
).
The primary objective of this paper was to document the events of pollen and anther development in Nelumbo lutea using LM, TEM, and SEM. Furthermore, the details of pollen and anther ontogeny in Nelumbo were compared to those of Nymphaeales and lower eudicots, primarily Platanus, in an effort to provide additional insight into systematic and phylogenetic interpretations.
MATERIALS AND METHODS
Floral buds of Nelumbo lutea extend above the water surface on long peduncles and are easily accessible. Within the buds, numerous stamens surround a central, cone-shaped receptacle in which the gynoecium is embedded. The stamens of Nelumbo consist of a thin filament, an elongate anther, and a vegetative tip appendage that has thermogenic properties (capable of raising bud and floral temperatures an average of 2.3°–4.8°C above ambient; see Schneider, Williamson, and Whitenberg, 1990
, and references therein). Anthers were measured from the base of the thermogenic tip to the top of the filament.
Ten floral buds were collected from Lilypons aquatic nursery in Brookshire, Texas, and an additional 49 buds were collected from Lake Springfield in Springfield, Missouri. Anthers were dissected from the buds in the field and were fixed in 3% glutaraldehyde (in 0.2 mol/L phosphate buffer, pH 7.4) for 24 h and then buffer-washed at least four times. Specimens were postfixed in 1% osmium tetroxide (in 0.2 mol/L phosphate buffer, pH 7.4) for 3 h and buffer-washed four times.
The anthers for LM and TEM were dehydrated in a series of ethanol and acetone rinses and then gradually infiltrated and embedded in Spurr epoxy resin. Embedded anthers were sectioned on an ultramicrotome with glass or diamond knives. Thick sections (850 nm) were stained with either Richardson's Stain (Azure II and Methylene Blue) or Toluidine Blue O and examined/imaged with bright-field and differential interference contrast illumination on an Olympus BHS Compound Light Microscope. Thin sections (90–100 nm) were collected on 1 x 2 mm slot grids and dried on formvar-support films (Rowley and Moran, 1975
). Grids were stained with 1% potassium permanganate (0–2 min), 1% uranyl acetate (10 min), and lead citrate (8 min; Venable and Coggeshall, 1965
) and then examined/imaged using a JEOL JEM-100SX transmission electron microscope at 80 kV.
Anthers for SEM were dehydrated in a graded ethanol series, critical point dried, and mounted onto aluminum stubs with collodial graphite. To view morphological features of the anther wall and the locular contents, some anthers (while in 70% ethanol) were either transversely fractured with a double-edged razor blade at room temperature, or were frozen in liquid nitrogen and then fractured. Additional anthers were transversely fractured after critical point drying. To view individual pollen grains, dried anthers were macerated using a syringe needle and mounted onto aluminum stubs with double-sided adhesive tape. All stubs were sputter-coated with gold-palladium and evaluated/imaged using a JEOL JSM-6100 scanning electron microscope at 5 kV.
RESULTS
Within a single floral bud, anther and pollen development typically vary in timing among the stamen whorls (personal observation). Furthermore, pollen development is not synchronous within individual anthers, but varies along the length of the individual anthers. The results presented below have been divided into sections based on the following major stages of pollen ontogeny: sporogenous tissue, microspore mother cells, tetrads, free spores, and mature pollen grains.
Sporogenous tissue stage
The sporogenous tissue completely fills the locular space. Individual cells tightly abut and have somewhat polygonal shapes (Figs. 1–2). The cell cytoplasm stains densely with chromatic stains (Fig. 1), and the nuclei are relatively large (Figs. 1–2). Anthers in the sporogenous stage range in length from 2.5 to 7 mm. The tapetum is undifferentiated at this stage (Fig. 1).
|
|
Anthers in the microspore mother cell stage range in length from 8 to 11 mm. The tapetal cells tightly abut, are multinucleate, and contain abundant endoplasmic reticulum (Figs. 3–4, 9). The tapetal cells have well-defined primary cell walls at this stage (Figs. 4, 9). Large vacuoles are also present in many tapetal cells. These vacuoles contain an electron-dense substance resembling a locular matrix that surrounds the microspore mother cells (Fig. 9) and often completely fills the locular space (Figs. 4, 9). Another, more dense substance is also present in the locules. This second locular matrix is concentrated in regions of contact between adjacent microspore mother cells (Fig. 10), as well as between microspore mother cells and tapetal cells.
Tetrad stage
The majority of the tetrads occur in a tetrahedral arrangement (Figs. 11–14), but a small proportion have been documented in a tetragonal configuration (Fig. 12). The tetragonal tetrads were not found in all anthers, but rather occurred in specific anthers. The microspore mother cell coat (Figs. 14–16) and underlying callose (Figs. 11–17) are persistent through the duration of the tetrad stage. Both layers surround the entire tetrad, whereas only callose separates individual members of the tetrad (Figs. 12, 14). In early tetrads, the microspore plasmalemma is tightly appressed against the callose (Fig. 15). At the middle tetrad stage, the microspore plasmalemma pulls away from the callose and a primexine begins to develop (Fig. 16). The primexine has a lamellar appearance and uniformly surrounds each of the four microspores within the tetrads. In late tetrads, the primexine thickens and numerous electron-dense procolumellae become distinct and span the height of the primexine (Fig. 17).
|
Free spore stage
Breakdown of the callose wall marks the transition from the tetrad stage to the free spore stage. In the earliest free spore stage, anthers contain microspores still in a tetrad arrangement, but with residual callose present between free spores (Figs. 18–20). During this stage, the initial tectum forms over the primexine in nonapertural regions. In surface view, the tectum of early free spores appears as a loose reticulum (Figs. 21–22). In transverse section, the tectal elements are widely spaced, a foot layer is established in the lowermost region of the primexine, and columellae begin to form on the procolumellae (Fig. 23).
|
Anthers in the early free spore stage range in length from 11 to 15 mm. By this stage, the locular matrix has disintegrated and free spores completely fill the locular space. During the early free spore stage the tapetum begins to dissociate; the primary cell walls begin to degrade and individual cells initially pull apart (Fig. 25). The cytoplasm of the tapetal cells remains relatively dense and contains pre-orbicular bodies and abundant lipid globules (Fig. 25).
At the middle free spore stage, tectum deposition continues in nonapertural regions and forms a more reticulate surface sculpture. Deposition does not occur uniformly in all grains, as patchy areas of a more dense ornament may form (Figs. 26–27). At nonapertural walls, more columellae form and widen, the foot layer becomes much thicker, and endexine lamellae develop (Fig. 29). At the apertural regions, the columellae widen, additional foot layer is deposited, and the endexine lamellae thicken and anastomose (Fig. 28). Furthermore, the apertures widen (Fig. 26). At both apertural and nonapertural regions, the primexine begins to dissociate and a layer of minute granules develops intermixed with the endexine lamellae (Figs. 28–29).
|
|
Mature pollen grain stage
The majority of mature pollen grains are tricolpate (Figs. 35, 38) and have a uniformly dense reticulate ornamentation (Figs. 35–36). The nonapertural wall is characterized by a well-developed ectexine, a compressed layer of endexine lamellae and granules, and a two-layered intine (Fig. 39). In surface view, the apertures are intectate, but have well-defined ectexine elements present (Fig. 37). The endintine is thicker at apertural regions and causes the entire pollen wall to bulge slightly (Figs. 38, 40).
|
|
|
This is the first study to comprehensively examine pollen and anther ontogeny in Nelumbo, as well as investigate any aspect of pollen development in Nelumbo lutea using electron microscopy. The major events of sporoderm and tapetal ontogeny are summarized in Fig. 49 and specific developmental characters are discussed below. Furthermore, new data on pollen and anther development are interpreted regarding their phylogenetic implications.
|
; Schmid, Eberwein, and Hesse, 1996
). At the tetrad stage of N. lutea, the primexine is distributed completely and uniformly around the individual microspores, and there does not appear to be accumulation of endoplasmic reticulum at potential aperture sites. This coincides with Flynn and Rowley's (1971a)
report that the primexine at the tetrad stage did not indicate future aperture positions in Nelumbo nucifera. The first evidence of aperture formation detected in N. lutea was in the earliest free spores, where apertures appear as indentations at the equatorial regions and are the sites of the first identifiable endexine lamellae (Fig. 49). The current study provides confirmation of the post tetrad establishment of apertures in Nelumbo, as early reports of this in N. nucifera (Flynn and Rowley, 1971a
; see also Rowley, 1975
) have not been widely recognized.
Observations of the two tetrad configurations and co-occurring monoaperturate and triaperturate pollen in Nelumbo lutea complement those of Kuprianova (1979)
, who documented the same in Nelumbo nucifera. Blackmore, Stafford, and Persson (1995) also reported boat-shaped monoaperturate grains in N. nucifera, however, the monoaperturate grains of N. lutea found in the present study were spheroidal, as are the common triaperturate grains. Kuprianova (1979)
has suggested that the co-occurring aperture types in Nelumbo could be an important transition in angiosperms, from monoaperturate to triaperturate pollen. However, as demonstrated in this study, several other aperture types also exist in N. lutea and have also recently been reported in N. nucifera (Borsch and Wilde, 1999
).
An alternative explanation to Kuprianova's (1979)
transitional hypothesis is that aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus. Borsch and Wilde (1999)
have also suggested that aperture variation in Nelumbo may be due to weak control of the developmental processes that govern aperture formation. These authors further suggest that such plasticity in aperture control mechanisms may be a primitive condition and that this plesiomorphic state may be present in Nelumbo (Borsch and Wilde, 1999
). Although this postulate complements the developmental hypothesis posed in the present paper, the degree to which aperture variation in Nelumbo can be attributed to phylogeny vs. ontogeny requires additional study.
Exine ontogeny
The primexine is the location for sporopollenin deposition of the exine. Although procolumellae form during the late tetrad stage, exine development does not occur until the free spore stage. As the free spore stage progresses, the ectexine layers mature more or less simultaneously; the tectal elements thicken, the columellae widen, and the foot layer thickens (Fig. 49). Some sporopollenin of the tectum may be deposited late in the free spore stage from tapetal orbicules, thereby contributing to the reticulate morphology. This may not occur uniformly across the surface of the pollen, as isolated patches of varying tectum density are found in some free spores.
Initiation of the endexine occurs after initial formation of the ectexine in the early free spore stage, as in many angiosperms (e.g., Blackmore and Barnes, 1990
). However, distinguishing between endexine and foot layer is often difficult, especially at apertural regions, where these layers are thickened, noncontinuous, and often anastomose. Furthermore, the endexine and foot layer commonly have similar electron densities, an occurrence related to staining properties that has been noted in other taxa as well (e.g., Weber, 1998
; El-Ghazaly, Swedish Museum of Natural History, personal communication). When the two layers exhibited similar staining densities in N. lutea pollen, the endexine lamellae were identified by the presence of white lines.
At the middle free spore stage, a two-zoned granular layer forms below the endexine lamellae. This layer appears to compress the lamellae against the foot layer. The granules persist into the mature pollen stage, but the layer is reduced in thickness (Fig. 49). It is possible that the granular layer is another component of the endexine. Similar granules have been identified in other angiosperms and found to be acetolysis resistant, although not definitively described as endexine (e.g., Huysmans, El-Ghazaly, and Smets, 1998
). Alternatively, such a granular layer has been suggested to be an intine precursor (see below).
Intine ontogeny
The only stage in which an intine layer was detected was in fully mature pollen grains, located within dehiscent or near dehiscent anthers. Furthermore, the intines documented were well developed and two layered (Fig. 49). Consequently, it is presumed that the events of intine development occur rapidly. As discussed above, it is possible that the granular layer below the endexine lamellae, first detectable in the free spore stage, could be a precursor to the intine. A layer of similar appearance has been described as an intine precursor in Liriodendron chinense (hlemsl.) Sarg (Gabarayeva, 1996
).
Locule and anther wall ontogeny
The locular matrix present at the microspore mother cell and tetrad stages can be abundant and often fills the locular space. This matrix material surrounds the microspore mother cells or tetrads and appears to suspend the cells within the anther locule. It is not clear whether or not the more dense matrix present between adjacent cells is a different substance or whether it is the same matrix material found throughout the locules, but has a different ultrastructure as a result of fixation influences. The abundant matrix material is presumed to be secreted by the tapetum, as tapetal cells have large vacuoles containing a substance that is similar in ultrastructure and electron density. A locular matrix described as "colloidal" was identified by Farr (1922)
in Nelumbo lutea, but it was reported to occur only in the microspore mother cell stage. Following the tetrad stage, individual free spores and pollen grains occupy the entire locular space.
The tapetum is first distinguishable at the microspore mother cell stage and is of the secretory type. Tapetal cells retain a similar morphology throughout the microspore mother cell and tetrad stages. Tapetal degradation occurs at the free spore stage, during which cell walls break down and numerous orbicules are synthesized and secrected (Fig. 49). A secretory tapetum has also been described in N. nucifera based on studies using LM (Batygina et al., 1980
; Batygina and Shamrov, 1983
).
Endothecial thickenings are clearly U-shaped in Nelumbo lutea and were observed only in locules containing mature pollen grains (i.e., with complete intines). Endothecial cells with early to intermediate secondary wall thickenings were not detected at earlier ontogenetic stages. Consequently, it is presumed that the cell walls thicken rapidly during the latest free spore or earliest mature pollen grain stages. Previously, only Moseley (1958)
has described U-shaped endothecial wall thickenings in Nelumbo nucifera. Other studies have described the endothecial thickenings of N. nucifera as both "feeble" (Khanna, 1965
) and "fibrous," but masked by tannins (Gupta and Ahluwalia, 1979
); however, it was not clear from these studies whether the thickenings were U-shaped. The abundant tannins described by Gupta and Ahluwalia (1979)
in N. nucifera may be related to the fixatives used (formalin-acetic acid-alcohol and Nawaschin's fluid); tannins were not detected in the current study of N. lutea.
Phylogenetic implications
As discussed above, Nelumbo has been phylogenetically allied with both Nymphaeales and more recently with lower eudicots, especially Platanus. Therefore, comparisons of pollen ontogeny among Nelumbo and these groups may provide new insight into phylogenetic relationships. Six published studies have examined ultrastructural aspects of pollen development in Nymphaeales (water lilies). These include investigations of two genera of Nymphaeaceae: Nymphaea (Gabarayeva, 1991
; Rowley, Gabarayeva, and Walles, 1992
; Gabarayeva and Rowley, 1994
; Gabarayeva and El-Ghazaly, 1997
) and Nuphar (Flynn and Rowley, 1971b
; Takahashi, 1992
), although only limited developmental stages and characters have been reported for Nuphar. Only one published study has focused on pollen ontogeny in Platanus (Suarez-Cervera, Marquez, and Seoane-Camba, 1995
).
Pollen development in Nelumbo shares some similarities with that of both water lilies and Platanus; however, the ontogenetic sequence of the genus is different in many ways. Aperture formation occurs in the free spore stage in Nelumbo, which is later than that in both Nymphaea mexicana A. Gray (Gabarayeva and El-Ghazaly, 1997
) and Platanus acerifolia (Aiton) Willdenow (Suarez-Cervera, Marquez, and Seoane-Camba, 1995
). In the pollen of both N. mexicana and P. acerifolia, initiation of the apertures is apparent at the tetrad stage. Aperture number and position are also characters that do not clearly link Nelumbo to either water lilies or Platanus. Platanus pollen is triaperturate, with three equatorially positioned colpi, as are the majority of Nelumbo pollen grains. In contrast, the occurrence of monoaperturate pollen in Nelumbo, in which the aperture encircles the grain, correlates with the pollen of Nymphaea, Ondinea, Barclaya, and Euryale (Nymphaeaceae; e.g., Walker, 1976
; Osborn, unpublished data
). However, as discussed above, aperture variation in Nelumbo may be related to the late ontogenetic timing of aperture establishment and not necessarily informative in a strict phylogenetic context. Therefore, this character alone may be difficult to compare with that of other taxa until more information is available about aperture development.
Comparisons of exine morphology and architecture are also relevant. The tectum of Nelumbo pollen is reticulate, like that of Platanus and many other lower eudicot taxa (Suarez-Cervera, Marquez, and Seoane-Camba, 1995
; Blackmore, Stafford, and Persson, 1995
), whereas the pollen of water lilies has a variety of nonreticulate ornamentation patterns (e.g., verrucate, scabrate, psilate, spinose, and striate; Walker, 1976
; Osborn, Taylor, and Schneider, 1991
; Takahashi, 1992
; Gabarayeva and El-Ghazaly, 1997
). Regarding the infratectal layer, the columellate infratectum of Nelumbo has been considered more derived than the historically described granular infratectum of water lilies (e.g., Walker, 1976
) and thereby more similar to that of Platanus and other lower eudicots. More recent studies, however, have documented a columellate infratectum in the pollen of Brasenia and Cabomba (Cabombaceae; Osborn, Taylor, and Schneider, 1991
), as well as in Nymphaea pollen (Nymphaeaceae; Gabarayeva and Rowley, 1994
; Gabarayeva and El-Ghazaly, 1997
). The columellae of these three water lily taxa are not as distinct as those of Nelumbo; in particular, the columellae of Nymphaea are somewhat irregularly shaped. The synchronous timing of ectexine development and the subsequent formation and ultrastructure of endexine lamellae in Nelumbo pollen are similar to that in both Nymphaea and Platanus pollen. The granular layer that forms beneath the lamellae in Nelumbo also occurs during pollen ontogeny in Nymphaea (Gabarayeva and El-Ghazaly, 1997
), but not in Platanus (Suarez-Cervera, Marquez, and Seoane-Camba, 1995
).
Most recent research concerning the systematic position of Nelumbo has been based on molecular characters and has placed Nelumbo among lower eudicots (e.g., Chase et al., 1993
; Soltis et al., 1997
; Nandi, Chase, and Endress, 1998
; Qiu et al., 1998). However, the historical association of Nelumbo with Nymphaeales has also recently been supported based on analyses using character compatibility (Meacham, 1994
). Unfortunately, results from the present study of Nelumbo do not clearly link the genus to either lower eudicots or to Nymphaeales. The new data on pollen and anther ontogeny in Nelumbo, however, do fill an important gap in understanding this interesting plant, and they provide the opportunity for additional, comprehensive comparisons. Further clarification of the systematic position of Nelumbo will be aided by more investigations of pollen development in a greater number of other lower eudicot and nymphealean taxa.
FOOTNOTES
1 The authors thank Norman and Jean Youngsteadt (Springfield, Missouri), Benjamin Gutman (Duke University), as well as Ranessa Cooper and Bradford Day (Truman State University) for assistance with field collection; Lilypons (Brookshire, Texas) for access to plant material; and Stephen Blackmore (The Natural History Museum, London) and Thomas Borsch (Botanisches Institute und Botanischer Garten der Universität Bonn) for critically reviewing an early draft of this paper. This study was supported in part by Truman State University (Biology Alumni Research Stipend and Science Division Student Research Grant to SSK; Faculty Research Grant to JMO); Tri-Beta, National Biological Honor Society (Student Research Grant to SSK); and the National Science Foundation (Research Experience for Undergraduates; NSF-BIR-9424233). Acknowledgement is also made to the donors of The Petroleum Research Fund, administered by the ACS, for partial support of this research (ACS-PRF 29004-GB8). 
2 Current: Department of Plant Biology, Arizona State University, Tempe, Arizona 85287.
3 Author for correspondence (e-mail: josborn{at}truman.edu
).
LITERATURE CITED
Barthlott, W., C. Neihnuis, R. Jetter, T. Bourauel, and M. Riederer. 1996 Waterlily, poppy, or sycamore: on the systematic position of Nelumbo. Flora 191: 169–174.
Batygina, T. B., and I. I. Shamrov. 1983 Embryology of the Nelumbonaceae and Nymphaeaceae: pollen grain structure (some peculiar features of correlated development of the pollen grain and anther wall). Botanicheskii Zhurnal 68: 1177–1183.
———, T. I. Kravtsova, and I. I. Shamrov. 1980 Comparative embryology of some representatives of the orders Nymphaeales and Nelumbonales. Botanicheskii Zhurnal 65: 1071–1087.
Blackmore, S., and S. H. Barnes. 1990 Pollen wall development in angiosperms. In S. Blackmore and R. B. Knox [eds.], Microspores: evolution and ontogeny, 173–192. Academic Press, London.
———, and P. R. Crane. 1988 The systematic implications of pollen and spore ontogeny. In C. J. Humphries [ed.], Ontogeny and systematics, 83–115. Columbia University Press, New York, NY.
———, P. Stafford, and V. Persson. 1995 Palynology and systematics of Ranunculiflorae. Plant Systematics and Evolution (Suppl.) 9: 71–82.
Borsch, T., and W. Barthlott. 1994 Classification and distribution of the genus Nelumbo Adans. (Nelumbonaceae). Beiträge zur Biologie der Pflanzen 68: 421–450.
———, and V. Wilde. 1999 Pollen variability within species, populations, and individuals. In M. Harley, S. Blackmore, and C. Morton [eds.], Pollen and spores: morphology and biology. Royal Botanic Gardens, Kew, in press.
Chase, M. W., et al. 1993 Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Annals of the Missouri Botanical Garden 80: 528–580. [CrossRef][ISI]
Collinson, M. E. 1980 Recent and Tertiary seeds of the Nymphaeaceae sensu lato with a revision of Brasenia ovula (Brong.) Reid and Chandler. Annals of Botany 46: 603–632.
Cronquist, A. 1988 The evolution and classification of flowering plants. New York Botanical Garden, Bronx, NY.
Donoghue, M. J., and J. A. Doyle. 1989 Phylogenetic analysis of angiosperms and the relationships of Hamamelidae. In P. R. Crane and S. Blackmore [eds.], Evolution, systematics, and fossil history of the Hamamelidae, 17–45. Clarendon Press, Oxford.
Farr, C. H. 1922 The meiotic cytokinesis of Nelumbo. American Journal of Botany 9: 296–306.
Flynn, J. J., and J. R. Rowley. 1971a The primexine of Nelumbo nucifera. Experientia 27: 227–228.
———, and ———. 1971b Wall microtubules in pollen grains. Zeiss Informatics 76: 40–45.
Gabarayeva, N. I. 1991 Patterns of development in primitive angiosperm pollen. In S. Blackmore and S. H. Barnes [eds.], Pollen and spores: patterns of diversification, 257–268. Clarendon Press, Oxford.
———. 1996 Sporoderm development in Liriodendron chinense (Magnoliaceae): a probable role of the endoplasmic reticulum. Nordic Journal of Botany 16: 307–323. [ISI]
———, and G. El-Ghazaly. 1997 Sporoderm development in Nymphaea mexicana (Nymphaeaceae). Plant Systematics and Evolution 204: 1–19. [CrossRef][ISI]
———, and J. R. Rowley. 1994 Exine development in Nymphaea colorata (Nymphaeaceae). Nordic Journal of Botany 14: 671–691. [ISI]
Goleniewska-Furmanowa, M. 1970 Comparative leaf anatomy and alkaloid content in the Nymphaeaceae. Monographiae Botanicae 31: 1–55.
Gupta, S. C., and R. Ahluwalia. 1979 The anther and ovule of Nelumbo nucifera—a reinvestigation. Journal of the Indian Botanical Society 58: 177–182.
Huysmans, S., G. El-Ghazaly, and E. Smets. 1998 Pollen wall, tapetum and orbicule development in Rondeletia odorata (Rubiaceae). Pollen and spores: morphology and biology, Abstracts, 11. Royal Botanic Gardens, Kew.
Ito, M. 1986 Studies in the floral morphology and anatomy of Nymphaeales. IV. Floral anatomy of Nelumbo nucifera. Acta Phytotaxonomica et Geobotanica 37: 82–96.
———. 1987 Phylogenetic systematics of the Nymphaeales. Botanical Magazine, Tokyo 100: 17–35. [CrossRef][ISI]
Khanna, P. 1965 Morphological and embryological studies in Nymphaeaceae. II. Brasenia schreberi Gmel. and Nelumbo nucifera Gaertn. Australian Journal of Botany 13: 379–387. [CrossRef]
Kristen, U. 1971 Licht- und elektronenmikroskopische Untersuchungen zur Entwicklung der Hydropoten von Nelumbo nucifera Gaertn. Berichte der Deutschen Botanischen Gesellschaft 84: 211–214.
Kuprianova, L. A. 1979 On the possibility of the development of tricolpate pollen from monosulcate. Grana 18: 1–4.
———, and V. F. Tarasevich. 1983 Pollen morphology of recent and fossil species of the genus Nelumbo (Nelumbonaceae). Botanicheskii Zhurnal 68: 137–146.
Les, D. H., D. K. Garvin, and C. F. Wimpee. 1991 Molecular evolutionary history of ancient aquatic angiosperms. Proceedings of the National Academy of Sciences, USA 88: 10119–10123.
Meacham, C. A. 1994 Phylogenetic relationships at the basal radiation of angiosperms: further study by probability of character compatibility. Systematic Botany 19: 506–522. [CrossRef][ISI]
Meyer, N. R. 1964 Palynological studies in Nymphaeaceae. Botanicheskii Zhurnal 49: 1421–1422.
Moseley, M. F. 1958 Morphological studies of the Nymphaeaceae. I. The nature of the stamens. Phytomorphology 8: 1–29.
———, and N. W. Uhl. 1985 Morphological studies of the Nymphaeaceae sensu lato. XV. The anatomy of the flower of Nelumbo. Botanische Jahrbücher für Systematik, Pflanzengeschichte und Pflanzengeographie 106: 61–98.
Nandi, O. I., M. W. Chase, and P. K. Endress. 1998 A combined cladistic analysis of angiosperms using rbcL and non-molecular data sets. Annals of the Missouri Botanical Garden 85: 137–212.
Osborn, J. M., T. N. Taylor, and E. L. Schneider. 1991 Pollen morphology and ultrastructure of the Cabombaceae: correlations with pollination biology. American Journal of Botany 78: 1367–1378. [CrossRef][ISI]
Qui, Y-L., M. W. Chase, S. B. Hoot, E. Conti, P. R. Crane, K. J. Systma, and C. R. Parks. 1998 Phylogenetics of the Hamamelidae and their allies: parsimony analyses of nucleotide sequences of the plastid gene rbcL. International Journal of Plant Sciences 159: 891–905.
Rao, T. A., and B. C. Banerjee. 1979 On foliar sclereids in the Nymphaeaceae sensu lato and their use in familial classification. Proceedings of the Indian Academy of Science 88: 413–422.
Rowley, J. C., and D. T. Moran. 1975 A simple procedure for mounting wrinkle-free sections on formvar-coated slot grids. Ultramicroscopy 1: 151–155. [CrossRef][Medline]
Rowley J. R. 1975 Germinal aperture formation in pollen. Taxon 24: 17–25. [CrossRef]
———, N. Gabarayeva, and B. Walles. 1992 Cyclic invasion of tapetal cells into loculi during microspore development in Nymphaea colorata (Nymphaeaceae). American Journal of Botany 79: 801–808. [CrossRef][ISI]
Schmid, A. M., R. K. Eberwein, and M. Hesse. 1996 Pattern morphogenesis in cell walls of diatoms and pollen grains: a comparison. Protoplasma 193: 144–173. [CrossRef][ISI]
Schneider, E. L., P. S. Williamson, and D. C. Whitenberg. 1990 Hot sex in water lilies. Water Garden Journal 6: 41–51.
Skawinska, K. 1985 Some new and rare pollen grains from Neogene deposits at Ostrzeszów (south-west Poland). Acta Palaeobotanica 25: 107–118.
Soltis, D. E., et al. 1997 Angiosperm phylogeny inferred from 18S ribosomal DNA sequences. Annals of the Missouri Botanical Garden 84: 1–49.
Suarez-Cervera, M., J. Marquez, and J. Seoane-Camba. 1995 Pollen grain and Ubisch body development in Platanus acerifolia. Review of Palaeobotany and Palynology 85: 63–84.
Takahashi, M. 1992 Development of spinous exine in Nuphar japonicum De Candolle (Nymphaeaceae). Review of Palaeobotany and Palynology 75: 317–322. [CrossRef]
Ueno, J., and S. Kitaguchi. 1961 On the fine structure of the pollen walls of angiosperms. I. Nymphaeaceae. Journal of Biology, Osaka City University 12: 83–90.
Venable, J. H., and R. Coggeshall. 1965 A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology 25: 407.
Walker, J. W. 1976 Evolutionary significance of the exine in the pollen of primitive angiosperms. In I. K. Ferguson and J. Muller [eds.], The evolutionary significance of the exine, 251–308. Academic Press, London.
Weber, M. 1998 The detection of pollen endexines—with special reference to Araceae pollen. Pollen and spores: morphology and biology, Abstracts, 49. Royal Botanic Gardens, Kew.
Williamson, P. S., and E. L. Schneider. 1993 Nelumbonaceae. In K. Kubitzki [ed.], The families and genera of vascular plants, 470–473. Springer-Verlag, Berlin.
Zavada, M. S. 1991 Determining character polarities in pollen. In S. Blackmore and S. H. Barnes [eds.], Pollen and spores: patterns of diversification, 239–256. Clarendon Press, Oxford.
Zetter, R., and C. Keri. 1989 Investigation of pollen grains of the genus Nelumbo from an Upper Miocene locality of Burgenland (Austria). Annalen des Naturhistorischen Museums, Wien 90, serie A: 111–118.
This article has been cited by other articles:
|
|
 |
|
  M. L. Taylor, B. L. Gutman, N. A. Melrose, A. M. Ingraham, J. A. Schwartz, and J. M. Osborn Pollen and anther ontogeny in Cabomba caroliniana (Cabombaceae, Nymphaeales) Am. J. Botany, April 1, 2008; 95(4): 399 - 413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. BANKS, S. FEIST-BURKHART, and B. KLITGAARD The Unique Pollen Morphology of Duparquetia (Leguminosae: Caesalpinioideae): Developmental Evidence of Aperture Orientation Using Confocal Microscopy Ann. Bot., July 1, 2006; 98(1): 107 - 115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L Taylor and J. M Osborn Pollen ontogeny in Brasenia (Cabombaceae, Nymphaeales) Am. J. Botany, March 1, 2006; 93(3): 344 - 356. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
