Novel features of Equisetum arvense spermatozoids: insights into pteridophyte evolution


  • K. S. Renzaglia,

    1. Plant Biology Department and Center for Systematic Biology, Southern Illinois University, Carbondale, IL 62901, USA;
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  • S. B. Dengate,

    1. Plant Biology Department and Center for Systematic Biology, Southern Illinois University, Carbondale, IL 62901, USA;
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  • S. J. Schmitt,

    1. Integrated Microscopy and Graphics Expertise, Southern Illinois University, Carbondale, IL 62901, USA;
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  • J. G. Duckett

    Corresponding author
    1. School of Biological Sciences, Queen Mary, University of London E1 4NS, UK
      Author for correspondence: J. G. Duckett Tel: +44 (0)20 7 882 3294 Fax: +44 (0)20 7 983 0973 Email:
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Author for correspondence: J. G. Duckett Tel: +44 (0)20 7 882 3294 Fax: +44 (0)20 7 983 0973 Email:


  •  To characterize structural diversity within Equisetum and among pteridophytes, architectural features of the sperm cell are described here in a second subgenus of Equisetum, a divergent basal group in the fern clade.
  •  Transmission electron microscopy observations of prereleased spermatozoids of Equisetum arvense were correlated with three-dimensional scanning electron microscopy images of swimming cells.
  •  The mature spermatozoid completes a helix of approximately 2.5 revolutions. At the cell anterior is a complex multilayered locomotory apparatus with staggered flagella. Mitochondria (elongated–rounded) are aggregated near the locomotory apparatus and organelles extend along the cell length. The spline contains up to 300 microtubules and wraps in part around the long cylindrical nucleus. In swimming sperm cells, the anterior of the cell remains tightly coiled while the posterior relaxes and extends in a trailing fashion.
  •  Spermatozoids of Equisetum arvense are smaller than those of Equisetum hyemale but structurally similar, except for nuclear shape. Conservation of cellular features suggests recent radiation of the genus. Equisetum spermatozoids share several critical features with ferns, including Psilotum, and support monophyly of a fern–Equisetum assemblage. Entry of the male gametes of Equisetum in their entirety into the archegonial venters indicates possible biparental inheritance of chloroplast and mitochondrial genomes.


Contemporary descriptions of the architecture of sperm cells in pteridophytes have revealed a complexity and degree of diversity that is unparalleled in other groups of eukaryotes (Renzaglia et al., 2000; Renzaglia & Garbary, 2001). Male gametes in pteridophytes range from relatively simple, slightly coiled biflagellated cells (lycophytes) to highly elaborate helical cells with multiple flagella (ferns). A primary conclusion that has emerged from these studies is that structural and developmental features of motile male gametes provide a remarkable window on the evolutionary history of land plants: one from which a robust reconstruction of phylogeny as well as details of cellular modifications that have accompanied cladogenesis can be inferred.

The seminal studies by Duckett (1973) and Duckett & Bell (1977) on Equisetum hyemale L. elucidated striking similarities in male gametogenesis among Equisetum and leptosporangiate ferns. Cladistic analyses based solely on spermatogenesis repeatedly have supported nesting of Equisetum within a monophyletic fern assemblage (Garbary et al., 1993; Mishler et al., 1994; Maden et al., 1997; Renzaglia et al., 2000; Renzaglia & Garbary, 2001). This evolutionary affinity has been reinforced by morphological analyses (Kenrick & Crane, 1997; Renzaglia & Garbary, 2001), and molecular analyses using a single gene (Duff & Nickrent, 1998) or multiple genes (Nickrent et al., 2000; Pryer et al., 2001). As a morphologically distinct genus in the fern clade with an evolutionary history that dates back to the mid-Devonian (Kenrick & Crane, 1997), Equisetum is a central taxon in understanding pteridophyte diversification. Morphological homogeneity in this monotypic family (Equisetaceae) suggest relatively recent diversification of the 15 extant species (Hauke, 1974). More difficult to document than structural diversity at the organism level is variation in cellular design and development. With the wealth of available data on gamete structure in land plants, a study of sperm cells in a second species of Equisetum would provide comparative information in assessing conservation of cellular features.

Equisetum hyemale is a member of the putatively basal subgenus Hippochaete (Hauke, 1993) and remains the sole species of this phylogenetically significant genus in which the fine structure of motile gametes has been documented (Duckett, 1973; Duckett & Bell, 1977). In this study, we describe the details of the structural organization in mature male gametes of a second species of Equisetum, Equisetum arvense L., representing the putatively more derived subgenus Equisetum. Comparisons between the two species of Equisetum and among a wide range of embryophytes provide information on the degree of infrageneric variation in spermatozoid architecture and allow the identification of phylogenetically informative characters. With correlation of three-dimensional scanning electron microscopy (SEM) images and ultrastructural observations, as well as the addition of ferrocyanide to the fixation cocktail, several novel anatomical features of Equisetum sperm cells have surfaced. Cladistic analyses based on mature sperm characters and the entire developmental process in a wide range of archegoniates, including Equisetum, have been presented elsewhere (Renzaglia et al., 2000; Renzaglia & Garbary, 2001).

Materials and Methods

Strobili of Equisetum arvense were collected on April 5 and 6, 1996 at Alto Pass, in Union County, IL, USA. A voucher specimen was deposited in the herbarium of KSR. Spores were sown on Parker–Thompson nutrient medium and maintained at room temperature under constant fluorescent lights until antheridia were observed (approx. 70 d). Tissue was regularly subcultured to ensure continuous antheridial production. Antheridial tissue was harvested over the ensuing 6 months and prepared for transmission electron microscopy (TEM) and SEM as follows.

SEM preparation

Antheridial lobes were excised from the gametophyte and placed in a droplet (c. 100 µl) of 0.01 m phosphate buffer, pH 7.2, on a dental wax plate, covered to retard evaporation, and left overnight at room temperature to facilitate release of sperm. Gametophytic tissue was removed and the droplet was placed in a 12-ml centrifuge tube containing 1 ml 2% glutaraldehyde in 0.05 m sodium cacodylate buffer, pH 7.4. The tube was shaken gently and the spermatozoid suspension was fixed for 1 h at room temperature. The fixed sperm cells were rinsed three times (10 min each) in 5 ml 0.05 m sodium cacodylate prior to postfixing for 20 min in 2 ml 4% aqueous osmium tetroxide. To facilitate solution changes, the spermatozoids were concentrated by centrifugation at 100 g. for 5 min. Following centrifugation, the specimens were rinsed twice in 5 ml demineralized distilled water (DDW) (10 min), once each in 25%, 50% and in 75% ethanol (10 min), twice in 100% ethanol (10 min), and then twice in 100% hexamethyl disilizane (HMDS; 10 min). Sperm cells in HMDS were pipetted onto a glass coverslip affixed to a standard, dried 20 min in a 60°C oven prior to coating with c. 390 nm palladium–gold. Specimens were viewed using a Hitachi S570 SEM.

TEM preparation

Antheridial lobes were excised from the gametophyte and placed in 2.5% glutaraldehyde in 0.05 m phosphate buffer, pH 7.2, for 18 h at 4°C. Specimens were rinsed three times (15 min each) in 0.05 m cacodylate buffer prior to postfixing for 2 h at room temperature in aqueous 2% osmium tetroxide with 1.5% potassium ferrocyanide. Fully fixed specimens were rinsed three times in DDW (10 min each), dehydrated through 25%, 50%, 75% and 95% ethanol (20 min each), rinsed twice in 100% ethanol (20 min each), then rinsed three times in 100% propylene oxide (15 min each). Specimens were infiltrated in 25%, 50% and 75% in low viscosity resin (diluted with propylene oxide) for 12 h each at 4°C and soaked in three changes of pure resin for 8 h each at room temperature prior to polymerization for 48 h at 60°C. Gold/silver sections were cut on a diamond knife, collected on formvar films using slot (1 × 2 mm) grids, stained with 2% uranyl acetate (12 min) followed by basic lead citrate (5 min) prior to viewing in a Hitachi HF7100.


General cell organization

Figure 1 shows reconstructions illustrating the overall shape and relationships among the major cellular components of prereleased and swimming spermatozoids of Equisetum arvense. The orientation and terminology related to micrographic data that follow are most readily understood by reference to these reconstructions.

Figure 1.

Line drawings showing the location and orientation of major cellular features of the Equisetum arvense spermatozoid. (a) Three-dimensional drawing showing the overlap between the anterior lamellar strip and posterior of the nucleus. Numbers indicate number of plates in lamellar strip at specific locations. (b) Top view of the leading edge of the cell showing fibrous band as a rim outside the anterior mitochondrion along the first 1.5 coils. The anterior mitochondrion is broadest where it extends beyond the fibrous band at the tip. (c) Lamellar strip and nucleus as in (a) with overlain spline. Lines in the spline indicate the longitudinal axes of its component microtubules. The portion of the spline that folds around the inner (ventral) surface of the nucleus is arrowed (cf. d). (d) Three-dimensional drawing showing the external architecture of a swimming cell with flagella omitted. Labels identify prominent cellular components recognizable in this surface view, and anterior, posterior, dorsal and ventral surfaces of the cell. Lines in the spline indicate the longitudinal axes of component microtubules. AM, anterior mitochondrion; B, band composed of fibrous and striated material that encircles the cell anterior; CM, cytoplasmic mass; D, dorsal; LS, lamellar strip; N, nucleus; NB, anterior nuclear beak; S, spline; VE, ventral.

The mature sperm cell of Equisetum arvense completes a sinistral helix of approximately 2.5 revolutions (Fig. 1c,d). The locomotory apparatus, including approximately 54 flagella and the multilayered structure with lamellar strip, occupies the anterior 1.75 cell gyres (Fig. 1a,b). The prominent nucleus is elongated, tapered at both ends and occupies the posterior half of the cell (Fig. 1a). A narrow anterior portion of the nucleus overlaps the locomotory apparatus for two-thirds revolution (Fig. 1a,c). A fibrous band outlines the anterior-most limit of the cell and runs parallel to the locomotory apparatus and large subtending anterior mitochondrion (Fig. 1b). The cell is bound throughout by a wide band of parallel microtubules (MTs) commonly known as the spline (Figs 1c,d and 2a). The spline extends just inside the plasmalemma along the dorsal (outside) surface of the cell, and provides a structural framework upon which the organelles are arranged. A portion of the spline folds around the nucleus and partially sheaths the ventral (inside) nuclear surface (Figs 1c,d and 2a). Organelles are scattered along the ventral surface of the cell adjacent to the spline (Figs 1d and 2a).

Figure 2.

Mature spermatozoid. The leading edge of each cell is directed toward the top of the page. (ai) Longitudinal section through a spermatozoid enclosed in an antheridium showing successive half gyres (1–6) from anterior to posterior. Multilayered structure, anterior mitochondrion, basal bodies and fibrous band, occupy the leading edge of the first 1.5 revolutions. Along the trailing edge of half revolutions three to five, the nucleus is completely or partly encircled by spline microtubules that separate it from the remainder of the cell, leaving an internal groove. Spline microtubules dramatically decrease in number and extend beyond the nucleus to the cell posterior (half gyre 6). Numerous elongated mitochondria traverse the cytoplasm between gyres 1–4. Plastids containing single starch grains are scattered through the central cytoplasm. Evidence of cytoplasmic deletion include abundant vesicles and a large cavity at the cell posterior. (aii). Enlargement of half gyre 6 at the posterior-most limit of the cell showing the few remaining spline microtubules. (b) Scanning electron micrograph of a swimming cell showing tightly coiled anterior and extension of the cell posterior. (c). Transmission electron micrograph of swimming cell comparable to (c). Description as in (ai). Flagellar orientation shifted from lateral in (ai) to obliquely longitudinal in (b) and (c). AM, anterior mitochondrion; ANT, cell anterior; B, band composed of fibrous and striated material that encircles the cell anterior; C, cytoplasm; F, flagellum; FB, fibrous portion of band; G, groove in cell where coils overlap; HC, hole in cytoplasm; IG, internal groove in cell where spline encircles nucleus on ventral surface; M, mitochondrion; MLS, multilayered structure; MT, microtubules; N, nucleus; NB, anterior nuclear beak; P, plastid; POS, cell posterior; S, spline; SG, starch grain; V, vesicles.

While confined to its nearly spherical boundaries within the antheridium, the mature spermatozoid is tightly coiled and compressed longitudinally (Figs 1a,c and 2a,i). In all directions, the diameter of the cell is approximately 10 µm. The flagella encircle the cell laterally, around the anterior coils; at the extreme cell anterior, they occupy grooves delimited by the overlapping posterior coils (Fig. 2ai). Upon liberation from the antheridium and with initiation of swimming (Figs 1d and 2b,c), the coils extend longitudinally such that the distance from anterior to posterior more or less doubles (compare Fig. 1c with 1d and Fig. 2ai with 2c). Measurements from SEM images reveal that swimming cells are, on average, 8.0 µm in diameter at the mid-region and up to 17 µm in length. Numerous organelles, especially mitochondria, and remnant cytoplasm, lie within the anterior coils, whereas a large vacuole/cavity occupies the inner region of the cell posterior (Fig. 2ai). Consequently, the cell anterior appears to be less extendable than the posterior; with the onset of motility, the anterior portion of the cell maintains a tight coil with a low pitch while the posterior gyre extends, assuming a steep pitch (Figs 1d and 2b). Flexibility of the cell posterior results in part from the nearly complete separation of the nucleus from adjacent cytoplasm by infolding of the spline and plasmalemma along the ventral nuclear surface, thus forming an internal groove that spirals around the cell (Fig. 2a,c). Flagella in motile spermatozoids are similarly shifted to an anterior–posterior orientation that parallels the pitch of the stretched posterior gyre (Fig. 2a,c). the direction of movement of the gametes is toward the anterior with the flagella directed rearwardly.

Anterior mitochondrion

The extreme tip of the spermatozoid contains a conspicuous anterior mitochondrion (AM) that projects slightly forward from the lamellar strip (LS) and anterior tip of the spline (Fig. 3a–c). From a broadened anterior (c. 1.0 µm diameter), the AM suddenly narrows after the first one-quarter gyre and then tapers gradually toward the posterior down to 0.25 µm for a total of just under 1.75 gyres (see Fig 1b). The lateral outline of the AM more or less conforms to that of the overlying LS (Figs 3b–d, 4g–j and 5b), with the LS extending slightly posterior to the AM (Fig. 5c,d).

Figure 3.

Organization of the locomotory apparatus and cell anterior. (a). Scanning electron micrograph of a swimming sperm showing the large anterior mitochondrion at the cell tip and approximately three half revolutions of the fibrous band into which the first row of flagella are inserted. Additional rows of flagella (arrowheads) are evident under and below the first row, between the fibrous band and the anterior beak of the nucleus. A ring around the base of each flagellum demarcates the boundary between the stellate pattern and axoneme (cf, b and 4a). (b) Section traversing the narrow anterior-most coil showing the broad anterior mitochondrion that extends in front of the lamellar strip. More posteriorly, the striated portion of the fibrous band and the staggered basal bodies overlie the spline. Arrows labelled 10 and 11 indicate plane of sections in (c) and (d), respectively. (c) Section of the cell anterior, perpendicular to that in (b) along the plane indicated by arrow labelled 10. The fibrous band delimits the anterior rim of the coils; the anterior mitochondrion extends slightly anterior to the multilayered structure. The lamellar strip narrows from 52 plates at the anterior (right side) to 39 plates within one-half revolution (left side). Infolding of the trailing edge of the spline (arrow) begins near the cell tip. (d) Section of the cell anterior, perpendicular to that in (b) along the plane indicated by arrows labelled 11. The anterior limit of the lamellar strip is approximately 44 plates wide. Dense membrane-bound tubular bodies are associated with the lamellar strip and anterior mitochondrion. (e) Transverse section of the lamellar strip showing typical substructure of the lamellae with upper and lower plates separated by a dense plate. Similar substructure is visible in (d). (f) Transverse section of the lamellar strip showing substructure that lacks differentiation of strata. Similar substructure is visible on the right side of (c). (g) Scanning electron micrograph of the anterior of a swimming cell showing the anterior-most basal body and a single row of staggered flagella that insert into the striated portion of the fibrous band around the leading edge of the cell. (h) Scanning electron micrograph of the anterior of a swimming sperm cell that is rotated approximately 90° to that in (g). The row of 24 flagella around the leading edge of the cell terminates at the posterior limit of the fibrous band (arrow). A narrow anterior beak of the nucleus is evident one gyre from the tip of the cell (arrowhead). Cytoplasm occurs in the groove between gyres. AM, anterior mitochondrion; ANT, cell anterior; ABB, anterior-most basal body; AF, anterior-most flagellum; B, band composed of fibrous and striated material that encircles the cell anterior; BB, basal body; C, cytoplasm; DB, dense body; F, flagellum; FB, fibrous portion of band; LS, lamellar strip; LL, lower lamel/ae; M, mitochondrion; MLS, multilayered structure; NB, anterior nuclear beak; PL, plug; R, ring; S, spline; SB, striated portion of fibrous band; SG, starch grain; UL, upper lamellae.

Figure 4.

The locomotory apparatus and organization of the cell anterior. (a) Longitudinal section of a basal body showing a short cartwheel region and elongated stellate pattern with a plug of electron-opaque material on either side. A ring of granular material surrounds the base of the axoneme, which begins with a short zone devoid of two central microtubules (arrowhead), followed by the 9 + 2 configuration. (b) Cross-sections of a cartwheel configuration, four stellate patterns and flagella overlying the spline. The cartwheel configuration is surrounded by a granular matrix (amorphous zone) while the stellate patterns are partly encircled by membrane-bound dense bodies similar to those associated with the spline and lamellar strip (cf. Fig. 3c,d) and those extending from the nuclear envelope (Fig. 5b). (c) Substructure of the fibrous band showing the striated component that overlies the spline along the anterior rim. Striations (arrowhead) show a periodicity of approximately 30 nm. (d) Scanning electron micrograph showing the spatial relationship between the fibrous band and anterior organelles. (e) Paradermal section along the outer rim of the cell showing the fibrous band, spline microtubules and lamellar strip. The angle of spline microtubules to plates of the lamellar strip varies from 26° to 28°. (f) Tangential section of the anterior coil showing a row of five basal bodies (arrowheads) that overlie the spline, are surrounded by the amorphous zone and are inserted into the striated band. The bracket delimits the entire band, including striated portion and fibrous portion. (g–j) Successive cross-sectional profiles of the cell from anterior to posterior showing spline and lamellar strip along the leading edge (top of micrographs). Along the trailing edge (bottom of micrographs), the spline forms a crozier that first encircles accessory mitochondria (g) and more posteriorly surrounds the anterior beak of the nucleus. The upper basal body in each figure is embedded into the striated portion of the fibrous band. Microtubule number increases from approximately 88 in (g) to 150 in (j). (j) The nuclear envelope contains electron-opaque material that extends from the nucleus along the inner surface of the spline (small arrow). Cylindrical membrane-bound dense bodies are associated with the spline and mitochondria (arrowheads). AM, anterior mitochondrion; AZ, amorphous zone; B, band composed of fibrous and striated material that encircles the cell anterior; BB, basal body; CW cartwheel; DB, dense body; G, groove in cell where coils overlap; LS, lamellar strip; M, mitochondrion MLS, multilayered structure; NB, anterior nuclear beak; PL, plug, R, ring; S, spline; SB, striated portion of fibrous band.

Figure 5.

Anterior to mid-region of the cell. (a) Longitudinal section of the anterior nuclear beak showing incompletely condensed chromatin and cylindrical nuclear inclusions surrounded by the spline. (b–d) Progressively more posterior cross-sectional profiles along the rear portion of the locomotory apparatus, which overlaps the broadened nucleus. The leading edge of each cell is oriented to the left of micrographs. (b) Section approximately 1.5 gyres from the anterior tip of the cell. The spline, consisting of approximately 200 microtubules, is the upper stratum of the multilayered structure, where it overlies the lamellar strip at the leading edge of the cell. At the trailing edge, the spline wraps around and encircles the nucleus. Numerous spherical mitochondria align along the spline between the multilayered structure and nucleus. Three basal bodies overlie the concave anterior part of the spline and are interspersed among amorphous granular material and membrane-bound dense bodies. An emergent stellate pattern and axonemes with 9 + 0 (arrowheads) are visible proximal to the 9 + 2 microtubule configurations. The condensed nucleoplasm is surrounded by a peripheral halo and contains a dense inclusion. The nuclear envelope has a large pore and branch (large arrow). The perinuclear space with electron-opaque material is connected by fine fibres to the spline (arrow). (c) Section showing spline of 220 microtubules, with subjacent narrow lamellar strip and overlying basal bodies (arrowheads). Note extension of the nuclear envelope (arrow) and membrane-bound dense bodies associated with the lamellar strip, basal bodies and spline. (d) Section approximately 1.75 gyres from the anterior tip of the cell showing the posterior limit of the lamellar strip underlying the spline of approximately 300 microtubules. The broadened nucleus, surrounded by spline, contains large, dense spherical inclusions. The nuclear envelope extends along the ventral surface of the spline (arrow). (e) Scanning electron micrograph of the anterior of a spermatozoid showing four irregular rows of basal bodies (arrows) adjacent to a broad nucleus, approximately one gyre from the tip of the nucleus. AM, anterior mitochondrion; AZ, amorphous zone; BB, basal body; DB, dense body; FB, fibrons band; H, halo around nucleoplasm; IG, internal groove in cell where spline encircles nucleus on ventral surface; LS, lamellar strip; M, mitochondrion; MLS, multilayered structure; N, nucleus; NB, anterior nuclear beak; NI, nuclear inclusion; S, spline; SB, striated portion of fibrous band; SG, starch grain; SP, stellate pattern.

Lamellar strip

The multilayered structure (MLS) comprises the spline and underlying lamellar strip (LS). If uncoiled and laid out flat, the shape of the LS is an extremely elongated, narrow triangle (cf. reconstructions in Duckett & Bell, 1977). With a broader curved anterior boundary, the LS gradually tapers to a blunt narrow distal tip (Fig. 1a). The lamellae that constitute the LS run longitudinally along its length (Fig. 1a). The LS plates number approximately 44 at the anterior extreme and rapidly increase to 59 within half of a gyre (Fig. 1a). From this level, there is a gradual decrease in plate number over the distal 1.25 revolutions down to approximately 10 at the posterior limit of the LS (Figs 1a, 3c,d, and 5b,c). Because the lamellae parallel the longitudinal axis of the LS, vertical differentiation of the plates is evident in cross-section of the LS. Over most of its length, the LS consists of three strata: two vertically differentiated series of lamellae in an alternate arrangement that are separated by an electron-opaque plate (Fig. 3c–e). The latter zone may simply represent the zone of overlap between upper and lower plate strata. Occasionally, the LS plates are not differentiated into vertical strata, but rather are continuous from top to bottom (Fig. 3f). These two substructural variants in the LS are frequently visible in the same cell (Fig. 3d). There does not appear to be any specific LS portion that exhibits the less frequent undifferentiated plate arrangement. Finger-like single membrane-bound bodies containing amorphous dense material are often associated with the LS (Fig. 3c,d). Spline MTs always overlie the LS on its dorsal surface. The angle between plates and spline MTs is highly variable, ranging between 23° and 43° (Fig. 4e). In general, the more acute angles are found in the posterior portion of the LS.

Anterior fibrous band

The lamellar strip and spline are overarched by a fibrous band that begins near the front of the cell and extends along the leading edge of the anterior rim (Figs 1b, 2ai,c and 3c,d). In longitudinal section, the band is differentiated into two zones: a striated region that overlies the spline (Figs 3b,g,h and 4c,f) and a nonstriated zone that extends lateral to the spline and LS (Figs 3h and 4e). The striations in the former are regularly spaced (30 nm) and connected by finer fibres (Fig. 4c). In cross section, these regions are evident as a fine granular zone that overlies the MLS (striated band) and a more opaque region containing larger granules (fibres in transection) and small tubular units (Figs 3c,d, 4g,j). Occasionally, these two regions are separated from each other by the MLS (Figs 2ai and 4i).

Basal bodies and flagella

Approximately 54 basal bodies and associated flagella are staggered along the upper portion of the two anterior cellular coils (Fig. 3g,h). In the mature cell, basal bodies retain their substructure (Fig. 4a,b). Each basal body consists of a short cartwheel region (Fig. 4a,b,f,g) and a more elongated transition zone with a well-defined stellate pattern (Figs 4a,b,f and 5b). Typically, the cartwheel region is linked to the spline by a granular amorphous matrix, the so-called amorphous zone, that often merges with the striated band (Figs 4b,f,g,i,j and 5b). In more distal regions, a network of elongated, branched, single membrane-bound dense bodies that contain fine granular material are frequently visible amongst and surrounding the basal bodies (Figs 4b and 5c). Similar structures are abundant around the LS and AM (Figs 3c,d and 5c). At the proximal and distal limits of the stellate pattern (as viewed in long section), dense plugs traverse the interior of the basal body (Figs 3b and 4a). A ring of electron-opaque granular material surrounds the MT triplets external to the distal plug (Figs 3b and 4a). In surface view, such rings are visible at the base of each axoneme, at the approximate level where the flagellum emerges from the cell (Fig. 3a,g,h). Between the stellate pattern and typical 9 + 2 axonemal configuration is a short zone that lacks the two central MTs (Figs 4a and 5b).

The anterior-most basal body/flagellum is inserted near the tip of the cell (Fig. 3b,g). From here, a single row of approximately 24 evenly spaced basal bodies is associated with the fibrous band along its entire length (Fig. 3a,b,g,h). A second row begins approximately one-half gyre from the cell tip and a third and fourth row of flagella are progressively added toward the cell posterior. Successive flagellar rows underlie each other and are staggered irregularly (Fig. 5e). In cross-section, basal bodies overlie the MLS and extend along a concave portion of the spline up to the nucleus (Figs 2ai, 3c, 4g–j and 5b–d). In the cell anterior, flagella may be positioned along a groove formed by overlapping successive revolutions (Figs 2ai,c and 4d).

Accessory mitochondria and plastids

In addition to the large AM, numerous small, spherical to elongated mitochondria occupy the interior of the cell adjacent to the LS and nucleus (Figs 2ai, 3b–d, 4g–j and 5b–d). These organelles lie among approximately 25 plastids containing one to three starch grains (Figs 2ai,c, 6a and 7c). Plastids are concentrated in the cell posterior (Fig. 6a) but are also found within the anterior coils (Figs 2ai,c and 5b). An elaborate membrane reticulum containing abundant small, rounded vesicles is evident adjacent to the plasmalemma in cytoplasmic regions not surrounded by spline (Fig. 2ai,c). These membranous networks decrease as maturation proceeds and likely are involved in elimination of excess cytoplasm.

Figure 6.

Mid- to posterior region of the nucleus. (a) Cross-section through a spermatozoid that parallels the nuclear coil and illustrates the internal groove where the spline encircles the nucleus. The nucleus is connected to the central cytoplasm away from spline (arrow). (b) Scanning electron micrograph of an extended swimming sperm cell showing the organization of mid to posterior of the cell. A large cytoplasmic mass remains to the left of the nucleus, which appears smooth because it is surrounded by spline microtubules. An internal groove is evident between the spline and cytoplasm. Arrows 33–36 identify approximate plane of section in parts (c–f), respectively. The posterior of the cell consists of few spline microtubules on the ventral cell surface that extend beyond the nucleus and progressively decrease in number, forming a posterior point (arrowhead) (cf. Figure 2ai). (c–f) Progressively more posterior cross-sectional profiles of the nucleus behind the locomotory apparatus. Approximate planes of section are indicated in (b). (c) Behind the locomotory apparatus, the nucleus is approximately 5.0 µm at its maximum width. A spline of 255 microtubules extends slightly to the left of the nucleus (large arrow) and infolds around the right-hand side, only partly encircling the ventral surface of the nucleus. The spline is intimately associated with the plasmalemma and the nuclear envelope, so that an internal groove forms adjacent to the ventral nuclear surface. Inside the nucleus, coarsely granular chromatin is surrounded by an electron-lucent halo. Several dense spherical inclusions are visible. Perinuclear material (arrowheads) is associated with the nuclear envelope and large pores (small arrow) are present in regions not associated with the spline. Osmiophilic cytoplasmic inclusions lie adjacent to the nucleus in this region. (d) The maximum nuclear width is reduced to approximately 4.5 µm and the spline width is 230 microtubules. Explanation of this figure is as in (c). Note the branch of the nuclear envelope along the left extension of the spline (large arrow). (e) The nucleus is narrow and flattened. Spline microtubules number approximately 120. (f) Near the cell posterior, the nucleus is highly reduced and the spline is irregular, consisting of approximately 30 microtubules on the ventral nuclear surface, outlining the internal groove. CI, cytoplasmic inclusion; CM, cytoplasmic mass; H, halo around nucleoplasm; IG, internal groove in cell where spline encircles nucleus on ventral surface; N, nucleus; NI, nuclear inclusion; P, plastid; S, spline.

Figure 7.

Details of the central cytoplasmic mass. (a) Abundant elongated mitochondria are interrupted by scattered plastids and osmiophilic cytoplasmic inclusions. (b) Scanning electron micrograph of an extended swimming spermatozoid showing the irregular outline of organelles, most likely mitochondria, along the length of the ventral surface of the cell, contrasting with the smooth surface overlying the nucleus. (c) Central cytoplasm with abundant mitochondria and plastids, each with one to three starch grains. CI, cytoplasmic inclusion; CM, cytoplasmic mass; IG, internal groove in cell where spline encircles nucleus on ventral surface; M, mitochondrion; N, nucleus; P, plastid; S, spline; SG, starch grain.


The nucleus commences approximately one revolution from the anterior tip of the cell (Figs 1a,c,d, 3h, 4d,h) and it overlaps the posterior portions of the LS and AM for two-thirds of a gyre (Figs 1a, 2ai, 4h–j and 5b–d). From a narrow cylindrical anterior beak (Figs 4h and 5a), the nucleus broadens toward its mid-region (Figs 4i,j, 5b–d, and 6c,d) and constricts to a flattened ribbon at the rear of the cell (Fig. 6e,f). In cross-section, this organelle typically assumes a triangular to semicircular outline (Figs 4i,j and 5b–d). This is partly due to the fact that the spline MTs wrap laterally around the nucleus (see below). Nucleoplasm consists of uniformly granular chromatin surrounded by an electron-transparent halo adjacent to the envelope (Figs 4j, 5b,c and 6c–e). Scattered throughout the nucleoplasm are large spherical to cylindrical inclusions of variable diameters, each of which is surrounded by an electron-transparent halo (Figs 5b,d and 6c–e). These inclusions tend to be aggregated in the nuclear beak and around the nuclear periphery, especially in the angles outlined by the spline (Figs 5a,b,d and 6e). A tangential section along the nuclear beak reveals alternating regions of condensed chromatin and cylindrical inclusions (Fig. 5a). Similar electron-opaque bodies in the cytoplasm resemble these spheroidal nuclear inclusions (Fig. 6c–e).

Portions of the nucleus not delimited by spline MTs are often irregular in outline and contain modified nuclear pores (Figs 2c and 6c–e). In most profiles, the perinuclear space contains an electron-opaque matrix (Fig. 5b–d). With compaction and elongation, the nuclear diameter dramatically decreases and there is a concomitant reduction of nuclear volume and surface area. Excess nuclear envelope appears to be eliminated through evagination of the matrix-filled envelope along the ventral spline surface (Fig. 5b–d). A membranous network similar to the perinuclear material extends over both surfaces of the spline and underlies or partially cradles the basal bodies (Figs 4a,j and 5b,c).

Spline organization and cellular architecture

The spline provides a structural framework that covers the dorsal cellular surface and cradles part of the ventral surface. A large mass of cytoplasm, including plastids, mitochondria, vesicles, and spheroidal bodies, aggregates on the side of the nucleus that is not enclosed by spline (Figs 6a and 7a,c). In swimming spermatozoids, the cytoplasmic mass extends along the inner cellular region (Figs 1d, 2b,c, 6b and 7b).

From a narrow anterior, the spline progressively increases in diameter as it wraps around the cell and diverges downward from the horizontal axis of the LS at an angle of approximately 25° (Fig. 4e). It reaches its widest diameter of c. 300 MTs in the broad mid-region of the nucleus (Fig. 5d). MTs are progressively lost from the spline as the nucleus decreases in diameter down to the cell posterior. A small, pointed cytoplasmic zone extends beyond the posterior nuclear limit (Fig. 2aii,b).

The width (number of MTs) and organization of the spline are seen most clearly in cross-section. Figures 3c,d, 4g–j, 5b–d, 6c–f represent a series of cross-sectional profiles of the spline from anterior to posterior. At the anterior of the cell where it overlies only the LS and AM, just behind the leading edge of the AM (Fig. 3c), the spline comprises approximately 10 MTs (Fig. 3d). MTs are added progressively on the side of the spline away from the fibrous band. When the spline contains approximately 30 MTs, the margin away from the LS begins to form a hook that wraps around the accessory mitochondria (Fig. 4g). Towards the cell posterior, this crosier surrounds most of the nucleus (Figs 4h–j, 5b–d and 6c–e). The plasmalemma is connected to the spline by fine fibres (arrow, Fig. 5b) and delimits the cell boundary; thus, where the nucleus is ensheathed by the spline, it is set apart from the remainder of the cytoplasm and an internal groove is formed (Figs 2ai,c, 4i,j, 5b–d and 6c–f). As noted above, it is this spatial separation that partially individualizes coils and enables the posterior of the cell to stretch during swimming (Fig. 2b,c). The MTs at the posterior extreme of the cell are reduced down to those outlining the groove, (i.e. none remain along the plasmalemma on the dorsal surface, Figs 2aii and 6f).


The most striking finding from the present study is that male gamete structures of Equisetum arvense and E. hyemale (Duckett & Bell, 1977) are remarkably similar. Such conservation in gamete structure parallels that among the leptosporangiate ferns Pteridium, Onoclea, and Ceratopteris, but stands in stark contrast to the immense variation in the Lycopsida (Duckett, 1975; Kotenko, 1990; Maden et al., 1996, 1997; Renzaglia et al., 1998; Renzaglia & Garbary, 2001). The primary difference between the two horsetail gametes is one of size: in E. hyemale the cell extends 3.25 revolutions and the locomotory apparatus occupies 2.5 anterior gyres, compared with a total helix of 2.5 with 1.75 anterior gyres occupied by the locomotory apparatus in Equisetum arvense. The larger cell of E. hyemale is correlated with a greater number of flagella, which in our study was based on maximum counts for both species in SEM images. Spermatozoids of E. hyemale possess at least 80 flagella that are arranged singly at the cell anterior and increase up to six rows of flagella toward the posterior. In comparison, Equisetum arvense gametes contain at least 54 flagella that are similarly arranged around the cell anterior, with up to four rows of flagella near the posterior.

Nuclear shape is much the same in both species, but with a slightly more swollen mid-region in E. hyemale. The general nuclear shape in Equisetum gametes conforms to that of other pteridophytes in that it is narrow at the anterior end, broad in the middle and then abruptly tapers to a narrow posterior. However, as noted above, the mid-nuclear region is broader in Equisetum than in ferns. The nuclei of both horsetail spermatozoids contain peripheral dense inclusions but the aggregation of membrane-bound vesicles in nuclei of E. hyemale were not observed in Equisetum arvense.

The longer and broader spermatozoid nucleus in E. hyemale correlates with larger chromosomes in the subgenus Hippochaete (Manton, 1950). In contrast, published data on amounts of DNA suggest that these are higher in Equisetum arvense (28.4 pg) (Grime et al. 1988) than in E. hyemale (24.5 pg) (Bennett & Leitch, 2001). However, the most recent determinations, using improved and more reliable protocols, indicate a significantly higher amount of DNA in E. hyemale than in Equisetum arvense (M. D. Bennett & I. J. Leitch, unpublished) in line with the karyological and spermatozoid data. Manton (1950) suggests that the larger chromosomes in Hippochaete are indicative of a long-standing subgeneric division, a notion borne out by the occurrence of numerous intrasubgeneric but no genuine intersubgeneric hybrids (Duckett & Page, 1975; Duckett, 1975). However, as with traditional morphology, neither sperm dimensions nor chromosome sizes, nor amounts of DNA shed any new light on which is the more primitive subgenus.

The present study reveals a number of unique membrane-bound organelles associated with the locomotory apparatus and nuclear envelope that were highlighted by the osmium-ferrocyanide fixation and overlooked in earlier observations of E. hyemale. Most striking are the extensive tubular to flattened cisternae containing an electron-opaque material and the similar dense material filling the perinuclear space. Because these membrane arrays are continuous with the nuclear envelope extending on either side of the spline up to the lamellar strip, a likely interpretation is that they are a repository for excess nuclear envelope that is eliminated during nuclear metamorphosis. Careful scrutiny of the published micrographs of E. hyemale also indicates their presence, although they lack the dense contents produced by osmium-ferrocyanide fixation. This protocol also illustrates the lack of pores in the nuclear envelope immediately adjacent to the spline, a feature that appears to be ubiquitous in archegoniate sperm cells (Myles & Hepler, 1977; Myles et al., 1978). The finger-like cisternae associated with the lamellar strip in Equisetum arvense are almost certainly homologous to the sheets of similarly located endoplasmic reticulum (ER) in E. hyemale. During fertilization in ferns, major changes occur in the structures associated with the lamellar strip (Duckett & Bell, 1972; Fasciati et al., 1994a; Fasciati et al., 1994b). The possibility that these organelles in Equisetum contain macromolecules involved in fertilization invites further investigation.

In gross architecture, spermatozoids of Equisetum, Psilotum and ferns are readily distinguished from those of lycophytes and bryophytes in that they contain multiple flagella (36 or more) and numerous plastids and mitochondria (Renzaglia & Garbary, 2001). Ultrastructural studies of Psilotum sperm cells reveal a locomotory apparatus similar in many respects to those of Equisetum and ferns (Renzaglia et al., 2001). Comparisons among Equisetum, Psilotum and leptosporangiate ferns uncover common behavioural and structural features of their spermatozoids. In these cells, the longitudinal axis of the coils (a line running down the centre of the coils) is parallel, to the direction in which the cells swim (Bilderback et al., 1973). In contrast, the direction of movement in bryophytes and Selaginella gametes is perpendicular to the longitudinal axis of the coils. Within the Equisetum-fern assemblage, the subcellular basis for this swimming orientation lies in the highly consistent spatial organization of individual components of the locomotory apparatus. The lamellar strip is a long narrow ribbon that wraps around the anterior coils and, unlike in bryophytes, it reaches its maximum structural complexity in the mature cell. Constituent lamellae are aligned more or less parallel to the longitudinal axis of the lamellar strip. The band of parallel microtubules that constitute the spline is oriented at a 25–45° angle to the plates of the LS. This angle determines the pitch of the helix and, together with cell size, fixes the number of revolutions in the fully differentiated cell. Basal bodies overlie the MLS along the anterior coils and are roughly lined up in parallel with spline MTs; their constituent axonemes are directed slightly obliquely toward the cell posterior. This alignment establishes the direction of motility (Bilderback et al., 1973). In addition, the wide spline (150–300 MTs) ensheaths the nucleus (except in Marsilea) and is involved in separation of cellular coils. This latter feature clearly differentiates the male gamete of Psilotum, Equisetum and ferns from those of Lycopodiaceae (Renzaglia & Garbary, 2001; Renzaglia et al., 2001).

Other cellular features unique to Equisetum and ferns include an overlap between a narrow anterior region of the nucleus and the locomotory apparatus. The nucleus extends beyond the locomotory apparatus to the cell posterior, or nearly so. Between the large anterior mitochondrion and nucleus, in the region of overlap, numerous small accessory mitochondria lie beneath the spline MTs. Such accessory mitochondria are absent from Marsilea (Myles & Bell, 1975) and Angiopteris (Renzaglia & Garbary, 2001) spermatozoids. During spermiogenesis, a granular matrix, the so-called amorphous zone (Vaughn et al., 1993), overlies the spline and is interspersed among the basal bodies. This matrix is derived from similar electron-opaque material associated with the reorganizing blepharoplast of the young spermatid. Both matrices contain the calcium-binding protein centrin and both are implicated in nucleation and organization of MTs (Vaughn & Harper, 1998; Klink & Wolniak, 2001). During reorientation and migration of centrioles/basal bodies in the nascent spermatid, the transition region forms on the distal end of the basal bodies. This transition zone contains a stellate pattern that elongates prior to anchoring of the basal body and growth of the flagellar axoneme. As a consequence, unlike the situation in bryophytes and lycophytes, the stellate pattern is positioned internal to a plug of dense material that traverses the base of the flagellar shaft and delimits the boundary of the cell body. The flagella emerge from the cell with a short region of 9 + 0, before they give way to the 9 + 2 axonemal arrangement of MTs (Duckett, 1975).

A variety of electron-opaque bands encircle the anterior rim of multiflagellated gametes of pteridophytes (Renzaglia & Garbary, 2001). Such structures are involved in securing the basal bodies in a fixed position in the cell, thereby determining the angle and direction of divergence of the flagellar axonemes. By nature of their position along the tightly coiled leading edge of these cells, the electron-dense bands ostensibly provide rigid but somewhat elastic structural support that maintains coil integrity at the cell anterior. This idea is supported by the fact that the tighter anterior coils, compared with posterior coils not bordered by such bands, are maintained even after the spermatozoid becomes motile and is released from the confining spherical boundary of the cell in which it develops. In Equisetum and Ophioglossum the coil-delimiting band exhibits a striated substructure (Renzaglia & Garbary, 2001). This microanatomy is reminiscent of the striated roots or rhizoplast of green algal motile cells (Van der Hoek et al., 1995). However, unlike the rhizoplast, which contains centrin (Vaughn & Harper, 1998), the striated band of Equisetum and the osmiophilic crest and ridge of Ceratopteris lack this protein (Vaughn & Renzaglia, 1993; Vaughn et al., 1993; Hoffman & Vaughn, 1995).

Notwithstanding the numerous structural commonalties that unify male gametes of Equisetum and leptosporangiate fern gametes, there are highly distinctive traits that define both groups. The most notable are the general dimensions of the cell, and especially the nuclear shape. Spermatozoids of leptosporangiate ferns are more streamlined than those of Equisetum, that is, in cross-sectional profile, the coils are flattened. In the mid- to posterior region of Equisetum spermatozoids, the prominent nucleus is quite broad. The anterior coils, although they exhibit a similar arrangement of organelles along the spline, contain numerous mitochondria and plastids. In motile gametes, this abundance of cytoplasm ‘fuses’ the anterior coils and prevents complete extension compared with the more protracted posterior coils. Moreover, the present observations clearly demonstrate that, on release of the gamete from the antheridium, organelles along the inner regions of the nucleus compact down along the ventral nuclear surface and become integral components of the motile gamete. In fact, male gametes of Equisetum pass down the archegonial necks into the venter in their entirety (J. G. Duckett, unpublished). These features of Equisetum gametes are even more exaggerated in Psilotum sperm cells, where successive coils are inflexible owing to the occurrence of abundant organelles, especially the massive, central nucleus (Renzaglia et al., 2001). Similarly the compact organization of lycophyte spermatozoids (Renzaglia et al., 2000; Renzaglia & Garbary, 2001) indicates that their entire organelle complement is almost certainly retained during motility. In leptosporangiate ferns, including Marsilea, virtually all extraneous cytoplasm is eliminated in mass during some phase of sperm motility and at the latest at the mouth of the archegonial neck (Bell et al., 1971; Sears, 1980; Fasciati et al., 1994a, 1994b). The anterior coils of fern gametes are therefore completely individualized and, as such, they enable the flexible helix to elongate along its entire length. An accessory band of up to 42 microtubules is a prominent structure that is restricted to leptosporangiate ferns and Angiopteris, and whose presence may be associated with the full-scale elimination of cytoplasm and separation of coils at the cell anterior. This microtubular band overlies the MLS at the cell anterior and provides a rigid framework in which to anchor components of the locomotory apparatus and to maintain integrity of the narrow, ribbon-shaped anterior coils.

Turning to bryophytes, in Sphagnum the spermatozoid plastid is jettisoned during motility (Manton, 1957) and in the liverwort Sphaerocarpos (Diers, 1967) the posterior mitochondrion and the plastid are both lost by the time the male gametes enter the archegonial neck. However, similar loss of organelles in other liverworts and bryalean mosses is unlikely since here the plastid and posterior mitochondrion are compressed against the spline and nucleus, respectively. Regardless of the fate of the posterior mitochondria and spermatozoid plastids, entry of the anterior mitochondrion, which is firmly anchored to the spline, into the egg is almost certainly universal in archegoniates.

These considerations of the fate of spermatozoid organelles during motility have major implications for the possible inheritance of paternal plastid and mitochondrial genomes. In turn, phylogenetic reconstructions based on organellar molecular data must take account of whether the inheritance of chloroplast and mitochondrial genomes in a particular group is material or biparental. In ferns Gastony & Yatskievych’s (1992) elegant demonstration of purely maternal inheritance of the chloroplast and mitochondrial genomes from sequence data is completely in line with cytological studies showing exclusion of the sperm plastids in the archegonial necks and rapid digestion of all the paternal mitochondria in the egg cytoplasm (Duckett & Bell, 1971, 1972; Fasciati et al., 1994a,b). However, Gastony and Yakskievych do not rule out the possibility of some leakiness in maternal inheritance, as is well documented in angiosperms (Sears, 1980; Medgyesy et al., 1986). It now becomes pertinent to explore paternal organelle inheritance in groups with (Equisetum, Psilotum, lycophytes, bryalean mosses and hornworts) and without (Sphagnum, hepatics and ferns) a complete male gamete complement of mitochondria and plastids. For such studies, Equisetum is particularly inviting since intergametophytic crossing is widespread in nature (Soltis et al., 1988) and can be readily performed in culture (Duckett, 1979).

Other features that differentiate the sperm cells of Equisetum from those of ferns are more subtle, subcellular details. These include the complex interconnected endomembrane system in Equisetum discussed earlier, and the prominent nuclear envelope, with a distinct perinuclear matrix. Unlike mature gametes of most archegoniates, but similar to those of Lycopodiaceae, basal body substructure is not modified during development in Equisetum. In ferns, the central cartwheels disappear and the basal bodies become occluded with an electron-opaque substance similar to the material of the amorphous zone (Vaughn et al., 1993). Finally, large, abundant nuclear inclusions, as occur in Equisetum, have not been described in ferns but find counterparts in the nuclei of Psilotum (Renzaglia et al., 2001) and Lycopodiaceae (Maden et al., 1996, 1997). These inclusions are also absent in bryophytes, Selaginella and Isoëtes, and as far as we know have not been described in animal sperm. Further studies are now required to ascertain whether these inclusions reflect unusual forms of DNA packing perhaps related to the high chromosome numbers in these plants.

It has long been recognized that the angle between the LS plates and longitudinal axes of the spline MTs is instrumental in establishing the layout of the cellular coils (Bell et al., 1971). In ferns, this angle typically ranges from 35 to 45° (Duckett, 1975), in bryophytes it is a constant 45° (Renzaglia & Duckett, 1988) and in lycophytes the angle is highly variable, ranging from 45° to 90° (Maden et al., 1996, 1997; Renzaglia & Garbary, 2001). Based on the present study of Equisetum arvense and after careful evaluation of the micrographs in Duckett (1973), it appears that the spline–LS angle in Equisetum deviates from those reported in all other archegoniates in that it ranges from 23° to 43°. Among archegoniates, such acute angles have been reported only in Psilotum and Equisetum and may well be diagnostic of spermatozoids of basal groups in the fern clade (Renzaglia et al., 2001).

Consistent with numerous phylogenetic analyses based on spermatogenesis (Garbary et al., 1993; Maden et al., 1997; Renzaglia et al., 2000; Renzaglia & Garbary, 2001), the present study supports a sphenopsid-fern clade (Moniliformoses of Kenrick & Crane, 1997). This relationship was strongly reinforced by a series of molecular analyses (Duff & Nickrent, 1999; Nickrent et al., 2000; Renzaglia et al., 2000), culminating in the most recent combined multigene and morphological analysis by Pryer et al. (2001). Conservation of the detailed architectural features among male gametes in Equisetum and ferns points to the utility of characters relating to spermatogenesis in inferring phylogenetic relationships among plants.

We now propose that comparative data from spermatogenesis can provide additional insights into phylogenetic events during land-plant evolution. Diversity among male gamete structure in lycophytes may be interpreted as indicative of deep (most likely Palaeozoic) divergences in this group and between homosporous and heterosporous ferns. By contrast, the remarkable similarities in male gamete architecture between the two subgenera of Equisetum suggest a much more recent subgeneric split, though the genus itself is almost certainly Palaeozoic. This is clearly in line with the northern hemisphere rather than Gondwanaland distribution of Equisetum. Bryophytes present a similar picture. Here highly divergent blepharoplast architecture within the Metzgeriales, a group anchored in the southern hemisphere (Renzaglia, 1982), underlines wide, and most likely very ancient, generic discontinuities, whereas uniformity in the blepharoplasts of hornworts (again a most ancient group) suggests recent origins for the genera (Ranzaglia & Duckett, 1991). These broad summations clearly call for an expansion and new directions in morphological and molecular analyses to include sampling of multiple taxa in key plant groups.


This study was supported by NSF grant DEB-9527735 to KSR. We thank Hilarie Dee Gates for the illustrations in Fig. 1a and c, Gayleen A. Cochran for technical assistance, and Dr John Bozzola at the Center for Electron Microscopy for assistance and use of the facility.