The Structure and Development of Haustorial Placentas in Leptosporangiate Ferns Provide a Clear‐cut Distinction Between Euphyllophytes and Lycophytes

Abstract

This light and electron microscope study revealed that leptosporangiate ferns have highly distinctive gametophyte–sporophyte junctions characterized by sporophytic haustoria, the absence of intraplacental spaces and degenerating cells, and the early appearance of wall ingrowths in both generations. Other notable cytological features are highly pleomorphic plastids and mitochondrial aggregates in the gametophytic placental cells. Close similarities with the gametophyte–sporophyte junctions in Tmesipteris and major differences from those of homosporous lycophytes are in line with the placement of psilophytes and ferns in the same clade and distance both from lycophytes. A smooth interface between the two generations in Azolla suggests a clear‐cut discontinuity between homosporous and heterosporous ferns, although this is the only heterosporous fern investigated to date. Similarities between the gametophyte–sporophyte junctions of leptosporangiate ferns and hornworts, when balanced against differences between them, are considered more likely the result of parallel evolution rather than homology.

INTRODUCTION

Detailed comparative studies of the gametophyte–sporophyte junction have revealed major differences between all the major groups of bryophytes (for reviews, see Ligrone et al., 1993; Frey et al., 2001) and have thereby provided highly significant data for phylogenetic analysis. The structure and ontogeny of pteridophyte placentas is poorly known. Recent textbooks base their accounts of pteridophyte embryology exclusively on light microscope studies dating from the first half of the twentieth century (Goebel, 1905; Campbell, 1918; Schoute, 1938). The foot, a lateral development of the early embryo, is described as the site of exchange between gametophyte and sporophyte but subcellular details are lacking. Ultrastructural studies focused on oogenesis (review by Sheffield and Bell, 1987), apogamy (Menon and Bell, 1981; Lal and Narag, 1985) and early events after fertilization (Duckett and Bell, 1972) do not extend to the development of the foot and placenta.

Thus, in the fern clade [the Monilliformopses including psilophytes, sphenopsids, leptosporangiate and heterosporous ferns of Pryer et al. (2001) and the Euphyllophytina which also embraces seed plants (Kenrick and Crane, 1997)], specific information on the placenta is limited to a mention of transfer cells that is without micrographic evidence in Equisetum (Gunning and Pate, 1974), brief accounts of transfer cells in both generations in a handful of ferns (Adiantum, Pellaea, Pteridium and Polypodium; Gunning and Pate, 1969; Khatoon, 1986) and a description of haustorial sporophytic cells with wall ingrowths in Tmesipteris (Holloway, 1918; Frey et al., 1994a, b).

In Polypodium and Adiantum the wall ingrowths develop very early in both gametophyte and sporophyte generations, even before the expansion of the first leaf, elongation of the root and differentiation of the first xylem. This is precisely the stage when the young sporophytes are most dependent on their parent gametophytes for nutrients. Accumulation of starch in sporophyte cells, but not those of the gametophytes, probably reflects sugar translocation from the latter. Unlike mosses and liverworts, but similar to hornworts, all the above placentas lack mucilage‐containing intraplacental spaces and dead and collapsed cells are also absent. Ligrone et al. (1993) suggested that these differences might be related to the transient nature of sporophytic dependence on the gametophyte in ferns.

Although still fragmentary, published data on lycophyte junctions are perhaps a little more informative than those on ferns. In the foot region of Lycopodium appressum (Peterson and Whittier, 1991), the two generations are separated by a thin layer of electron‐dense material, and both develop coarse labyrinthine wall ingrowths. In contrast in Lycopodium cernuum, the interface between the two generations, which also both develop wall ingrowths, is the lower part of the embryonic axis derived from the suspensor. The foot develops as a lateral appendage and produces a mycorrhizal protocorm (Duckett and Ligrone, 1992). Isoëtes stands alone amongst pteridophytes investigated to date in the possession of a placental space containing collapsed gametophytic cells (Hilger et al., 2002). There are no wall ingrowths or interdigitation of the cells between the two generations. The profound differences between the placentas of these three lycopods, like those between their spermatozoids (Renzaglia et al., 2000) almost certainly reflects the considerable antiquity of the group and suggest that other variations in placental morphology still await discovery.

In the fern clade even tentative inferences would seem premature. However, Frey et al. (2001) suggested that haustorial sporophytic cells in hornworts and Tmesipteris indicate two separate lineages of pteridophytes in land‐plant evolution, namely hornworts and psilophytes with intermingling of sporophyte and gametophyte cells, and lycophytes, horsetails and ferns with a smoother contact zone between the two generations. The former condition is considered the more primitive. Frey et al. did, however, temper these radical ideas by noting that putative homology in the gametophyte–sporophyte junctions in lycophytes and ferns does not accord with other recent views of separate lycophyte and euphyllophyte lineages (Kenrick and Crane, 1997; Pryer et al., 2001). Resolution of this conflict clearly requires a much more incisive analysis of the structure and development of fern placentas and critical comparisons with hornworts. The results of such a study embracing four leptosporangiate ferns including both primitive (Gleichenia) and advanced (Ceratopteris) taxa and the heterosporous water fern Azolla are presented here.

MATERIALS AND METHODS

Gametophytes of Athyrium and Gleichenia, bearing young sporophytes at different stages of development were collected from shady roadside banks in the Cameroon Highlands, Peninsula Malaysia below stands of mature plants of Athyrium spp. and Gleichenia spp. Individual specimens were identified to the generic level from the morphology of the young sporophytes and the presence (Athyrium) or absence (Gleichenia) of gametophytic trichomes. Young sporophytes of Pteridium aquilinum (L.) Kuhn and Ceratopteris thalictroides (L.) Brongn. were produced by flooding axenically grown gametophyte cultures provided by K. S. Renzaglia.

Young sporophytes and adjacent gametophytic tissues were fixed with 2 % glutaraldehyde + 1 % formaldehyde (freshly prepared from paraformaldehyde) + 0·5 % tannic acid in 0·05 m sodium phosphate buffer, pH 7 for 2 h at room temperature followed by 1 % osmium tetroxide in the same buffer overnight at 4 °C. The samples were then dehydrated through a graded ethanol series and embedded in low‐viscosity resin via propylene oxide. Germinating female sporocarps of Azolla caroliniana, already fixed in 3 % glutaraldehyde and embedded in Epon, were provided by G. A. Peters. Thin sections were cut with a diamond knife and sequentially stained with methanolic uranyl acetate and basic lead citrate, then observed with a Jeol 100C transmission electron microscope. Sections (0·5 µm thick) were stained with 0·5 % toluidine blue in borax and photographed under differential interference contrast optics with a Leitz Ortholux microscope.

OBSERVATIONS

Light microscopy

The foot of all four genera of homosporous ferns in the present study is a slightly bulbous structure 0·4–0·5 mm in diameter (Fig. 1A and B). The placentas are likewise the same in the four genera and comprise elongate haustorial sporophyte cells growing into the closely adjacent gametophyte cells (Fig. 1C–E). At the light microscope level intercellular spaces between the two generations are rarely evident. The placental cells of both generations have a distinctive cytology very different from the highly vacuolated cells in the central region of the foot and the other gametophyte cells. The sporophytic haustoria develop prior to the emergence of the first leaf. In Pteridium these contain dense cytoplasm packed with elongate plastids and a large vacuole towards the centre of the foot (Fig. 1C). Sub sequently the plastids are filled with starch and dispersed throughout the cells (Fig. 1D). The gametophytic cells adjacent to the haustoria remain highly vacuolated but after the emergence of the first leaf their plastids also accumulate starch. Wall ingrowths appear first in gametophytic placental cells along the flanks and the tips of the young sporophytic haustoria (Fig. 1C) but later these also appear within the haustoria themselves (Fig. 1D). The same ontogenetic changes occur in the placentas of Gleichenia and Adiantum but in Ceratopteris (Fig. 1E) gametophytic wall ingrowths are less prominent and extensive sporophytic wall labyrinths develop before the accumulation of starch in the plastids.

Fig. 1. Gametophtye–sporophyte junctions in ferns. Light micrographs of 0·5 µm toluidine blue stained, longitudinal sections. A, Gleichenia sp., mature bulbous foot with interdigitating sporophyte and gametophyte cells. B, Portion of mature foot of Pteridium. C, Pteridium, young sporophytic haustorial cell containing numerous plate‐like plastids. Wall ingrowths (arrowed) are restricted to the adjacent gametophyte cells. D, Pteridium, old sporophytic haustorial cells with wall ingrowths (arrowed) and amyloplasts. E, Ceratopteris, young sporophytic haustorial cells with extensive wall ingrowths (arrowed). F and G, Azolla. F, Germinating female sporocarp showing a young sporophyte with the first leaf lobe beginning to develop on the female gametophyte. The gametophyte tissues growing beyond the confines of the megaspore are highly vacuolated. In contrast the megaspore remains packed with food reserves layed down before germination. The ovoid sporophyte–gametophyte junction is arrowed. G, Detail of the junction from F showing a smooth boundary between the two generations both comprising highly vacuolated cells. g, Gametophyte; ll, leaf lobe; me, megaspore; p, plastid; s, sporophyte; st, starch. Bars = 100 µm (A, B and F); 10 µm (C–E); 25 µm (G).

In striking contrast to the leptosporangiate ferns described above, the foot of Azolla is ovoid (Fig. 1F and G) and there is no interdigitation between the cells of the two generations. The sporophytic placental cells contain dense cytoplasm, whereas those of the gametophyte are highly vacuolate.

Electron microscopy

Sporophyte haustoria.

Prior to the emergence of first juvenile leaf and the elongation of the haustoria in all four genera, the sporophytic placental cells contain numerous vacuoles (Fig. 2A). The cytoplasm contains aggregates of mitochondria, with dense stroma and packed with swollen cristae, together with elongate plastids containing small grana, clusters of plastoglobuli and scattered vesicles in the peripheral stroma. The nucleus is centrally located, ovoid to spherical with a diameter of 6–8 µm.

Fig. 2.Pteridium, transmission electron micrographs showing details of the sporophytic placental cells. A, Young sporophytic placental cell at the onset of wall ingrowth formation in the adjacent gametophyte. Note the aggregates of elongate starch‐free plastids with small grana and ovoid mitochondria. B, Onset of starch formation in the plastids. C, The side walls of a developing sporophytic haustorium lined with mitochondria adjacent to gametophytic walls ingrowths. D, Fully developed sporophyte haustorial cell containing numerous starch‐filled plastids and occasional wall ingrowths (arrowed) at the tip. Note the much more extensive gametophytic wall ingrowths and the small intercellular space (ic) between the two generations. E, Detail of the finger‐like sporophytic wall ingrowths from D. F, Old junction with extensive wall labyrinths in both generations and the sporophytic plastids packed with starch. g, Gametophyte; m, mitochondrion; p, plastid; s, sporophyte; st, starch. Bars = 5 µm (A, B, D and F); 2 µm (C and E).

As the haustoria begin to grow between the adjacent gametophytic cells, which, by this time, possess extensive wall ingrowths (Fig. 2B) the mitochondria and plastids disperse in the tip cytoplasm, which contains scattered profiles of endoplasmic reticulum (ER). Away from the tips of the haustoria mitochondria often line the walls of the sporophytic cells (Fig. 2C). Small starch grains begin to appear in the plastids which otherwise retain their elongate shape (Fig. 2B and C). In contrast the plastids in fully developed haustorial cells (Fig. 2D) are greatly distended with starch, though grana may still be discerned. Finger‐like wall ingrowths begin to appear around the tips of the haustoria (Fig. 2D and E). By the time the first leaf is 1–2 cm in length (Fig. 2F) these ingrowths are as extensive as those in the gametophyte. Starch now completely fills the plastids (Figs 2F and 3A). At first these are mainly situated in the tips of the haustoria, subsequently they disperse along the lateral walls, which in places lack wall ingrowths (Fig. 3A). By the emergence of the second leaf sporophytic wall ingrowths have developed around the upper parts of the foot where gametophytic wall ingrowths are lacking (Fig. 3B). Ultimately the placental cells of both generations become highly vacuolated (Fig. 3B). Small, mucilage‐filled lacunae (Fig. 3C) are occasionally visible between the cells of the two generations.

Fig. 3.Pteridium. A and B, Details of old placentas. A, Basal region of a haustorial cell showing massive starch grains in the sporophyte and wall ingrowths restricted to the gametophyte. B, Highly vacuolated placental cells in both generations. C, Intercellular space between the two generations. D–H, Details of young gametophytic placental cells: D, aggregation of free ribosomes; E, aggregation of variously shaped plastid profiles; F and G, highly pleomorphic plastids; H, extensively lobed nucleus. g, Gametophyte; n, nucleus; s, sporophyte; st, starch. Bars = 5 µm (A–C); 1 µm (D–H).

Gametophytic placental cells.

Wall labyrinths appear much earlier in the gametophytic placental cells (Fig. 2B) and their cytological differentiation is very different from the sporophyte. Prior to the development of the sporophytic haustoria the gametophytic placental cells contain aggregations of free ribosomes (Fig. 3D) and clusters of highly irregular plastid profiles with single thylakoids, sheets of osmiophilic globuli and occasional starch grains. Serial sections reveal that most of these profiles are linked to form a single multilobed organelle. Slightly older cells contain elongate mitochondria (Fig. 3F) and remarkably pleiomorphic plastids (Fig. 3F and G). These comprise narrow sheet‐like regions and more swollen portions containing peripheral reticulum, aggregations of osmiophilic globuli and occasional starch grains. The nucleus (Fig. 3H) is also highly lobed.

Starch accumulation in the sporophytic placental cells is accompanied by a remarkable series of changes in the ER on the gametophyte side (Fig. 4A–E). Single profiles of ER become increasingly distended (Fig. 4A) with some parts containing finely granular material others, coarser, denser deposits. Subsequently the ER fragments into clusters of microbodies with finely granular contents (Fig. 4B) and more pleiomorphic vacuoles with dense contents often interspersed with lipid droplets (Fig. 4C). As the first leaf emerges the cytoplasm contains an ovoid to spherical nucleus alongside numerous electron‐opaque tannin deposits (Fig. 4D). Ultimately these fuse to form a single large tannin‐filled vacuole (Fig. 4E).

Fig. 4.Pteridium, sporophytic placental cells: A, profiles of dilated endoplasmic reticulum with dense contents; B, aggregation of microbodies; C, pleomorphic vacuoles with granular contents and lipid bodies; D, aggregation of tannin vesicles adjacent to an ovoid nucleus; E, old gametophyte cells with large tannin vacuoles; F and G, stacks of mitochondria. er, Endoplasmic reticulum; l, lipid bodies; n, nucleus; t, tannin vacuoles. Bars = 5 µm (E); 2 µm (A–D and F); 1 µm (G).

Perhaps the most notable feature of the older gametophytic placental cells is the behaviour of the mitochondria (Figs 4F and G and 5C and D). These come together to form conspicuous stacks. Throughout the stacks the constituent mitochondria have swollen saccate cristae and osmiophilic globuli scattered in the stroma. As can be seen from Figs 4F and G and 5C and D similar stacks occur in all four genera studied, and placental ontogeny, as detailed above for Pteridium, is more or less the same. Minor differences include less digitate gametophytic wall ingrowths in Athyrium (Fig. 5A) and Gleichenia (Fig. 5B) than in Pteridium and very extensive labyrinthine walls in the fully developed sporophytic haustoria of Ceratopteris (Fig. 5G). The mitochondria in this last genus (Fig. 5F) have less swollen and more scattered cristae than in the other three.

Fig. 5. A and B, Gametophytic wall ingrowths in Athyrium and Gleichenia. Note the elongate starch‐free plastid in B. C–E, Stacks of mitochondria in old gametophytic placental cells of Gleichenia, Athyrium and Ceratopteris. F and G, Sporophytic placental cells of Ceratopteris showing numerous mitochondria and labyrinthine wall ingrowths. H and I, Azolla placental cells showing wall ingrowths in both generations. g, Gametophyte; n, nucleus; p, plastid; s, sporophyte. Bars = 2 µm (B, H and I); 1 µm (A and C–G).

Figure 5H and I illustrates the key features of the Azolla placenta. Wall ingrowths are equally well developed on both sides of a smooth junction that lacks intercellular spaces. Otherwise the cytology of the cells is like that of typical parenchyma cells. Both generations contain approximately spherical nuclei, ovoid plastids with small starch grains, scattered mitochondria and large vacuoles.

DISCUSSION

This study, on four widely separate members of the leptosporangiate ferns, reveals that the group has a highly distinctive gametophyte–sporophyte junction. The key features are: (a) single celled sporophytic haustoria closely interdigitating with the adjacent gametophyte cells; (b) absence of significant intraplacental spaces; (c) absence of degenerating cells at the junction; (d) the early appearance of wall ingrowths in both generations with those in the gametophyte appearing first; (e) major ontogenetic changes in the cytology of the gametophyte cells; (f) reversion of the placental cells to a highly vacuolated condition following nutritional independence of the sporophyte but without occlusion of the wall ingrowths as occurs in Lycopodium and many bryophytes (Ligrone et al., 1993).

Overall, fern placentas appear to be remarkably similar to those in Tmesipteris apart from the restriction of wall ingrowths to the gametophytic cells in the latter (Frey et al., 1994a, b), although details of the cytoplasmic organization in the latter still require further investigation. They are also very different from those of lycophytes. This new information clearly reinforces the inclusion of psilophytes and ferns in the euphyllophyte clade and distances both from the lycophytes. Similar data are now required for eusporangiate ferns and Equisetum to see whether they also possess haustorial sporophytic cells. A careful search for these structures in possible gametophyte–sporophyte junctions in Devonian Rhyniopsida (Frey et al., 1997) would also be useful to strengthen the claim that this represents the basal condition in the euphyllophytes.

At first sight, fern and Tmesipteris placentas appear very similar to those of hornworts (Ligrone et al., 1993). Commonalities include the bulbous shape of the foot, sporophytic haustoria, mitochondrial aggregations in the gametophyte cells, early development of wall ingrowths, pleomorphic gametophytic plastids with rudimentary grana and starch accumulation in the sporophytic plastids. However, the list of differences is similarly extensive. Sporophytic wall ingrowths are absent in hornworts (and Tmesipteris). The haustorial cells are branched and often septate in hornworts, which also possess large mucilage‐filled intraplacental spaces often containing proteinaceous crystals and degenerating gametophyte placental cells. Taken together the differences between these haustorial placentas point to parallel evolution rather than homology. This conclusion is supported by the presence of a superficially similar placenta in Diphyscium which, uniquely in mosses, possesses a foot covered with multicellular outgrowths that penetrate deeply into the adjacent gametophytic tissues (Ligrone et al., 1993). It is therefore concluded that earlier descriptions of fern placentas were based on inadequate information. Although these did correctly record wall ingrowths in both generations, the interface between the two generations was erroneously described as smooth. Thus, the interesting notion of two distinct pteridophyte lineages proposed by Frey et al. (2001), on the basis of placenta morphology no longer appears valid, all the more so, when set against other far more robust phylogenies based on multiple independent morphological and molecular characters (Renzaglia et al., 2000; Pryer et al., 2001).

The smooth junction between the two generations and the absence of unusual cytoplasmic features (these are closely similar cytologically to meristematic cells; Barlow et al., 1982) represents a clear‐cut discontinuity between Azolla and leptosporangiate ferns. The placental wall ingrowths are less extensive than those in the leaf cavity trichomes (Duckett et al., 1975; Calvert et al., 1985). Major differences also occur between the placentas of homosporous (Lycopodium) and heterosporous (Isoëtes) lycophytes (Hilger et al., 2002). It would now be interesting to see if other heterosporous ferns have similar junctions to Azolla and whether Selaginella and Phylloglossum, the male gametes of which are very different from those of Isoëtes (Renzaglia et al., 2000), have similarly diverse placentas.

Apart from phyletic implications, this study has also brought to light several unusual cytological features of fern placental cells, some reminiscent of bryophytes, others unique. Most striking is the contrast between the structure and ontogeny of the sporophyte and gametophyte plastids. The former change from discoidal and starch‐free, to ovoid or spherical and distended with starch grains. The latter, whilst the placental activity is maximal, become highly pleomorphic, most likely being derived from the fragmentation of a giant multilobed organelle. However, following nutritional independence of the sporophyte, they revert to simple ovoid structures. Highly pleomorphic multilobed single plastids characterize the sporophytic placental cells in the liverwort Haplomitrium (Ligrone et al., 1993) and similar plastid ontogeny has been recorded previously in meristematic and differentiating vascular parenchyma of both ferns and lycophytes (Duckett et al., 1996) and in sporogenesis in Pteridium (Sheffield and Bell, 1987). In bryophytes pleomorphic plastids are in general more frequent in sporophytic placental cells, and starch is equally distributed in both generations.

Equally remarkable are the stacks of mitochondria that develop in the gametophyte placental cells of ferns. Similar stacks occur in the gametophyte placental cells of some hornworts, and mitochondrial aggregates have also been noted in the differentiating cortical and vascular tissues of Tmesipteris and Psilotum (Fineran and Ingerfeld, 1985) but these are linked by numerous fine fibrils which were not observed in the placental cells. The functional significance of mitochondrial aggregation is unknown (for a review, see Duckett et al., 1977).

This study provides evidence that large tannin‐filled vacuoles, frequently observed in ferns, derive from distended ER cisternae. Microbodies, which have a similar origin to tannin vacuoles are also frequent in gametophyte placental cells. Aggregations of ribosomes and pleomorphic nuclei indicate that the functioning of gametophyte placental cells requires profound cytological reorganization recalling the occurrence of similar events in fern oogenesis and sporogenesis (Sheffield and Bell, 1987).

ACKNOWLEDGEMENTS

The authors thank Dr G. A. Peters for the kind gift of Azolla sporocarps, Dr Karen S. Renzaglia for the Ceratopteris cultures and Keith Pell and Paul Fletcher (QMUL) for skilful technical assistance. The observations were, in part, performed at the CISME (University of Naples ‘Federico II’, Italy), technical staff of which are gratefully acknowledged.

Received: 26 March 2003; Returned for revision: 21 May 2003; Accepted: 10 June 2003