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Structure and Development |
2Department of Plant Biology and Center for Systematic Biology, Southern Illinois University, Carbondale, Illinois 62901-6509 USA; 3Micro-Imaging and Analysis Center, Southern Illinois University, Carbondale, Illinois 62901-4402 USA 4Department of Biology, Vanderbilt University, Nashville, Tennessee 37235-1565 USA
Received for publication June 13, 2000. Accepted for publication November 21, 2000.
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ABSTRACT |
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36 flagella that are inserted into the cell by basal bodies. Subtending the basal bodies is the multilayered structure, which consists of a long narrow lamellar strip and an overlying band of microtubules. An elongated anterior mitochondrion underlies the multilayered structure. Additional amyloplasts and mitochondria are aggregated along the anterior coil in association with the locomotory apparatus, while a fibrous band encircles the leading edge of the cell. Salient features of this cell, including details of the locomotory apparatus, structure and position of organelles, and arrangement of the spline, are shared by spermatozoids of Equisetum and ferns (including eusporangiate and leptosporangiate taxa). Thus, this study provides morphological support for the hypothesis that Psilotum nudum is a member of an assemblage that includes ferns and Equisetum. However, the less streamlined architecture of Psilotum gametes and the lack of architectural features shared with any specific taxon examined to date suggest that Psilotum is an early divergent fern, with relatively remote affinities to Ophioglossaceae and Equisetaceae.
Key Words: Equisetum • fern • plant sperm cell • Psilotum • pteridophyte • spermatogenesis • ultrastructure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In the interim, the traditional tenet that the Psilotaceae is a separate and early divergent tracheophyte lineage (Wagner, 1977
) has been challenged by molecular evidence that suggests that these plants are members of the fern clade. In contrast to the contention that Psilotum and Tmesipteris are filicopsid ferns (Bierhorst, 1977
; White, 1977
), molecular analyses have repeatedly supported a sister relationship between Psilotaceae and Ophioglossaceae (Manhart, 1994, 1995
; Hasebe et al., 1995
; Pahnke et al., 1996
; Wolf, 1997
; Wolf et al., 1998
; Nickrent et al., 2000
). Morphological evaluation of this hypothesis is limited to the inclusion of general structural features of Psilotaceae in more comprehensive phylogenetic analyses (Stevenson and Loconte, 1996
; Kenrick and Crane, 1997
; Wolf et al., 1998
; Rothwell, 1999
). Since the 1970s and the works of Bierhorst (1971, 1977)
, Rouffa (1978)
and Siegert (1973)
, few new developmental, anatomical, or ultrastructural studies have been undertaken that shed light on the position of this phylogenetically intriguing plant taxon among tracheophytes (see Takiguchi, Imaichi, and Kato, 1997
).
With their complicated architecture and immense variability, motile sperm cells have emerged as a rich source of phylogenetic information (Garbary et al., 1993
; Maden et al., 1997
; Renzaglia, Bernhard, and Garbary, 1999
; Renzaglia et al., 2000
; Renzaglia and Garbary, 2001
). Such data are useful in phylogenetic analyses as well as in providing a foundation for evaluation of cellular modifications that accompanied the evolution of terrestrial plants. Surprisingly little is known about the gross structure of sperm cells in key pteridophyte lineages, including Psilotaceae. This undoubtedly reflects difficulties in acquiring natural collections of the subterranean gametophytes of these plants coupled with the challenges associated with successfully propagating sexually mature gametophytes in the laboratory.
Although spermatozoids of Psilotum were described as multiflagellated (Lawson, 1917
), the precise organizational features of this motile cell remain unknown. Thus, we undertook this combined TEM (transmission electron microscope) and SEM (scanning electron microscope) investigation of the spermatozoid of Psilotum nudum with two goals in mind. First, we endeavored to describe the detailed organization of the mature sperm cell. This entailed interpretation of two-dimensional TEM images correlated with three-dimensional SEM images. An artistic model of the whole cell was constructed based on these observations. Secondly, comprehensive comparisons of Psilotum spermatozoids with those of other pteridophytes were undertaken to identify features that may shed light on the placement of Psilotum in the embryophytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Transmission electron microscopy
Antheridial tissue was fixed in 4% glutaraldehyde in 0.05 mol/L cacodylate buffer for 2 h at room temperature and then overnight at 4°C. The tissue was washed three times over 2 h in cacodylate buffer (0.05 mol/L, pH 7.2), postfixed in 2% OsO4 in same buffer, rinsed in water, and en bloc stained in 2% aqueous uranyl acetate (UA) for 16 h at 4°C. After dehydration in a graded ethanol or acetone series, the material was infiltrated with a 1:1 mix of Spurrs/Polybed resin and cured at 65°C for 16 h. Specimens were thick sectioned and stained with 1% toluidine blue and monitored for presence of antheridia. The specimens with antheridia in the appropriate stage of development were thin sectioned and poststained with ethanolic UA and basic lead citrate for 5 min each. Observations were made on an Hitachi H500 TEM.
Scanning electron microscopy
A portion of a gametophyte with antheridia was dissected and placed on a drop of water on a glass slide. Once swimming sperm were observed, the solution containing sperm was pipetted off and transferred to a 12-mL polypropylene test tube. An excess of 3% glutaraldehyde in 0.05 mol/L cacodylate buffer, pH 7.2, was added and the cells were fixed for 1 h at room temperature and then placed overnight in a refrigerator at 4°C. The solution was centrifuged at 500 rpm for 3 min and the glutaraldehyde was discarded. The pellet was rinsed three times for 10 min in 0.05 m cacodylate buffer, with resuspension and centrifugation (500 rpm for 3 min) at the beginning and end of each rinse. The pelleted cells were resuspended in 2% OsO4 and postfixed for 1 h at room temperature and then centrifuged. The pellet was rinsed three times at room temperature for 10 min each in deionized-distilled water, and centrifuged after each rinse. The cells were resuspended and dehydrated in 25, 50, 75, 95, and 100% ethanol for 10 min each at room temperature, and pelleted by centrifuging after each step. The ethanol supernatant was removed and 5 mL of hexamethyldisilazane (HMDS) was added, mixed, and the solution was centrifuged immediately. Because retention of greater numbers of individual spermatozoids is more easily controlled with chemical dehydration using HMDS and cell preservation is satisfactory, this technique was preferred over traditional critical point drying techniques (Rumph and Turner, 1998
). After centrifugation, the HMDS supernatant was pipetted off and the specimens were applied to a stub, air-dried, sputter coated with 40 nm of Au/Pd using a Denton Desk II Vacuum Sputter Coater (Denton, Moorestown, New Jersey, USA) and viewed in an Hitachi S570 SEM (Hitachi, San Jose, California, USA).
Cell reconstruction
A three-dimensional reconstruction of the released spermatozoid was assembled after examination of >500 TEM micrographs correlated with 40 SEM images. This artistic model is based on average dimensions and thus represents the best model of a typical spermatozoid.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2.0 revolutions. The anteriormost coil is nearly horizontal in orientation, while the posterior revolution exhibits an increasingly steeper slope to a nearly vertical orientation at the cell terminus (Figs. 1, 3, 9). The locomotory apparatus extends along the anterior of the cell for 1.0–1.5 gyres and is subtended by a large, single anterior mitochondrion. Sperm cells contained within the antheridium are more tightly coiled (Fig. 4) than preserved motile cells (Figs. 1–3). Upon release of the gamete and commencement of motility, the anteriormost portion relaxes, consequently releasing the coils from 1.5 to 1.0 revolutions. The broad nucleus is the prominent organelle and, along with multiple plastids and mitochondria, it extends from the locomotory apparatus to the cell posterior. A parallel band of up to 190 microtubules (spline) that is positioned directly internal to the plasmalemma binds the outer cellular coils (dorsal cell surface). The trailing edge (side away from the cell anterior) of the spline wraps in part around the nucleus, forming a nuclear groove that extends most of the length of the cell (Figs. 2–4). Reconstruction of the cell from a ventral aspect thus shows the posterior portion of the nucleus and locomotory apparatus (Fig. 34). To illustrate the nuclear shape and nuclear groove, the numerous organelles that line the ventral nuclear surface have been omitted from this illustration.
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40° (Figs. 7, 9).
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1.5 µm in width) and gradually tapers toward the posterior (0.7 µm in width) (Figs. 11, 35). The anterior mitochondrion subtends the LS along its entire length. Because the lamellar plates compact late in development, vertical zonation in the LS is visible only in immature spermatozoids. Maximum complexity of the lamellar strip is revealed in diagonal sections of the MLS in mid-stage spermatids (Fig. 12). Constituent lamellae are traversed by a dense central plate and subtended by a rod-like layer, components of which appear ovoid in outline. Individual plates of the LS are more difficult to discern in cross section of the spline microtubules (Fig. 13). In such a view, connectors that are often three-pronged are readily apparent between microtubules and LS.
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36 flagella is positioned 1.5–2.0 µm from the cell anterior (Fig. 14). A single row of 22 flagella extends along the leading edge of the cell to within 2.0 µm of the posterior limit of the lamellar strip (Figs. 15, 16, 35). A second row of 14 flagella underlies the upper row beginning at the fifth or sixth flagellum from the cell anterior and terminating before the last flagellum in the upper row (Figs. 15, 35). Typically, flagella diverge at a 40° to 45° angle from the longitudinal axis of the lamellar strip. However, this angle decreases toward the posterior of the locomotory apparatus so that the posterior most flagella may run parallel to the LS plates (Figs. 16, 35). The flagella coil around the cell in a sinistral fashion (Fig. 16).
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Nucleus and spline
The nucleus is the prominent cellular component and is responsible for the external appearance of the cell (Figs. 1, 2). The nucleoplasm consists of electron-opaque granular material with scattered, more or less spherical, inclusions of variable dimensions (Figs. 2, 30, 32). The outer boundary of the nucleus is delineated by the spline and the angle of orientation of spline microtubules defines the shape of the nucleus on the dorsal side of the cell. A series of sections from the anterior to the posterior of the cell that reveal the entire band of spline microtubules is instrumental in visualizing the spatial relationship among locomotory apparatus, nucleus, and spline (Figs. 25–30).
At the extreme anterior of the cell, the MLS and anterior mitochondrion extend in front of the nucleus and the first six basal bodies are anchored in a staggered fashion over the upper side of the MLS (Fig. 25). In Fig. 25, the staggered insertion is evidenced by sections of individual basal bodies that are progressively more posterior from the leading edge (top) to the trailing edge (bottom of micrograph) of the cell. From this anterior region toward the cell posterior, basal bodies are added in an alternating fashion in two rows over the MLS (Figs. 26–30, see above for description of arrangement of basal bodies).
The spline increases in width toward the cell posterior from 38 microtubules (MTs) (Fig. 25) to nearly 190 MTs (Figs. 30 and 33, right-hand side). As MTs are added, the spline extends beyond the lamellar strip and over the nucleus (Figs. 26–30). Overlap between the nucleus and MLS with the subtending anterior mitochondrion is more extensive in anterior (Figs. 26–28, and left arrow in Fig. 33) than in posterior (Figs. 29, 30, and right arrow in Fig. 33) regions of the cell. With addition of MTs, the spline forms a crosier that projects into the massive nucleus and imposes a spiraled groove along most of the nuclear length (Figs. 3, 26–34). The broad outer portion of the spline frames the dorsal surface of the cell and narrows posteriorly (Figs. 1, 3, 32). In the anterior and mid-regions, the nucleus fills the inner coils of the cell, i.e., the coils are not separated from each other. At the cell posterior, a narrow portion of the nucleus extends beyond the MLS and follows the terminal strip of spline, which coils up under and encircles the nucleus and cell terminus (Figs. 31, 32, 34). Random gaps between bands of MTs are visible in the spline where it overlies the nucleus (Figs. 1, 3, 32, 33).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Renzaglia, Dengate, and Bernhard, 1998
; Renzaglia, Bernhard, and Garbary, 1999
), two species of Equisetum (Duckett and Bell, 1977
; Renzaglia et al., 2001
), Angiopteris (Renzaglia et al., 2000
; Renzaglia and Garbary, 2001)
, a variety of filicopsid ferns (Duckett, 1975
; Myles and Hepler, 1977
; Kotenko, 1990
), Ginkgo (Gifford and Lin, 1975
; Gifford and Larson, 1980
; Li, Wang, and Knox, 1989
) and cycads (Norstog, 1967, 1968, 1977, 1990
; Norstog and Nicholls, 1997
). The present study provides the first detailed description of the multiflagellated male gamete of a member of the Psilotaceae. The organization and salient features of the spermatozoid of Psilotum are summarized in the three-dimensional reconstruction presented in Fig. 34. Comparisons with other archegoniates reveal that the sperm cell of Psilotum nudum shares basic architectural features with Equisetum and ferns. Spermatozoids produced by these plants are elongated along a sinistral helix with the locomotory apparatus and associated anterior mitochondrion occupying the anterior coil(s). The compacted nucleus begins near the cell anterior where it overlaps the locomotory apparatus and extends to the cell terminus. The nucleus is broadest in the mid-region of the cell, and multiple organelles, especially plastids and mitochondria, line the inner nuclear surface. The broad spline wraps around the outer nuclear surface along its trailing edge and thus separates coils or forms a spiraled internal groove. This attribute of the spline is unique to the sperm cells of these plants.
Motile gametes of seed plants differ markedly from those described above in that they are ovoid with a spherical, uncondensed nucleus (Gifford and Lin, 1975
; Gifford and Larson, 1980
; Norstog and Nicholls, 1997
; Renzaglia and Garbary, 2001
). Although the locomotory apparatus coils around the cell anterior, there is no specialized anterior mitochondrion associated with the multilayered structure (Norstog, 1967
; Li, Wang, and Knox, 1989
). Organelles are abundant and scattered throughout the extensive cytoplasm. The spline forms a cage around the cell and exhibits no specific association with the nucleus (Norstog, 1968
). These fundamental architectural features serve to distinguish motile gametes of seed plants from those of Psilotum, Equisetum, and ferns.
A number of specific features of the locomotory apparatus likewise are shared by and unique to Psilotum, Equisetum, and ferns. The locomotory apparatus consists of an elongated multilayered structure (MLS) with over 30 staggered flagella inserted into the cell at 29–45° to the MLS. In contrast, this angle is 16° in Zamia (Norstog, 1974
), 45–90° in lycophytes, and 45° in bryophytes (Renzaglia and Garbary, 2001)
. As in all embryophytes, the MLS comprises the lamellar strip with overlying spline. The lamellar strip in Psilotum, Equisetum, and ferns is highly elongated and gradually tapers in width from front to back. The plates of the LS roughly parallel its longitudinal axis. Basal bodies are arranged in rows (two to four) over the MLS (Myles, Southworth, and Hepler, 1978
; Marc and Gunning, 1986
). The dense plate in the middle of the lamellar strip of Psilotum is reminiscent of a similar structure in Equisetum (Renzaglia et al., 2001)
.
The location of the stellate pattern of the basal body internal to the basal plate and flagellar ring is also diagnostic of Psilotum, Equisetum, and ferns. In most lycopsids, the stellate pattern is positioned both internal and external to these flagellar landmarks (Renzaglia and Maden, 2000)
, while in bryophytes the stellate pattern is restricted to the flagellar shaft (Renzaglia and Duckett, 1988
). Also diagnostic of fern, Equisetum, and Psilotum male gametes, but apparently lacking in lycophytes, is the existence of a dense amorphous matrix that overlies the MLS and surrounds the basal bodies. In Ceratopteris, this so-called amorphous zone contains the protein centrin and is involved in organization of MT constituents of the locomotory apparatus (Vaughn, Sherman, and Renzaglia, 1993
; Hoffman and Vaughn, 1995
; Vaughn and Harper, 1998
). The homology of this region among these pteridophytes awaits further biochemical characterization with immunocytochemical methodologies.
In association with the locomotory apparatus at the anterior end of multiflagellated sperm cells of ferns, Equisetum, and Psilotum are intricate structures that likely are involved in maintaining coil integrity and controlling flagellar orientation. Typically, a band of fibrous material runs parallel to the locomotory apparatus and delineates the leading edge of the cell. In Equisetum and Ophioglossum (Renzaglia and Garbary, 2001)
, this band includes a fibrous as well as a well-developed striated component, while in Psilotum, Angiopteris, Osmunda, Onoclea, Ceratopteris, Platyzoma, and Pteridium cross striations have not been observed in any portion of the band (Duckett, 1975
; Doonan, Lloyd, and Duckett, 1986
; Kotenko, 1990
; Mainwaring, 1997
; Renzaglia et al., 2000
). In Botrychium, the anterior band is highly elaborate and composed of electron dense, amorphous material, i.e., fibers are not differentiated. In addition to the anterior fibrous band, sperm cells of homosporous leptosporangiate ferns and Angiopteris (Renzaglia et al., 2000)
possess an accessory band of microtubules that overlies the MLS and into which many of the basal bodies are anchored. With cellular maturation, this microtubular band and associated materials compact and flatten, thus contributing to the intensive streamlining of these cells.
In comparison to sperm cells of Ophioglossaceae, Equisetum and Psilotum, those of leptosporangiate ferns and Angiopteris are highly streamlined (Renzaglia et al., 2000
; Renzaglia and Garbary, 2001)
. Indeed, in these plants, elimination of extraneous cytoplasm and compaction of all cellular components results in spiraled cells that are ribbon-shaped. In cross-sectional profile each coil is compressed dorsiventrally and is spatially separated from contiguous coils. In Botrychium (K. S. Renzaglia, unpublished data) and Equisetum (Renzaglia et al., 2001)
, streamlining and individualization of coils also occur, but the nucleus is a broad cylinder with a swollen mid-region. Nevertheless, the nucleus in these sperm cells is bordered by spline MTs and assumes the helical configuration of this microtubular skeleton. In comparison, sperm cells of Psilotum are more ovoid and the massive nucleus occupies the inner coil region. Cell coiling is evident primarily in the arrangement of the locomotory apparatus and the sloping spline microtubules, including the nuclear groove that is evident in Figs. 2, 3, and 34.
In general, sperm cell organization is more variable in Psilotum, Equisetum, and Ophioglossaceae than in Angiopteris and Filicopsida. The MLS is subtended by a single large anterior mitochondrion but numerous accessory mitochondria are randomly positioned near the nucleus at the cell anterior. In Angiopteris, no accessory mitochondria exist at the cell anterior while in most Filicopsida, accessory mitochondria are orderly aligned along the spline MTs (Duckett, 1975
; Kotenko, 1990
; Mainwaring, 1997
). In addition, sperm cells of Equisetum and Psilotum have variable numbers and positions of plastids and mitochondria that are distributed along the cell length. Even the angle of orientation of spline microtubules to lamellar plates is more variable (29–45°) in these sperm cells than in filicopsid gametes (40°).
Specialization of male gametes in filicopsid ferns involved elongation and compaction of all cellular components, especially the nucleus. Concomitantly, cell organization has become more precise with organelles aligned along the framework of the spline microtubules and adjacent to the nucleus. Cellular coiling has likewise increased from nearly 2.5 revolutions in Angiopteris and Osmunda to 3–6 coils in most taxa (Duckett, 1975
; Kotenko, 1990
; Mainwaring, 1997
). The exception is Marsilea which produces sperm cells that coil over 10 gyres (Myles and Hepler, 1977
). Because the nucleus occupies more volume of the cell than any other organelle it is probable that modifications in sperm cell architecture were in part directed by increases in genome size and especially ploidy level (Renzaglia, Rasch, and Pike, 1995
). Certainly, constraints imposed on naked motile cells in a terrestrial environment due to increased size could have a profound influence on swimming hydrodynamics. Negotiating passage through the narrow neck canal of the archegonium undoubtedly provided additional constraints on the architecture of these cells.
Evolutionary inferences may be posited based on comparative data accumulated to date on multiflagellated male gametes of pteridophytes. First, multiflagellated gametes evolved in land plants subsequent to the radiation of bryophyte and lycophyte groups, and prior to diversification of the remaining tracheophytes (subdivision Euphyllophytina, division Tracheophyta; Raubeson and Jansen, 1992
; Kenrick and Crane, 1997
). Ultrastructural similarities among motile gametes of Psilotum, Equisetum, and ferns are numerous, suggesting conservation of fundamental architectural features through the millions of years following cladogenesis. These collective characteristics in sperm cell microanatomy support the notion that Psilotum, Equisetum, and ferns share a common ancestry. Indeed, cladistic analyses based solely on spermatogenesis provide resolution of a fern clade that includes Psilotum, Equisetum, Angiopteris, and Filicopsida (Moniliformopses sensu Kenrick and Crane, 1997
) (Renzaglia et al., 2000
; see Renzaglia and Garbary, 2001
for character list and data matrix). Psilotum and Equisetum are basal and paraphyletic within this monophyletic assemblage. Thus, data from spermatogenesis do not support a sister group relationship between Psilotum and Ophioglossaceae as suggested by recent molecular phylogenies (Wolf, 1997
; Wolf et al., 1998
; Renzaglia et al., 2000
; Nickrent et al., 2000; Pryer et al., 2001
). Similarly, male gametogenesis does not support the contention that Psilotaceae is the basalmost branch of euphyllophytes as suggested by recent morphological analyses (Stevenson and Loconte, 1996
; Rothwell, 1999
). Deviations from the more streamlined architecture of Botrychium, Equisetum, Angiopteris, and Filicopsida, and the relatively simple design of spermatozoids of Psilotum (low flagellar number and coiling) support the concept that the Psilotaceae is a remote, basal lineage of ferns. Further analysis of this interpretation must await detailed examination of spermatogenesis in Tmesipteris, as well as continued studies of all aspects of the biology of these organisms.
Studies of spermatogenesis in plants provide an unparalleled opportunity to compare complicated developmental processes among plant groups and to identify historical changes at the cellular level. Continued scrutiny of this morphogenetic system will enable the precise determination of evolutionary modifications in these complex motile cells and will continue to elucidate informative data for evaluation of plant phylogeny.
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FOOTNOTES |
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DEDICATION: We dedicate this manuscript to the memory of two inspirational botanists of the twentieth century: Warren Herb Wagner and David W. Bierhorst. May the interest they excited in Psilotum and basal pteridophytes be kept alive for the millennia to come. 
5 Author for reprint requests (renzaglia{at}plant.siu.edu
; FAX: 618-453-3441).
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