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The ultrastructure of the exine of the megaspores in two palaeozoic seed-like structures
Review of Palaeobotany and Palynology, 63 (1990): 137-152
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
The Ultrastructure of the Exine of the Megaspores in two Palaeozoic Seed-like Structures A L A N R. H E M S L E Y
Departments of Biology and Geology, Royal Holloway and Bedford New College (University of London), Egham, Surrey TW20 OEX (England) (Received August 30, 1989; revised and accepted November 30, 1989)
Abstract Hemsley, A.R., 1990. The ultrastructure of the exine of the megaspores in two Palaeozoic seed-like structures. Rev. Palaeobot., Palynol., 63: 137-152. The exine of Cystosporites devonieus Chaloner and Pettitt (1964), a Devonian seed-megaspore, and Didymosporites seottii Chaloner (1958), a Carboniferous fern tetrad, are investigated using SEM, TEM and laser scanning microscopy. C. devonieus is shown to have a homogeneous exine structure and in this respect is different from all other members of this genus (lycopod seed megaspores), which have a fibrous exine. The genus Spermasporites is erected to accommodate Devonian, Cystoporites-like tetrads with a homogeneous exine. The new combination Spermasporites devonieus (Chaloner et Pettitt) Hemsley has been made. The exine of the functional spores of Didymosporites is also simple in structure but the bag of tapetal material surrounding the tetrad is complex and is suggested to be analogous, and possibly homologous, with the tapetal membrane of gymnosperms.
Introduction Cystosporites devonicus a n d Didymosporites scottii are t w o fossil spore species of different age a n d b o t a n i c a l affiliations, w h i c h h a v e i m p o r t a n t i m p l i c a t i o n s with r e g a r d to the origin of h e t e r o s p o r y and the e v o l u t i o n of the seed m e g a s p o r e syndrome. B o t h c a n be seen to r e p r e s e n t stages in a h y p o t h e t i c a l s e q u e n c e of spore e v o l u t i o n c u l m i n a t i n g in the gymnosperm seed ( C h a l o n e r a n d Pettitt, 1987). This s t u d y deals with u l t r a s t r u c t u r a l i n v e s t i g a t i o n s of the exines of t w o spore t e t r a d s t h a t h a v e a t t a i n e d an ovule-like g r a d e o f evolution, one with two f u n c t i o n a l m e m b e r s o f the t e t r a d (Didymosporites scottii), the o t h e r with o n l y one (Cystosporites devonicus). N e w d a t a from S E M and T E M c o n t r i b u t e s to the u n d e r s t a n d ing of the e n c l o s i n g s t r u c t u r e s as well as the m e g a s p o r e s themselves, 0034-6667/90/$03.50
Cystosporites devonicus is a n ellipsoidal Upper D e v o n i a n spore r e a c h i n g a r o u n d 2 mm in length. The w i d t h is v a r i a b l e due to p l e a t i n g of the exine in c o m p r e s s i o n b u t is u s u a l l y a r o u n d 0.3 mm. The m e g a s p o r e is s u r m o u n t e d by t h r e e t i g h t l y adpressed, a b o r t e d spores w h i c h conceal the t r i r a d i a t e m a r k , f o r m i n g a cap-like s t r u c t u r e . The s u r f a c e of b o t h f u n c t i o n a l a n d a b o r t e d spores is s m o o t h to g r a n u l a r with u n d u l a t i o n s w h i c h m a y r e p r e s e n t the impressions of n u c e l l a r a n d t a p e t a l cells. The exine u l t r a s t r u c t u r e h a s been described as finely g r a n u l a r a n d h o m o g e n e o u s t h r o u g h o u t as seen by light m i c r o s c o p y ( C h a l o n e r a n d Pettitt, 1964). A t h i n m e m b r a n e s u r r o u n d i n g the entire t e t r a d has been r e p o r t e d by P e t t i t t a n d Beck (1968), who o b t a i n e d the m e g a s p o r e s from inside the fossil seed. Cystosporites devonicus was based on a number of specimens obtained from U p p e r D e v o n i a n
© 1990 Elsevier Science Publishers B.V.
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material from Canada, showing a large "fertile" megaspore and three small "aborted" members of the tetrad (Chaloner and Pettitt, 1964). In this respect it resembled the Carboniferous spore genus Cystosporites (Schopf, 1938) known to be produced by Lepidocarpon. Spores matching the description of C. devonicus have been reported from several localities and may all have been produced by similar plants (Chaloner and Pettitt, 1963; Chaloner and Pettitt, 1964; Mortimer and Chaloner, 1967). Cystosporites devonicus occupies a significant position with regard to the origin of the enclosed megaspore and the evolution of the seed habit. In their description of the cupulate organ Archaeosperma arnoldii, Pettitt and Beck (1968) refer to C. devonicus and suggest that the dispersed spore of A. arnoldii would be assignable to that species. Gillespie et al. (1981) do not mention C. devonicus in their discussion of mid Devonian cupules and ovular structures, but it seems likely (from their illustrations and description)that their ovular megaspores are comparable to this dispersed species. Seed megaspores described by FaironDemaret and Scheckler (1987) in their redescription of Moresnetia zalesskyi would also be included within this taxon. The exine ultrastructure of C. devonicus has not previously been investigated, The spores of Stauropteris burntislandica Bertrand (1909), an enigmatic fern from the late Lower Carboniferous of Scotland, were first reported by R. Scott (1908) in her description of the sporangium Bensonites fusiformis although at the time this was believed to be some form of glandular structure. The sporangium was later shown to be borne by the fern-like plant S. burntislandica (Chodat, 1912). In his detailed survey of the plant, Surange (1952) described the megasporangium and showed clearly the presence of two (presumably functional) megaspores. He also commented on the observation by B.D. Harrison on the occurrence of a small spore in association with the two large spores and concluded from this that the contents of the sporangium derived from a single tetrad. Some debate ensued regarding the nature of the sporangial contents, i.e. how many mega-
A.R. HEMSLEY
spores were present and how many small spores also occupied the sporangial chamber. In a description of the vascular system and sporangia of Stauropteris burntislandica, Lacey et al. (1957)suggested that there may have been as many as 6 megaspores per sporangium although their illustrations are inconclusive on this matter. No mention of small, aborted spores was made; however, these authors implied that some abortion had occurred, by their belief that the contents of the sporangium were perhaps derived from two initial tetrads. Chaloner (1958) demonstrated the existence of two (functional) megaspores and two smaller (aborted) spores. The spore tetrad in the dispersed state was given the name Didymosporites scottii Chaloner, this name being used in following discussion. (To comply with current usage, an orthographic change is made here to the specific epithet of this species.) The ultrastructure of the exine of Didymosporites scottii was briefly investigated by Pettitt (1966b). He found that the exine of specimens obtained from a coal macerate lacked any significant structure. Specimens of Stauropteris berwickensis Long (1966), from the late Tournaisian deposits of Berwickshire (Scott et al., 1984), also appear to have borne sporangia containing pairs of megaspores (and presumably a pair of small spores) and these would probably be indistinguishable in the dispersed state from tetrads produced by S. burntislandica. Material and methods Sporangial contents referable to Bensonites fusiformis were extracted from samples of Pettycurlimestone collected byW.G. Chaloner in 1952 on the foreshore of the Pettycur locality, Fifeshire, Scotland. The site is described by Rex and Scott (1987). The age of the Pettycur limestone is now considered to be late Vis~an (see Scott et al., 1984). The spores, together with the surrounding structures, are found as dispersed units within a matrix containing plentiful macrofossil remains of
Stauropteris burntislandica. Samples of limestone were dissolved in 10% HC1. The organic material was washed with
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
distilled water and separated using various grade sieves such that particles measuring between 50 and 500 ~m were retained. Spore tetrads enclosed in their surrounding envelope were then picked from the macerate. Tetrads for SEM were attached to standard stubs using glue tabs and air dried. These were then sputter coated with gold/palladium before viewing with a Cambridge S100 SEM. Specimens of Cystosporites devonicus were obtained by H F maceration of material from the Acanthodon Beds, Escuminac Formation, Scauminac Bay, Quebec, Canada (the type locality and horizon of that species). The rock sample containing the spores discussed here (number V63163) was collected by W. GrahamSmith in 1937 and was kindly lent for study by the British Museum (Natural History). This specimen is considered conspecific with the spore tetrad described by Chaloner and Pettitt (1964). Tetrads of spores for observation by SEM were4)repared by the same process as was used for Didymosporites scottii, Tetrads of both spore species for TEM investigation were soaked in 40~/o H F for 48 hours, neutralised and transferred to acetone, These were then introduced to a 50% mixture of Spurr resin (hard mix - - see Spurr, 1969) with acetone for 24 hours before being transferred to 100~/o resin for a further 8 hours. The preparation was placed under vacuum for a few minutes to remove remaining air and then the resin was polymerised at 70°C for 24 hours, Thin sections of between 80 and 100 nm thickness were cut using glass knives. The sections were collected on formvar-coated copper grids and viewed with a Hitachi H-600 TEM. Thick sections of around 0.5 ~m were cut for observation by light and Zeiss laser scanning microscope (LSM)(see Scott, 1989). Results Cystosporites devonicus: Description and
remarks The spore tetrad of Cystosporites devonicus measures between 1.5 and 2 mm in length and
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around one third of this in width (the width of the functional megaspore). The flattened functional spore is oval in outline and considerably elongated. The aborted spores comprise only one tenth of the total length of the tetrad, being about 150 ~m long and about 120 pm in maximum width (Plate I, 1, 3, 5). The three aborted spores adhere tightly to each other and to the apex of the functional spore. Arcuate ridges are present on the functional megaspore around the contact areas. No indication of a membrane surrounding the entire tetrad (as discussed by Pettitt and Beck, 1968) has been found in this study. The surface of both spore types is uneven and irregular (Plate I, 2). In detail, the surface consists of small granules, variously linked and fused (Plate I, 4). These granules measure 2 to 3 Ira1 in diameter. The surface is often interrupted by pyrite and shows indications of degradation. Folds occur along the length of the functional megaspore and are the result of the compression of an object that was probably circular in cross section. The hardness, state of preservation and density of the exine of C. devonicus made this spore difficult to section. The exine of both the aborted and functional megaspores is homogeneous to finely granular in structure (Plate II, 1-4). The aborted spores have a thin exine (Plate II, 4) and a narrow lumen. The exine appears to be fused between adjacent spores although this may be a result of the age and preservation of the spore. The exine of the functional megaspore is much thicker than that of the aborted spores (Plate II, 1), up to 60 pxn as compared to 15 to 20 lJm. No lumen is present, a result which, like the fusion of the exine of the aborted spores, suggests that certain changes in exine composition and structure have occurred. This is similar to the situation reported by Hemsley (1989) in the spores of Parka decipiens, where there is no cavity to indicate the original position of the lumen. Observations of knife damage on certain sections suggest that although the spore exine appears structurally homogeneous, it may be varied in its composition, some parts of
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A.R. HEMSLEY
PLATE I
ULTRASTRUCTUREOF EXINEOF MEGASPORESIN SEED-LIKESTRUCTURES
the exine being softer than others (Plate II, 3). No evidence has been found of surrounding membranes or of any differentiation toward the middle of the spore indicating the presence of a basal lamina.
Comparison with other species Cystosporites devonicus has an unstructured exine and while this may partly be a result of preservation, it seems unlikely that all indications of a complex exine ultrastructure would have been lost. The absence of any indication of a fibrous exine organisation, makes Cystosporites devonicus markedly different from other members of that genus. Other species of Cystosporites are large seed megaspores derived from lepidocarp sporangia, such as Lepidocarpon and Achlamydocarpon. The exine ultrastructure of some species of Cystosporites has been investigated by Pettitt (1966b), Taylor (1974), Taylor and Brack-Hanes (1976) and Brack-Hanes (1981). In general, the exine has a fibrous, open structure, occasionally covered by a more solid outer layer which may consist of residual tapetal debris. The absence of a fibrous coat, and indeed, the absence of any fibrous structures within the exine of Cystosporites devonicus conflicts with the diagnosis of Cystosporites (Schopf, 1938). However, the overall form of the tetrad, an elongate functional megaspore with three small aborted spores attached to the apex is similar in appearance to the Carboniferous species of Cystosporites (Chaloner and Pettitt, 1964).
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C. verrucosus was cited by Chaloner and Pettitt as an example of a seed megaspore within the genus Cystosporites which did not possess a fibrous exine (and being therefore similar to C. devonicus). However, in a study of Caudatocorpus, Brack-Hanes (1981) showed that the seed megaspores from this cone (Lagenicula saccata syn. Cystosporites verrucosus) did indeed possess a fibrous exine, thus weakening the similarity of C. devonicus to any other Cystosporites species. The exine of the aborted spores of Cystosporites devonicus is thinner than the functional megaspore exine and in this respect is unlike that of other aborted seed megaspores (Taylor, 1974; Taylor and Brack-Hanes, 1976; Brack-Hanes, 1981). This perhaps suggests that the functional spore of C. devonicus enlarges before significant sporopollenin deposition occurs rather than deposition of exine units followed by expansion as proposed by Taylor (1974) for the seed megaspore obtained from Achlamydocarpon. Delayed sporopollenin deposition, if taken to an extreme, would allow time for the complete degeneration of aborted megaspores in the tetrad such that only the functional megaspore would receive a coating, as is the case in modern gymnosperms (Pettitt, 1969). In view of the different exine structure, different age and probably different origin (gymnosperm rather than lycopod) of these spores, it would seem reasonable to follow the suggestion of Pettitt (1970) and erect a new genus for this species.
PLATEI
Cystosporites devonicus (SEM) 1. The complete tetrad of C. devonicus dominated by the single functional and elongate megaspore. The aborted spores remain attached at the apex (right). 2. The functional megaspore wall is uneven and apparently composed of smooth, fused granules. Much of the surface sculpturing is a result of the impression of the surrounding matrix. 3. The apex of the functional megaspore with the three aborted spores forming a tetradhedral "cap" at the apex. A fold in the spore wall is present along the length of the functional spore. 4. Detail of the surface of the functional megaspore showing granular components. 5. Detail of the aborted megaspores. The edges of the three spores are adjacent and form three sharply defined ridges with a distinct median groove. The body of each spore has collapsed leaving a triangular, concave depression.
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A.R. HEMSLEY
PLATE II
Cystosporites devonicus (TEM) 1. Outer part of the fertile megaspore exine. The structurally homogeneous exine is particularly hard. Knife marks and damage to the specimen result from heterogeneity in the composition of the exine. The black material on the surface of the spore (above) is the gold coating retained after SEM study. 2. Detail of the outer part of the exine showing its complete homogeneity. 3. The central part of the functional spore, presumably crossing the original position of the lumen. A change in composition at the centre of the spore is the only remaining evidence of the lumen. Cutting the spore perpendicular to the lumen results in a discontinuity (top) which may reflect a n internal interface between opposing parts of the wall. Additional markings (arrows) may represent similar interfaces between exine units. 4. The complete aborted spore exine is t h i n (around 10 ~m) and largely homogeneous with only a faint indication of the presence of exine units. The lumen can be seen bottom right.
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
Class PTERIDOSPERMOPSIDA incertae sedis
Genus Spermasporitesgen. nov.
Type species: Spermasporites devonicus (Chaloner and Pettitt) comb. nov. Etymology: From sperma, a seed, and -sporites, a suffix denoting a fossil spore, Diagnosis: Isolated, megaspore tetrads consisting of onelarge, elongated megaspore (presumably fertile) and three small, subtriangular ones (presumably abortive). Exine of fertile spore, frequently folded longitudinally, external surface granular but otherwise unornamented; aborted spores less evidently granular, Internal exine ultrastructure in both fertile and aborted spores, not differentiated into regions, homogeneous to finely granular, not fibrous, Spermasporites devonicus (Chaloner and Pettitt) comb. nov. et emend. (plates 1 and 2) syn. Cystosporites devonicus Pettitt and Chaloner (1964) Emended diagnosis: Large (fertile)megaspore, oval in outline, elongated 2550 ~m in length and 750 pm greatest width; shallow arcuate ridges present on large spore beyond area of contact with aborted spores; laesurae around 100 to 150 ~m in length, visible at apex when aborted spores are missing; surface, granular; exine up to 60 ~m in thickness. Small (aborted) spores subtriangular; shortest side (adjacent to fertile spore) 40 to 60 ~m in length; other sides 60 to 120 ~m long; surface, smooth to finely granular; exine 10 to 20 ~m in thickness, Holotype: V45428, Department of Palaeontology, British Museum (Natural History). Figures: Chaloner and Pettitt (1964) (plate 3, figs.l, 2. plate 4, fig.l). Type locality: Escuminac Formation, Scaumenac Bay, Quebec, Canada. Age: Upper Devonian. Didymosporites scottii: Description and
remarks The
three-dimensional
spore tetrads of Stauropteris burntislandica appear as ornam-
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ented ovoid structures around 450 l~n long and 210 pm in diameter (Plate III, 1). In most cases the outline of the enclosed megaspores is obscured by tapetal debris deposited as a saclike covering. If this outer coating is teased apart, the relatively smooth outer layer of the exine can be seen (Plate III, 2). Light microscopy reveals that the two aborted spores (which lie at right angles to the functional megaspores at the edge of their region of contact) bear a T-shaped triradiate mark (see Plate IV and Fig.l). The cross of the T separates the contact face linking these two spores from the larger faces in contact with the functional spores. The exine of the aborted spores appears transparent and structureless with conventional light microscopy (Plate IV, 2). Under the LSM, the surface ornamentation of the aborted spores (Plate IV, 3) appears to be granular and corresponds with the indications of ornamentation observed under SEM. The surface details of the outer sac structure and exine are seen most clearly with SEM (Fig.2 and Plate III, 3, 4) and the enclosing material shows considerable complexity with three distinct structural units. (1) Periclinal walls: these outermost units are large and more or less continuous, particularly around the equator of the functional spores. (2) Anticlinal walls: the periclinal walls are supported on straight to slightly sinuous anticlinal walls (10 to 20 ~m high and 2 to 4 ~m in width). These interconnect to form chambers. (3) Reticulum: the floor of the chambers is formed by a fine reticulum which is fused at the base to produce a solid innermost structure. Isolated megaspores are around 120 pm in diameter and from 130 to 180 ~m in length (Plate III, 5). The outermost spore in each SlJorangium is usually narrower and more pointed towards the distal pole. At low magnification, the megaspore exine appears smooth but more detailed observation reveals a weakly tuberculate ornamentation (Plate IV, 1). The megaspores are sharply folded, the folds demarcating collapsed areas which are generally
~ !
~i! ~'~¸'
~~
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
145
A
Complete
tetrad
20~m
B
F u n c t i o n a l s p o r e pair
C
D .,~. :. !.-..~;.
Aborte¢
spore pair
Contact
with
a b o r t e d spore
C o n t a c t s with functional s p o r e s
Fig.1. (A) The spore tetrad of Didymosporites is composed of two pairs of spores set at right angles. (B) The functional megaspores (the presumed distal one r a t h e r more conical t h a n the proximal) are barely attached to each other and retain no significant indication of the original position of the aborted spores. (C) The pair of aborted spores removed from the tetrad and rotated so t h a t the faces adjacent to the functional megaspores can be seen. A sharp ridge is retained on this face equivalent to the cleft between the two functional spores. (D) A single aborted spore showing the T-shaped triradiate mark t h a t results from this configuration of spores.
PLATE III
Didymosporites scottii (SEM) 1. A tetrad of Didymosporites surrounded by structured tapetal material (tapetal membrane). 2. Part of the tetrad showing the relatively smooth wall of a n exposed megaspore (left) and a n area of reticulate tapetal membrane. 3. The structure of the tapetal membrane. Polygonal chambers with a reticulate floor and thin walls (anticlinal walls) are shown without the covering structures (left) and with these structures (periclinal walls) (right). See also Fig.2. 4. Detail of the reticulate basal part of the tapetal structure and t h i n anticlinal walls. 5. A single functional megaspore removed from the tetrad. The spore has collapsed leading to sharp folds in the relatively thin walls. No indication of a triradiate mark is present.
3
r~
=:
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
147
~1 extensions
Llls
Tapetal membrane lls
0.4/~m Fig.2. A diagram illustrating the exine and surrounding material (the tapetal membrane of Pettitt) of a functional megaspore of Didymosporites scottii. All wall material is of similar electron density but is shown stippled (exine) and black (tapetal material) to emphasise the different structures. polygonal in shape. In places, the reticulate p o r t i o n of the o u t e r sac adheres to the e x i n e although usually (and particularly in the r e g i o n a d j a c e n t to the a r e a of c o n t a c t of the t w o f u n c t i o n a l m e g a s p o r e s ) , t h e r e is a g a p between this and the outer layer. No laesurae
are seen o n the large spores. The a b o r te d spores have a similar surface texture although t h i s t e n d s t o be m o r e p r o n o u n c e d t h a n i n t h e fertile spores. T h e y also lack the folding of the f u n c t i o n a l s p o r e s ( P l a t e IV, 4, 7). E a c h m a i n t a i n s a m o r e o r less s u b t r i a n g u l a r o u t l i n e a n d
PLATE IV
Did yrnosporites scotti i 1.
Detail of the surface of a functional megaspore wall. The surface features are irregular and slightly verrucate and may result in part from remnants of the tapetal membrane (SEM). 2. A transmitted light micrograph showing the two aborted megaspores. A narrow T-shaped triradiate mark can be seen on both spores. See also Fig.1. 3. Confocal scanning laser micrograph (LSM) of a single aborted spore. The plane of focus passes through the triradiate mark and reveals part of the proximal surface detail and the homogeneity of the exine material immediately beneath the surface. 4. Half of the spore tetrad showing the sharp folds present on the functional spore and a small, subtriangular aborted spore. Both are enclosed within the tapetal membrane (SEM). 5, 6 LSM micrographs of the tapetal membrane; confocal and confocal fluorescence respectively. The reticulate part of the tapetal structure runs top to bottom (left). Larger units forming the anticlinal and periclinal walls are to the right. The fine structure is just discernible using this microscopical technique. Fluorescence (6) is seen in many (but not all) outer structures and is of more general occurrence in the reticulate layer. 7. Detail of the aborted spore shown in 4. The surface ornament is apparent (SEM).
0~
ULTRASTRUCTUREOF EXINEOF MEGASPORESIN SEED-LIKESTRUCTURES
shows some indication of laesurae (the Tshaped mark). These spores are around 35 ~m along the polar axis and 30 ~m maximum width, With TEM the megaspore exine shows a fine granular matrix but is otherwise structureless (see Plate V, 1-3). The basal portion of the reticulate unit of the outer envelope often adheres closely to the exine and this may in part be responsible for the surface features of the spores (see Plate V, 4). At its thinnest (a perpendicular section) the exine is about 0.8 pm thick but in the region of the contact faces, reaches 1.6 ~m. One section shows structures which appear similar to the "multifoliate laminae" described in fern spores by Lugardon (1978) (Plate V, 2). No laesurae or apertures were encountered in any TEM sections or thick sections viewed by LSM. The structure of the outer envelope seen under TEM supports the interpretation from SEM (see Plate V, 5, 6; Fig.2). The outermost periclinal walls are variable in thickness. Gaps sometimes occur between these layers, generally towards the inner edge. Periclinal walls are between 0.5 and 3 pm in thickness and are joined at infrequent intervals by the underlying anticlinal walls which measure about 0.2 ~m in width. The base of the anticlinal walls interconnect with the upper layers of the reticulum. This appears as a thin lace-like structure with numerous cavities. The fused basal part of the reticulum often remains attached to the exine in dispersed specimens,
149
the anticlinal and periclinal units separating from the spore tetrad. There is no clear evidence t h a t the material surrounding the spore tetrad is composed of sporopollenin. However, these structures survive almost as much oxidation (up to 4 hours) in Schulze's solution as the spores, and observations of LSM fluorescence (see Scott, 1989) (Plate IV, 6), imply t h a t sporopollenin may be present as a component of this material, particularly in the reticulate layer which is most resistant to oxidation and has the greatest fluorescence. Both exine and the surrounding structures stain with safranin. The outer envelope of material in Bensonites originally referred to as the "spore bag" by Surange (1952) appears to have a more complex structure t h a n had previously been supposed. Chaloner (1958) believed this sac of "fibrous" material to be the remains of a tapetum and Pettitt (1966b) refers to the remains of this layer, obtained by oxidative maceration of coal, as thin and structureless. Tapetal debris frequently occurs around seed megaspores and megaspore membranes (Pettitt, 1966a)but such debris never approaches the complexity encountered in Bensonites. In most cases such remains are disorganised layers, globules, or homogeneous sheets. The structures found in the outer envelope of Bensonites are most similar to those found around the megaspores of the water fern Marsilea (Pettitt, 1966b; Southworth and Myles, 1984), in Ginkgo and cycads (Pettitt, 1970), and also around pre-
PLATE V
Didymosporites scottii (TEM) 1. The homogeneous exine of the functional megaspore. Equatorial TS. 2. Detail of the wall structure showing fine g r a n u l a r ultrastructure. Some indication of lamellate deposition is suggested toward the outside of the wall (arrows). 3. The homogeneous exine (bottom) gives way to the more heterogeneous tapetal material (above). Some suggestion of layering is present in the basal part of the tapetal membrane but this is less apparent in the upper parts of the reticulate region. 4. The wall frequently splits along the boundary between the basal part of the tapetal membrane (left) and the spore exine (right). 5. A section t h r o u g h a chamber within the tapetal membrane. The reticulate region (bottom) and anticlinal walls (arrows) occur b e n e a t h the thick overlying material (periclinal wall) which bears a n u m b e r of extensions. 6. Detail of the periclinal units. A single anticlinal wall and part of the reticulate region can be seen toward the bottom of the frame.
150
viously reported fossil megaspore membranes (Zimmerman and Taylor, 1970). Pettitt refers to the more organised structures found around seed megaspores as the "tapetal membrane" and restricts the term ~'megaspore membrane" to the sporopollenin layers believed to be homologous with the free-sporing megaspore exine. The structured outer envelope of Didymosporites may therefore be in this sense homologous with the tapetal membrane surrounding seed megaspores and it seems reasonable to assume that the chambers formed by periclinal walls, anticlinal walls and reticulum may have originally been occupied by tapetal cells (Pettitt, 1977). However, the reticulate base to these chambers is probably not the result of simple tapetal breakdown. The nature of the outer surface of the periclinal walls suggests a second overlying layer of tapetal cells or sporangial cells around which sporopollenin was only partially deposited, the periclinal units themselves having more of the features one might expect from tapetal degen° eration i.e. variable thickness and a lack of organisation (see Fig.2). No evidence has been found that the outer envelope leaves any impression on the exine as suspected by Chaloner (1958) (as seen in flattened spores from coal deposits) although in some cases, particularly around the equator of the functional spores, the reticulum adheres to the exine and traces of this can be identified under the light microscope. Similarly, the SEM has provided no evidence of a distinct triradiate mark on the functional megaspores reported by Surange (1952) and Lacey et al. (1957). Their observations may have resulted from sets of three folds which converge at a point close to the expected position of a triradiate mark. The triradiate mark (present on the aborted spores) may be lost in the expansion of the functional megaspores. The multiple megaspores observed by Lacey et al. have not been found in this study. Rather, I accept Chaloner's (1958) suggestion that there are really only two '~fertile" megaspores and that folding of the exine accounts for multiple encounters with the same spore wall
A.R. HEMSLEY
in any particular section. Nonetheless, my observations cannot entirely preclude the occurrence of more than two functional megaspores in the same sporangium, be it by failure to abort or the initial presence of two tetrads. In the 16 sporangia examined in this study, there were consistently only two large spores. The belief of Chaloner (1958) and Long (1966) that the Didymosporites tetrad was dispersed as a single unit within the sporangium, or at least within the outer envelope, is reinforced by this study. The complexity and sturdiness of the outer envelope show no preformed line of dehiscence. The dispersal of two supposedly functional megaspores within the same unit is most unusual and the nature of the spores within the spore tetrad remains unresolved. Chaloner (1958) argued that it is unlikely that the tetrads of Didymosporites consisted of both micro and megaspores because the occurrence of micro and megaspores in the same sporangium was unknown among living plants and if the tetrad was the unit of dispersal, recombination of segregated alleles would occur, such that a condition similar to apomixis would result. Several examples are now known among Recent and fossil plants where micro and megaspores are formed within the same sporangium. These include Isoetes (Goswami and Arya, 1968; Goswami, 1974), the fossils Barinophyton (Pettitt, 1965; Taylor and Brauer, 1983), Protobarinophyton (Cichan et al., 1984) and Chaleuria (Andrews et al., 1974). Although no modern pteridophytes are known to produce tetrads consisting of 2 functional microspores and 2 megaspores, such a situation does occur in some bryophytes where a size difference in the spores is associated with the sex of the gametophyte. In these plants, the 2 + 2 tetrad is found to be the result of segregation of sex chromosomes at meiosis (Vitt, 1968) and both types of spore produced are functional. It is possible that Stauropteris burntislandica demonstrated a similar syndrome. Although fertilization of gametes from the same tetrad would severely restrict recombination within the species, there are short term gains that may be ecologically advantage-
U L T R A S T R U C T U R E OF EXINE OF M E G A S P O R E S IN SEED-LIKE S T R U C T U R E S
ous. Species adopting this habit can be efficient colonisers of constant habitats, apomixis being common amongst weedy plants. However, if the environment is in a state of flux or selective pressures vary rapidly, then the species may be unable to make a rapid genotype response. A more precise understanding of the biology and ecology of Stauropteris burtislandica will require definitive information on the function of the large and small spores in view of the possibility t h a t the small spores may have produced microgametophytes, Discussion The two spore tetrads described here, together with the lycopod seed megaspores (Taylor, 1975; Taylor and Brack-Hanes, 1 9 7 6 ; Brack-Hanes, 1981), illustrate how a number of plant groups have attained a particular grade of evolution: the reduction of the number of functional megaspores in the sporangium to one (or two) and the enclosure of the functional spore within a sporangium which was indehiscent. It seems likely t h a t the differences in exine ultrastructure encountered in these megaspores reflect nutritional requirements and environmental responses about which we know very little. It is possible t h a t the homogeneous exine observed in Cystosporites devonicus is a function of its enclosure within a seed-like structure. The homogeneous exine in this species is significant since it counters the view t h a t megaspore exines (with their ultrastructural complexity) were incorporated in an unaltered form within indehiscent sporangia, Additionally, some other explanation is needed for the subsequent development of complex megaspore and tapetal membranes in later gymnosperm ovules. It appears t h a t in many ways Bensonites was comparable to an ovular structure, the major differences being the presence of two functional spores and lack of integuments. It has been suggested (Coulter, 1898) t h a t only when the sporangial content is restricted to one functional spore, can there be the correct
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conditions for dispersal of the sporangium as a unit, self fertilization (recombination within the same sporangium) being impossible. However, if it is assumed that both functional spores in the Didymosporites tetrad were strictly female then self fertilization would presumably be equally impossible. It is only if the small spores in the tetrad were functional microspores (male) that a problem would occur. Whatever the sporangial contents of Stauropteris burntislandica, it is clear that this plant had attained an evolutionary grade lying between that of Selaginella or Archaeopteris and a true seed. The features shared by the seed-like megaspores discussed here show a number of parallels which are perhaps to be expected in structures performing the same function. Possibly less evident is the probability that these structures are analogous, and perhaps homologous, between the major plant groups represented here. Acknowledgements I should like to t h a n k Prof. W.G. Chaloner and Dr. A.C. Scott for their advice and encouragement, the staff of the Electron Microscopy Unit (R.H.B.N.C.), the British Museum (Natural H i s t o r y ) f o r the loan of specimens and Carl Zeiss (Oberkochen) for the use of their Laser Scanning Microscope. I also t h a n k Prof. W.T. Stearn and Prof. D.L. Hawksworth for their advice regarding the new generic name. This work was undertaken whilst A.R.H. was in receipt of an N.E.R.C. research studentship which is here gratefully acknowledged. References Andrews, H.N., Gensel, P.G. and Forbes, W.H., 1974. An apparently heterosporous plant from the Middle Devo-
nian of New Brunswick. Palaeontology, 17: 387-408. Bertrand, P., 1909. ]~tude de la fronde des Zygopt~rid~es.
Lille: Danel. Brack-Hanes, S.D., 1981. On a lycopsid cone with winged spores. Bot. Gaz., 142: 294-304.
Chaloner, W.G., 1958. Isolated Megaspore Tetrads of Stauropterisburntislandica. Ann. Bot., 22: 197-204.
152 Chaloner, W.G. and Pettitt, J.M., 1963. A Devonian seed megaspore. Nature, 198: 808-809. Chaloner, W.G. and Pettitt, J.M., 1964. A seed megaspore from the Devonian of Canada. Palaeontology, 7: 29-36. Chaloner, W.G. and Pettitt, J.M., 1987. The Inevitable Seed. Bull. Soc. Bot. Fr., Actual. Bot., 134:39 49. Cichan, M.A., Taylor, T.N. and Brauer, D.F., 1984. Ultrastructural studies of in situ Devonian spores: Protobarinophytonpennsylvanicurn Brauer. Rev. Palaeobot. Palynol., 41: 167--75. Chodat, R., 1912. Le Bensonites fusiformis gland du Stauropteris burntislandica. Bull. Soc. Bot. Gen~ve, 3: 353 60. Coulter, J.M., (1898). The origin of gymnosperms and the seed habit. Bot. Gaz., 39: 161-178. Fairon-Demaret, M. and Scheckler, S.E., 1987. Typification and redescription of Moresnetia zalesskyi Stockmans 1948, an early seed plant from the Upper Famennian of Belgium. Bull. Inst. R. Sci. Nat. Belg. Sci. Terre, 57: 183-199. Gillespie, W.H., Rothwell, G.W. and Scheckler, S.E., 1981. The earliest seeds. Nature, 293:462 464. Goswami, H.K. 1974. Chromosome Studies in Natural populations of Isoetes pantii, with Heterosporous Sporangia. Cytologia, 40: 543-551. Goswami, H.K. and Arya, B.S., 1968. Heterosporous sporangia in Isoetes. Br. Fern Gaz., 10: 39-41. Hemsley, A.R., 1989. The ultrastructure of the spores of the Devonian plant Parka decipiens. Ann. Bot., 64: 359367. Lacey, W.S., Joy, K.W. and Willis, A.J., 1957. Observations on the Aphlebiae and Megasporangia of Stauropteris burntislandica P. Bertrand. Ann. Bot., 21: 621-25. Long, A.G., 1966. Some Lower Carboniferous fructifica, tions from Berwickshire, together with a theoretical account of the evolution of ovules, cupules, and carpels, Proc. R. Soc. Edinburgh, 64: 345-75. Lugardon, B., 1978. Comparison between pollen and pteridophyte spore walls. IV Int. Palynol. Conf., Lucknow (1976-77), 1: 199-206. Mortimer, M.G. and Chaloner, W.G., 1967. Devonian megaspores from the Wyboston borehole, Bedfordshire, England. Palaeontology, 10: 189-213. Oliver, F.W. and Scott, D.H., 1903. Lagenostoma lomaxi, the seed of Lyginodendron. Ann. Bot., 17: 625-629. Pettitt, J. M., 1965. Two Heterosporous plants from the Upper Devonian of North America. Bull. Br. Mus. (Nat. Hist.) Geol., 10:83 92. Pettitt, J. M., 1966a. A new interpretation of the structure of the megaspore membrane in some gymnospermous ovules. Bot. J. Linn. Soc., 59: 253-63. Pettitt, J. M., 1966b. Exine structure in some fossil and recent spores and pollen as revealed by light and
A.R. HEMSLEY electron microscopy. Bull. Br. Mus. (Nat. Hist.) Geol,, 13: 223-57. Pettitt, J. M., 1969. Pteridophytic features in some Lower Carboniferous seed megaspores. Bot. J. Linn. Soc., 62: 233-239. Pettitt, J. M., 1970. Heterospory and the origin of the seed habit. Biol. Rev., 45: 401-415. Pettitt, J. M., 1977. The megaspore wall in gymnosperms: Ultrastructure in some zooidogamous forms. Proc. R. Soc. London, B 195:497 515. Pettitt, J. M. and Beck, C.B., 1968. Archaeosperma arnoldii - - a cupulate seed from the upper Devonian of North America. Contrib. Mus. Paleontol. Univ. Mich., 10: 139 154. Rex, G.M. and Scott, A.C., 1987. The sedimentology, palaeoecology and preservation of the Lower Carboniferous plant deposits at Pettycur, Fife, Scotland. Geol. Mag., 124: 43-66. Schopf, J.M., 1938. Spores from the Herrin (N °. 6) Coal Bed in Illinois. Rep. Invest. Ill. St. Geol. Surv., 50:1 73. Scott, A.C., 1989. Geological applications of Laser Scanning Microscopy. Microsc. Anal., 10:17 19. Scott, A.C., Galtier, J. and Clayton, G., 1984. Distribution of anatomically-preserved floras in the Lower Carboniferous in Western Europe. Trans. R. Soc. Edinburgh E a r t h Sci. 75:311 340. Scott, R., 1908. On Bensonites fusiformis sp. nov. a fossil associated with Stauropteris burntislandica and on the sporangia of the latter. Ann. Botany, 22: 683-87. Southworth, D. and Myles, D.G., 1984. Ultraviolet absorbance spectra of megaspore and microspore walls of Marsilea vestita. Pollen Spores, 26: 481-488. Spurr, A.R., 1969. A low viscosity resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Surange, K.R., 1952. The morphology of Stauropteris burntislandica P. Bertrand and its megasporangium Bensonites fusiformis R. Scott. Phil. Trans. R. Soc. London, B 237: 73-91. Taylor, T.N., 1974. Scanning electron microscopy of fossil megaspores: Wall development. Scanning Electron Microsc. 1974 (IITRI): 359-366. Taylor, T.N. and Brack-Hanes, S.D., 1976. The structure and reproductive significance of the exine in fossil lycopod megaspores. Scanning Electron Microsc. 1976 (IITRI): 513- 518. Taylor, T.N. and Brauer, D.F., 1983. Ultrastructural studies of in situ Devonian spores: Barinophyton citrulliforme. Am. J. Bot., 70: 106-12. Vitt, D.H., 1968. Sex determination in mosses. Mich. Bot., 7: 195-203. Zimmerman, R.P. and Taylor, T.N., 1970. The ultrastructure of Paleozoic megaspore membranes. Pollen Spores, 12: 451-68.
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
The Ultrastructure of the Exine of the Megaspores in two Palaeozoic Seed-like Structures A L A N R. H E M S L E Y
Departments of Biology and Geology, Royal Holloway and Bedford New College (University of London), Egham, Surrey TW20 OEX (England) (Received August 30, 1989; revised and accepted November 30, 1989)
Abstract Hemsley, A.R., 1990. The ultrastructure of the exine of the megaspores in two Palaeozoic seed-like structures. Rev. Palaeobot., Palynol., 63: 137-152. The exine of Cystosporites devonieus Chaloner and Pettitt (1964), a Devonian seed-megaspore, and Didymosporites seottii Chaloner (1958), a Carboniferous fern tetrad, are investigated using SEM, TEM and laser scanning microscopy. C. devonieus is shown to have a homogeneous exine structure and in this respect is different from all other members of this genus (lycopod seed megaspores), which have a fibrous exine. The genus Spermasporites is erected to accommodate Devonian, Cystoporites-like tetrads with a homogeneous exine. The new combination Spermasporites devonieus (Chaloner et Pettitt) Hemsley has been made. The exine of the functional spores of Didymosporites is also simple in structure but the bag of tapetal material surrounding the tetrad is complex and is suggested to be analogous, and possibly homologous, with the tapetal membrane of gymnosperms.
Introduction Cystosporites devonicus a n d Didymosporites scottii are t w o fossil spore species of different age a n d b o t a n i c a l affiliations, w h i c h h a v e i m p o r t a n t i m p l i c a t i o n s with r e g a r d to the origin of h e t e r o s p o r y and the e v o l u t i o n of the seed m e g a s p o r e syndrome. B o t h c a n be seen to r e p r e s e n t stages in a h y p o t h e t i c a l s e q u e n c e of spore e v o l u t i o n c u l m i n a t i n g in the gymnosperm seed ( C h a l o n e r a n d Pettitt, 1987). This s t u d y deals with u l t r a s t r u c t u r a l i n v e s t i g a t i o n s of the exines of t w o spore t e t r a d s t h a t h a v e a t t a i n e d an ovule-like g r a d e o f evolution, one with two f u n c t i o n a l m e m b e r s o f the t e t r a d (Didymosporites scottii), the o t h e r with o n l y one (Cystosporites devonicus). N e w d a t a from S E M and T E M c o n t r i b u t e s to the u n d e r s t a n d ing of the e n c l o s i n g s t r u c t u r e s as well as the m e g a s p o r e s themselves, 0034-6667/90/$03.50
Cystosporites devonicus is a n ellipsoidal Upper D e v o n i a n spore r e a c h i n g a r o u n d 2 mm in length. The w i d t h is v a r i a b l e due to p l e a t i n g of the exine in c o m p r e s s i o n b u t is u s u a l l y a r o u n d 0.3 mm. The m e g a s p o r e is s u r m o u n t e d by t h r e e t i g h t l y adpressed, a b o r t e d spores w h i c h conceal the t r i r a d i a t e m a r k , f o r m i n g a cap-like s t r u c t u r e . The s u r f a c e of b o t h f u n c t i o n a l a n d a b o r t e d spores is s m o o t h to g r a n u l a r with u n d u l a t i o n s w h i c h m a y r e p r e s e n t the impressions of n u c e l l a r a n d t a p e t a l cells. The exine u l t r a s t r u c t u r e h a s been described as finely g r a n u l a r a n d h o m o g e n e o u s t h r o u g h o u t as seen by light m i c r o s c o p y ( C h a l o n e r a n d Pettitt, 1964). A t h i n m e m b r a n e s u r r o u n d i n g the entire t e t r a d has been r e p o r t e d by P e t t i t t a n d Beck (1968), who o b t a i n e d the m e g a s p o r e s from inside the fossil seed. Cystosporites devonicus was based on a number of specimens obtained from U p p e r D e v o n i a n
© 1990 Elsevier Science Publishers B.V.
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material from Canada, showing a large "fertile" megaspore and three small "aborted" members of the tetrad (Chaloner and Pettitt, 1964). In this respect it resembled the Carboniferous spore genus Cystosporites (Schopf, 1938) known to be produced by Lepidocarpon. Spores matching the description of C. devonicus have been reported from several localities and may all have been produced by similar plants (Chaloner and Pettitt, 1963; Chaloner and Pettitt, 1964; Mortimer and Chaloner, 1967). Cystosporites devonicus occupies a significant position with regard to the origin of the enclosed megaspore and the evolution of the seed habit. In their description of the cupulate organ Archaeosperma arnoldii, Pettitt and Beck (1968) refer to C. devonicus and suggest that the dispersed spore of A. arnoldii would be assignable to that species. Gillespie et al. (1981) do not mention C. devonicus in their discussion of mid Devonian cupules and ovular structures, but it seems likely (from their illustrations and description)that their ovular megaspores are comparable to this dispersed species. Seed megaspores described by FaironDemaret and Scheckler (1987) in their redescription of Moresnetia zalesskyi would also be included within this taxon. The exine ultrastructure of C. devonicus has not previously been investigated, The spores of Stauropteris burntislandica Bertrand (1909), an enigmatic fern from the late Lower Carboniferous of Scotland, were first reported by R. Scott (1908) in her description of the sporangium Bensonites fusiformis although at the time this was believed to be some form of glandular structure. The sporangium was later shown to be borne by the fern-like plant S. burntislandica (Chodat, 1912). In his detailed survey of the plant, Surange (1952) described the megasporangium and showed clearly the presence of two (presumably functional) megaspores. He also commented on the observation by B.D. Harrison on the occurrence of a small spore in association with the two large spores and concluded from this that the contents of the sporangium derived from a single tetrad. Some debate ensued regarding the nature of the sporangial contents, i.e. how many mega-
A.R. HEMSLEY
spores were present and how many small spores also occupied the sporangial chamber. In a description of the vascular system and sporangia of Stauropteris burntislandica, Lacey et al. (1957)suggested that there may have been as many as 6 megaspores per sporangium although their illustrations are inconclusive on this matter. No mention of small, aborted spores was made; however, these authors implied that some abortion had occurred, by their belief that the contents of the sporangium were perhaps derived from two initial tetrads. Chaloner (1958) demonstrated the existence of two (functional) megaspores and two smaller (aborted) spores. The spore tetrad in the dispersed state was given the name Didymosporites scottii Chaloner, this name being used in following discussion. (To comply with current usage, an orthographic change is made here to the specific epithet of this species.) The ultrastructure of the exine of Didymosporites scottii was briefly investigated by Pettitt (1966b). He found that the exine of specimens obtained from a coal macerate lacked any significant structure. Specimens of Stauropteris berwickensis Long (1966), from the late Tournaisian deposits of Berwickshire (Scott et al., 1984), also appear to have borne sporangia containing pairs of megaspores (and presumably a pair of small spores) and these would probably be indistinguishable in the dispersed state from tetrads produced by S. burntislandica. Material and methods Sporangial contents referable to Bensonites fusiformis were extracted from samples of Pettycurlimestone collected byW.G. Chaloner in 1952 on the foreshore of the Pettycur locality, Fifeshire, Scotland. The site is described by Rex and Scott (1987). The age of the Pettycur limestone is now considered to be late Vis~an (see Scott et al., 1984). The spores, together with the surrounding structures, are found as dispersed units within a matrix containing plentiful macrofossil remains of
Stauropteris burntislandica. Samples of limestone were dissolved in 10% HC1. The organic material was washed with
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
distilled water and separated using various grade sieves such that particles measuring between 50 and 500 ~m were retained. Spore tetrads enclosed in their surrounding envelope were then picked from the macerate. Tetrads for SEM were attached to standard stubs using glue tabs and air dried. These were then sputter coated with gold/palladium before viewing with a Cambridge S100 SEM. Specimens of Cystosporites devonicus were obtained by H F maceration of material from the Acanthodon Beds, Escuminac Formation, Scauminac Bay, Quebec, Canada (the type locality and horizon of that species). The rock sample containing the spores discussed here (number V63163) was collected by W. GrahamSmith in 1937 and was kindly lent for study by the British Museum (Natural History). This specimen is considered conspecific with the spore tetrad described by Chaloner and Pettitt (1964). Tetrads of spores for observation by SEM were4)repared by the same process as was used for Didymosporites scottii, Tetrads of both spore species for TEM investigation were soaked in 40~/o H F for 48 hours, neutralised and transferred to acetone, These were then introduced to a 50% mixture of Spurr resin (hard mix - - see Spurr, 1969) with acetone for 24 hours before being transferred to 100~/o resin for a further 8 hours. The preparation was placed under vacuum for a few minutes to remove remaining air and then the resin was polymerised at 70°C for 24 hours, Thin sections of between 80 and 100 nm thickness were cut using glass knives. The sections were collected on formvar-coated copper grids and viewed with a Hitachi H-600 TEM. Thick sections of around 0.5 ~m were cut for observation by light and Zeiss laser scanning microscope (LSM)(see Scott, 1989). Results Cystosporites devonicus: Description and
remarks The spore tetrad of Cystosporites devonicus measures between 1.5 and 2 mm in length and
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around one third of this in width (the width of the functional megaspore). The flattened functional spore is oval in outline and considerably elongated. The aborted spores comprise only one tenth of the total length of the tetrad, being about 150 ~m long and about 120 pm in maximum width (Plate I, 1, 3, 5). The three aborted spores adhere tightly to each other and to the apex of the functional spore. Arcuate ridges are present on the functional megaspore around the contact areas. No indication of a membrane surrounding the entire tetrad (as discussed by Pettitt and Beck, 1968) has been found in this study. The surface of both spore types is uneven and irregular (Plate I, 2). In detail, the surface consists of small granules, variously linked and fused (Plate I, 4). These granules measure 2 to 3 Ira1 in diameter. The surface is often interrupted by pyrite and shows indications of degradation. Folds occur along the length of the functional megaspore and are the result of the compression of an object that was probably circular in cross section. The hardness, state of preservation and density of the exine of C. devonicus made this spore difficult to section. The exine of both the aborted and functional megaspores is homogeneous to finely granular in structure (Plate II, 1-4). The aborted spores have a thin exine (Plate II, 4) and a narrow lumen. The exine appears to be fused between adjacent spores although this may be a result of the age and preservation of the spore. The exine of the functional megaspore is much thicker than that of the aborted spores (Plate II, 1), up to 60 pxn as compared to 15 to 20 lJm. No lumen is present, a result which, like the fusion of the exine of the aborted spores, suggests that certain changes in exine composition and structure have occurred. This is similar to the situation reported by Hemsley (1989) in the spores of Parka decipiens, where there is no cavity to indicate the original position of the lumen. Observations of knife damage on certain sections suggest that although the spore exine appears structurally homogeneous, it may be varied in its composition, some parts of
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A.R. HEMSLEY
PLATE I
ULTRASTRUCTUREOF EXINEOF MEGASPORESIN SEED-LIKESTRUCTURES
the exine being softer than others (Plate II, 3). No evidence has been found of surrounding membranes or of any differentiation toward the middle of the spore indicating the presence of a basal lamina.
Comparison with other species Cystosporites devonicus has an unstructured exine and while this may partly be a result of preservation, it seems unlikely that all indications of a complex exine ultrastructure would have been lost. The absence of any indication of a fibrous exine organisation, makes Cystosporites devonicus markedly different from other members of that genus. Other species of Cystosporites are large seed megaspores derived from lepidocarp sporangia, such as Lepidocarpon and Achlamydocarpon. The exine ultrastructure of some species of Cystosporites has been investigated by Pettitt (1966b), Taylor (1974), Taylor and Brack-Hanes (1976) and Brack-Hanes (1981). In general, the exine has a fibrous, open structure, occasionally covered by a more solid outer layer which may consist of residual tapetal debris. The absence of a fibrous coat, and indeed, the absence of any fibrous structures within the exine of Cystosporites devonicus conflicts with the diagnosis of Cystosporites (Schopf, 1938). However, the overall form of the tetrad, an elongate functional megaspore with three small aborted spores attached to the apex is similar in appearance to the Carboniferous species of Cystosporites (Chaloner and Pettitt, 1964).
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C. verrucosus was cited by Chaloner and Pettitt as an example of a seed megaspore within the genus Cystosporites which did not possess a fibrous exine (and being therefore similar to C. devonicus). However, in a study of Caudatocorpus, Brack-Hanes (1981) showed that the seed megaspores from this cone (Lagenicula saccata syn. Cystosporites verrucosus) did indeed possess a fibrous exine, thus weakening the similarity of C. devonicus to any other Cystosporites species. The exine of the aborted spores of Cystosporites devonicus is thinner than the functional megaspore exine and in this respect is unlike that of other aborted seed megaspores (Taylor, 1974; Taylor and Brack-Hanes, 1976; Brack-Hanes, 1981). This perhaps suggests that the functional spore of C. devonicus enlarges before significant sporopollenin deposition occurs rather than deposition of exine units followed by expansion as proposed by Taylor (1974) for the seed megaspore obtained from Achlamydocarpon. Delayed sporopollenin deposition, if taken to an extreme, would allow time for the complete degeneration of aborted megaspores in the tetrad such that only the functional megaspore would receive a coating, as is the case in modern gymnosperms (Pettitt, 1969). In view of the different exine structure, different age and probably different origin (gymnosperm rather than lycopod) of these spores, it would seem reasonable to follow the suggestion of Pettitt (1970) and erect a new genus for this species.
PLATEI
Cystosporites devonicus (SEM) 1. The complete tetrad of C. devonicus dominated by the single functional and elongate megaspore. The aborted spores remain attached at the apex (right). 2. The functional megaspore wall is uneven and apparently composed of smooth, fused granules. Much of the surface sculpturing is a result of the impression of the surrounding matrix. 3. The apex of the functional megaspore with the three aborted spores forming a tetradhedral "cap" at the apex. A fold in the spore wall is present along the length of the functional spore. 4. Detail of the surface of the functional megaspore showing granular components. 5. Detail of the aborted megaspores. The edges of the three spores are adjacent and form three sharply defined ridges with a distinct median groove. The body of each spore has collapsed leaving a triangular, concave depression.
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A.R. HEMSLEY
PLATE II
Cystosporites devonicus (TEM) 1. Outer part of the fertile megaspore exine. The structurally homogeneous exine is particularly hard. Knife marks and damage to the specimen result from heterogeneity in the composition of the exine. The black material on the surface of the spore (above) is the gold coating retained after SEM study. 2. Detail of the outer part of the exine showing its complete homogeneity. 3. The central part of the functional spore, presumably crossing the original position of the lumen. A change in composition at the centre of the spore is the only remaining evidence of the lumen. Cutting the spore perpendicular to the lumen results in a discontinuity (top) which may reflect a n internal interface between opposing parts of the wall. Additional markings (arrows) may represent similar interfaces between exine units. 4. The complete aborted spore exine is t h i n (around 10 ~m) and largely homogeneous with only a faint indication of the presence of exine units. The lumen can be seen bottom right.
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
Class PTERIDOSPERMOPSIDA incertae sedis
Genus Spermasporitesgen. nov.
Type species: Spermasporites devonicus (Chaloner and Pettitt) comb. nov. Etymology: From sperma, a seed, and -sporites, a suffix denoting a fossil spore, Diagnosis: Isolated, megaspore tetrads consisting of onelarge, elongated megaspore (presumably fertile) and three small, subtriangular ones (presumably abortive). Exine of fertile spore, frequently folded longitudinally, external surface granular but otherwise unornamented; aborted spores less evidently granular, Internal exine ultrastructure in both fertile and aborted spores, not differentiated into regions, homogeneous to finely granular, not fibrous, Spermasporites devonicus (Chaloner and Pettitt) comb. nov. et emend. (plates 1 and 2) syn. Cystosporites devonicus Pettitt and Chaloner (1964) Emended diagnosis: Large (fertile)megaspore, oval in outline, elongated 2550 ~m in length and 750 pm greatest width; shallow arcuate ridges present on large spore beyond area of contact with aborted spores; laesurae around 100 to 150 ~m in length, visible at apex when aborted spores are missing; surface, granular; exine up to 60 ~m in thickness. Small (aborted) spores subtriangular; shortest side (adjacent to fertile spore) 40 to 60 ~m in length; other sides 60 to 120 ~m long; surface, smooth to finely granular; exine 10 to 20 ~m in thickness, Holotype: V45428, Department of Palaeontology, British Museum (Natural History). Figures: Chaloner and Pettitt (1964) (plate 3, figs.l, 2. plate 4, fig.l). Type locality: Escuminac Formation, Scaumenac Bay, Quebec, Canada. Age: Upper Devonian. Didymosporites scottii: Description and
remarks The
three-dimensional
spore tetrads of Stauropteris burntislandica appear as ornam-
143
ented ovoid structures around 450 l~n long and 210 pm in diameter (Plate III, 1). In most cases the outline of the enclosed megaspores is obscured by tapetal debris deposited as a saclike covering. If this outer coating is teased apart, the relatively smooth outer layer of the exine can be seen (Plate III, 2). Light microscopy reveals that the two aborted spores (which lie at right angles to the functional megaspores at the edge of their region of contact) bear a T-shaped triradiate mark (see Plate IV and Fig.l). The cross of the T separates the contact face linking these two spores from the larger faces in contact with the functional spores. The exine of the aborted spores appears transparent and structureless with conventional light microscopy (Plate IV, 2). Under the LSM, the surface ornamentation of the aborted spores (Plate IV, 3) appears to be granular and corresponds with the indications of ornamentation observed under SEM. The surface details of the outer sac structure and exine are seen most clearly with SEM (Fig.2 and Plate III, 3, 4) and the enclosing material shows considerable complexity with three distinct structural units. (1) Periclinal walls: these outermost units are large and more or less continuous, particularly around the equator of the functional spores. (2) Anticlinal walls: the periclinal walls are supported on straight to slightly sinuous anticlinal walls (10 to 20 ~m high and 2 to 4 ~m in width). These interconnect to form chambers. (3) Reticulum: the floor of the chambers is formed by a fine reticulum which is fused at the base to produce a solid innermost structure. Isolated megaspores are around 120 pm in diameter and from 130 to 180 ~m in length (Plate III, 5). The outermost spore in each SlJorangium is usually narrower and more pointed towards the distal pole. At low magnification, the megaspore exine appears smooth but more detailed observation reveals a weakly tuberculate ornamentation (Plate IV, 1). The megaspores are sharply folded, the folds demarcating collapsed areas which are generally
~ !
~i! ~'~¸'
~~
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
145
A
Complete
tetrad
20~m
B
F u n c t i o n a l s p o r e pair
C
D .,~. :. !.-..~;.
Aborte¢
spore pair
Contact
with
a b o r t e d spore
C o n t a c t s with functional s p o r e s
Fig.1. (A) The spore tetrad of Didymosporites is composed of two pairs of spores set at right angles. (B) The functional megaspores (the presumed distal one r a t h e r more conical t h a n the proximal) are barely attached to each other and retain no significant indication of the original position of the aborted spores. (C) The pair of aborted spores removed from the tetrad and rotated so t h a t the faces adjacent to the functional megaspores can be seen. A sharp ridge is retained on this face equivalent to the cleft between the two functional spores. (D) A single aborted spore showing the T-shaped triradiate mark t h a t results from this configuration of spores.
PLATE III
Didymosporites scottii (SEM) 1. A tetrad of Didymosporites surrounded by structured tapetal material (tapetal membrane). 2. Part of the tetrad showing the relatively smooth wall of a n exposed megaspore (left) and a n area of reticulate tapetal membrane. 3. The structure of the tapetal membrane. Polygonal chambers with a reticulate floor and thin walls (anticlinal walls) are shown without the covering structures (left) and with these structures (periclinal walls) (right). See also Fig.2. 4. Detail of the reticulate basal part of the tapetal structure and t h i n anticlinal walls. 5. A single functional megaspore removed from the tetrad. The spore has collapsed leading to sharp folds in the relatively thin walls. No indication of a triradiate mark is present.
3
r~
=:
ULTRASTRUCTURE OF EXINE OF MEGASPORES IN SEED-LIKE STRUCTURES
147
~1 extensions
Llls
Tapetal membrane lls
0.4/~m Fig.2. A diagram illustrating the exine and surrounding material (the tapetal membrane of Pettitt) of a functional megaspore of Didymosporites scottii. All wall material is of similar electron density but is shown stippled (exine) and black (tapetal material) to emphasise the different structures. polygonal in shape. In places, the reticulate p o r t i o n of the o u t e r sac adheres to the e x i n e although usually (and particularly in the r e g i o n a d j a c e n t to the a r e a of c o n t a c t of the t w o f u n c t i o n a l m e g a s p o r e s ) , t h e r e is a g a p between this and the outer layer. No laesurae
are seen o n the large spores. The a b o r te d spores have a similar surface texture although t h i s t e n d s t o be m o r e p r o n o u n c e d t h a n i n t h e fertile spores. T h e y also lack the folding of the f u n c t i o n a l s p o r e s ( P l a t e IV, 4, 7). E a c h m a i n t a i n s a m o r e o r less s u b t r i a n g u l a r o u t l i n e a n d
PLATE IV
Did yrnosporites scotti i 1.
Detail of the surface of a functional megaspore wall. The surface features are irregular and slightly verrucate and may result in part from remnants of the tapetal membrane (SEM). 2. A transmitted light micrograph showing the two aborted megaspores. A narrow T-shaped triradiate mark can be seen on both spores. See also Fig.1. 3. Confocal scanning laser micrograph (LSM) of a single aborted spore. The plane of focus passes through the triradiate mark and reveals part of the proximal surface detail and the homogeneity of the exine material immediately beneath the surface. 4. Half of the spore tetrad showing the sharp folds present on the functional spore and a small, subtriangular aborted spore. Both are enclosed within the tapetal membrane (SEM). 5, 6 LSM micrographs of the tapetal membrane; confocal and confocal fluorescence respectively. The reticulate part of the tapetal structure runs top to bottom (left). Larger units forming the anticlinal and periclinal walls are to the right. The fine structure is just discernible using this microscopical technique. Fluorescence (6) is seen in many (but not all) outer structures and is of more general occurrence in the reticulate layer. 7. Detail of the aborted spore shown in 4. The surface ornament is apparent (SEM).
0~
ULTRASTRUCTUREOF EXINEOF MEGASPORESIN SEED-LIKESTRUCTURES
shows some indication of laesurae (the Tshaped mark). These spores are around 35 ~m along the polar axis and 30 ~m maximum width, With TEM the megaspore exine shows a fine granular matrix but is otherwise structureless (see Plate V, 1-3). The basal portion of the reticulate unit of the outer envelope often adheres closely to the exine and this may in part be responsible for the surface features of the spores (see Plate V, 4). At its thinnest (a perpendicular section) the exine is about 0.8 pm thick but in the region of the contact faces, reaches 1.6 ~m. One section shows structures which appear similar to the "multifoliate laminae" described in fern spores by Lugardon (1978) (Plate V, 2). No laesurae or apertures were encountered in any TEM sections or thick sections viewed by LSM. The structure of the outer envelope seen under TEM supports the interpretation from SEM (see Plate V, 5, 6; Fig.2). The outermost periclinal walls are variable in thickness. Gaps sometimes occur between these layers, generally towards the inner edge. Periclinal walls are between 0.5 and 3 pm in thickness and are joined at infrequent intervals by the underlying anticlinal walls which measure about 0.2 ~m in width. The base of the anticlinal walls interconnect with the upper layers of the reticulum. This appears as a thin lace-like structure with numerous cavities. The fused basal part of the reticulum often remains attached to the exine in dispersed specimens,
149
the anticlinal and periclinal units separating from the spore tetrad. There is no clear evidence t h a t the material surrounding the spore tetrad is composed of sporopollenin. However, these structures survive almost as much oxidation (up to 4 hours) in Schulze's solution as the spores, and observations of LSM fluorescence (see Scott, 1989) (Plate IV, 6), imply t h a t sporopollenin may be present as a component of this material, particularly in the reticulate layer which is most resistant to oxidation and has the greatest fluorescence. Both exine and the surrounding structures stain with safranin. The outer envelope of material in Bensonites originally referred to as the "spore bag" by Surange (1952) appears to have a more complex structure t h a n had previously been supposed. Chaloner (1958) believed this sac of "fibrous" material to be the remains of a tapetum and Pettitt (1966b) refers to the remains of this layer, obtained by oxidative maceration of coal, as thin and structureless. Tapetal debris frequently occurs around seed megaspores and megaspore membranes (Pettitt, 1966a)but such debris never approaches the complexity encountered in Bensonites. In most cases such remains are disorganised layers, globules, or homogeneous sheets. The structures found in the outer envelope of Bensonites are most similar to those found around the megaspores of the water fern Marsilea (Pettitt, 1966b; Southworth and Myles, 1984), in Ginkgo and cycads (Pettitt, 1970), and also around pre-
PLATE V
Didymosporites scottii (TEM) 1. The homogeneous exine of the functional megaspore. Equatorial TS. 2. Detail of the wall structure showing fine g r a n u l a r ultrastructure. Some indication of lamellate deposition is suggested toward the outside of the wall (arrows). 3. The homogeneous exine (bottom) gives way to the more heterogeneous tapetal material (above). Some suggestion of layering is present in the basal part of the tapetal membrane but this is less apparent in the upper parts of the reticulate region. 4. The wall frequently splits along the boundary between the basal part of the tapetal membrane (left) and the spore exine (right). 5. A section t h r o u g h a chamber within the tapetal membrane. The reticulate region (bottom) and anticlinal walls (arrows) occur b e n e a t h the thick overlying material (periclinal wall) which bears a n u m b e r of extensions. 6. Detail of the periclinal units. A single anticlinal wall and part of the reticulate region can be seen toward the bottom of the frame.
150
viously reported fossil megaspore membranes (Zimmerman and Taylor, 1970). Pettitt refers to the more organised structures found around seed megaspores as the "tapetal membrane" and restricts the term ~'megaspore membrane" to the sporopollenin layers believed to be homologous with the free-sporing megaspore exine. The structured outer envelope of Didymosporites may therefore be in this sense homologous with the tapetal membrane surrounding seed megaspores and it seems reasonable to assume that the chambers formed by periclinal walls, anticlinal walls and reticulum may have originally been occupied by tapetal cells (Pettitt, 1977). However, the reticulate base to these chambers is probably not the result of simple tapetal breakdown. The nature of the outer surface of the periclinal walls suggests a second overlying layer of tapetal cells or sporangial cells around which sporopollenin was only partially deposited, the periclinal units themselves having more of the features one might expect from tapetal degen° eration i.e. variable thickness and a lack of organisation (see Fig.2). No evidence has been found that the outer envelope leaves any impression on the exine as suspected by Chaloner (1958) (as seen in flattened spores from coal deposits) although in some cases, particularly around the equator of the functional spores, the reticulum adheres to the exine and traces of this can be identified under the light microscope. Similarly, the SEM has provided no evidence of a distinct triradiate mark on the functional megaspores reported by Surange (1952) and Lacey et al. (1957). Their observations may have resulted from sets of three folds which converge at a point close to the expected position of a triradiate mark. The triradiate mark (present on the aborted spores) may be lost in the expansion of the functional megaspores. The multiple megaspores observed by Lacey et al. have not been found in this study. Rather, I accept Chaloner's (1958) suggestion that there are really only two '~fertile" megaspores and that folding of the exine accounts for multiple encounters with the same spore wall
A.R. HEMSLEY
in any particular section. Nonetheless, my observations cannot entirely preclude the occurrence of more than two functional megaspores in the same sporangium, be it by failure to abort or the initial presence of two tetrads. In the 16 sporangia examined in this study, there were consistently only two large spores. The belief of Chaloner (1958) and Long (1966) that the Didymosporites tetrad was dispersed as a single unit within the sporangium, or at least within the outer envelope, is reinforced by this study. The complexity and sturdiness of the outer envelope show no preformed line of dehiscence. The dispersal of two supposedly functional megaspores within the same unit is most unusual and the nature of the spores within the spore tetrad remains unresolved. Chaloner (1958) argued that it is unlikely that the tetrads of Didymosporites consisted of both micro and megaspores because the occurrence of micro and megaspores in the same sporangium was unknown among living plants and if the tetrad was the unit of dispersal, recombination of segregated alleles would occur, such that a condition similar to apomixis would result. Several examples are now known among Recent and fossil plants where micro and megaspores are formed within the same sporangium. These include Isoetes (Goswami and Arya, 1968; Goswami, 1974), the fossils Barinophyton (Pettitt, 1965; Taylor and Brauer, 1983), Protobarinophyton (Cichan et al., 1984) and Chaleuria (Andrews et al., 1974). Although no modern pteridophytes are known to produce tetrads consisting of 2 functional microspores and 2 megaspores, such a situation does occur in some bryophytes where a size difference in the spores is associated with the sex of the gametophyte. In these plants, the 2 + 2 tetrad is found to be the result of segregation of sex chromosomes at meiosis (Vitt, 1968) and both types of spore produced are functional. It is possible that Stauropteris burntislandica demonstrated a similar syndrome. Although fertilization of gametes from the same tetrad would severely restrict recombination within the species, there are short term gains that may be ecologically advantage-
U L T R A S T R U C T U R E OF EXINE OF M E G A S P O R E S IN SEED-LIKE S T R U C T U R E S
ous. Species adopting this habit can be efficient colonisers of constant habitats, apomixis being common amongst weedy plants. However, if the environment is in a state of flux or selective pressures vary rapidly, then the species may be unable to make a rapid genotype response. A more precise understanding of the biology and ecology of Stauropteris burtislandica will require definitive information on the function of the large and small spores in view of the possibility t h a t the small spores may have produced microgametophytes, Discussion The two spore tetrads described here, together with the lycopod seed megaspores (Taylor, 1975; Taylor and Brack-Hanes, 1 9 7 6 ; Brack-Hanes, 1981), illustrate how a number of plant groups have attained a particular grade of evolution: the reduction of the number of functional megaspores in the sporangium to one (or two) and the enclosure of the functional spore within a sporangium which was indehiscent. It seems likely t h a t the differences in exine ultrastructure encountered in these megaspores reflect nutritional requirements and environmental responses about which we know very little. It is possible t h a t the homogeneous exine observed in Cystosporites devonicus is a function of its enclosure within a seed-like structure. The homogeneous exine in this species is significant since it counters the view t h a t megaspore exines (with their ultrastructural complexity) were incorporated in an unaltered form within indehiscent sporangia, Additionally, some other explanation is needed for the subsequent development of complex megaspore and tapetal membranes in later gymnosperm ovules. It appears t h a t in many ways Bensonites was comparable to an ovular structure, the major differences being the presence of two functional spores and lack of integuments. It has been suggested (Coulter, 1898) t h a t only when the sporangial content is restricted to one functional spore, can there be the correct
151
conditions for dispersal of the sporangium as a unit, self fertilization (recombination within the same sporangium) being impossible. However, if it is assumed that both functional spores in the Didymosporites tetrad were strictly female then self fertilization would presumably be equally impossible. It is only if the small spores in the tetrad were functional microspores (male) that a problem would occur. Whatever the sporangial contents of Stauropteris burntislandica, it is clear that this plant had attained an evolutionary grade lying between that of Selaginella or Archaeopteris and a true seed. The features shared by the seed-like megaspores discussed here show a number of parallels which are perhaps to be expected in structures performing the same function. Possibly less evident is the probability that these structures are analogous, and perhaps homologous, between the major plant groups represented here. Acknowledgements I should like to t h a n k Prof. W.G. Chaloner and Dr. A.C. Scott for their advice and encouragement, the staff of the Electron Microscopy Unit (R.H.B.N.C.), the British Museum (Natural H i s t o r y ) f o r the loan of specimens and Carl Zeiss (Oberkochen) for the use of their Laser Scanning Microscope. I also t h a n k Prof. W.T. Stearn and Prof. D.L. Hawksworth for their advice regarding the new generic name. This work was undertaken whilst A.R.H. was in receipt of an N.E.R.C. research studentship which is here gratefully acknowledged. References Andrews, H.N., Gensel, P.G. and Forbes, W.H., 1974. An apparently heterosporous plant from the Middle Devo-
nian of New Brunswick. Palaeontology, 17: 387-408. Bertrand, P., 1909. ]~tude de la fronde des Zygopt~rid~es.
Lille: Danel. Brack-Hanes, S.D., 1981. On a lycopsid cone with winged spores. Bot. Gaz., 142: 294-304.
Chaloner, W.G., 1958. Isolated Megaspore Tetrads of Stauropterisburntislandica. Ann. Bot., 22: 197-204.
152 Chaloner, W.G. and Pettitt, J.M., 1963. A Devonian seed megaspore. Nature, 198: 808-809. Chaloner, W.G. and Pettitt, J.M., 1964. A seed megaspore from the Devonian of Canada. Palaeontology, 7: 29-36. Chaloner, W.G. and Pettitt, J.M., 1987. The Inevitable Seed. Bull. Soc. Bot. Fr., Actual. Bot., 134:39 49. Cichan, M.A., Taylor, T.N. and Brauer, D.F., 1984. Ultrastructural studies of in situ Devonian spores: Protobarinophytonpennsylvanicurn Brauer. Rev. Palaeobot. Palynol., 41: 167--75. Chodat, R., 1912. Le Bensonites fusiformis gland du Stauropteris burntislandica. Bull. Soc. Bot. Gen~ve, 3: 353 60. Coulter, J.M., (1898). The origin of gymnosperms and the seed habit. Bot. Gaz., 39: 161-178. Fairon-Demaret, M. and Scheckler, S.E., 1987. Typification and redescription of Moresnetia zalesskyi Stockmans 1948, an early seed plant from the Upper Famennian of Belgium. Bull. Inst. R. Sci. Nat. Belg. Sci. Terre, 57: 183-199. Gillespie, W.H., Rothwell, G.W. and Scheckler, S.E., 1981. The earliest seeds. Nature, 293:462 464. Goswami, H.K. 1974. Chromosome Studies in Natural populations of Isoetes pantii, with Heterosporous Sporangia. Cytologia, 40: 543-551. Goswami, H.K. and Arya, B.S., 1968. Heterosporous sporangia in Isoetes. Br. Fern Gaz., 10: 39-41. Hemsley, A.R., 1989. The ultrastructure of the spores of the Devonian plant Parka decipiens. Ann. Bot., 64: 359367. Lacey, W.S., Joy, K.W. and Willis, A.J., 1957. Observations on the Aphlebiae and Megasporangia of Stauropteris burntislandica P. Bertrand. Ann. Bot., 21: 621-25. Long, A.G., 1966. Some Lower Carboniferous fructifica, tions from Berwickshire, together with a theoretical account of the evolution of ovules, cupules, and carpels, Proc. R. Soc. Edinburgh, 64: 345-75. Lugardon, B., 1978. Comparison between pollen and pteridophyte spore walls. IV Int. Palynol. Conf., Lucknow (1976-77), 1: 199-206. Mortimer, M.G. and Chaloner, W.G., 1967. Devonian megaspores from the Wyboston borehole, Bedfordshire, England. Palaeontology, 10: 189-213. Oliver, F.W. and Scott, D.H., 1903. Lagenostoma lomaxi, the seed of Lyginodendron. Ann. Bot., 17: 625-629. Pettitt, J. M., 1965. Two Heterosporous plants from the Upper Devonian of North America. Bull. Br. Mus. (Nat. Hist.) Geol., 10:83 92. Pettitt, J. M., 1966a. A new interpretation of the structure of the megaspore membrane in some gymnospermous ovules. Bot. J. Linn. Soc., 59: 253-63. Pettitt, J. M., 1966b. Exine structure in some fossil and recent spores and pollen as revealed by light and
A.R. HEMSLEY electron microscopy. Bull. Br. Mus. (Nat. Hist.) Geol,, 13: 223-57. Pettitt, J. M., 1969. Pteridophytic features in some Lower Carboniferous seed megaspores. Bot. J. Linn. Soc., 62: 233-239. Pettitt, J. M., 1970. Heterospory and the origin of the seed habit. Biol. Rev., 45: 401-415. Pettitt, J. M., 1977. The megaspore wall in gymnosperms: Ultrastructure in some zooidogamous forms. Proc. R. Soc. London, B 195:497 515. Pettitt, J. M. and Beck, C.B., 1968. Archaeosperma arnoldii - - a cupulate seed from the upper Devonian of North America. Contrib. Mus. Paleontol. Univ. Mich., 10: 139 154. Rex, G.M. and Scott, A.C., 1987. The sedimentology, palaeoecology and preservation of the Lower Carboniferous plant deposits at Pettycur, Fife, Scotland. Geol. Mag., 124: 43-66. Schopf, J.M., 1938. Spores from the Herrin (N °. 6) Coal Bed in Illinois. Rep. Invest. Ill. St. Geol. Surv., 50:1 73. Scott, A.C., 1989. Geological applications of Laser Scanning Microscopy. Microsc. Anal., 10:17 19. Scott, A.C., Galtier, J. and Clayton, G., 1984. Distribution of anatomically-preserved floras in the Lower Carboniferous in Western Europe. Trans. R. Soc. Edinburgh E a r t h Sci. 75:311 340. Scott, R., 1908. On Bensonites fusiformis sp. nov. a fossil associated with Stauropteris burntislandica and on the sporangia of the latter. Ann. Botany, 22: 683-87. Southworth, D. and Myles, D.G., 1984. Ultraviolet absorbance spectra of megaspore and microspore walls of Marsilea vestita. Pollen Spores, 26: 481-488. Spurr, A.R., 1969. A low viscosity resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Surange, K.R., 1952. The morphology of Stauropteris burntislandica P. Bertrand and its megasporangium Bensonites fusiformis R. Scott. Phil. Trans. R. Soc. London, B 237: 73-91. Taylor, T.N., 1974. Scanning electron microscopy of fossil megaspores: Wall development. Scanning Electron Microsc. 1974 (IITRI): 359-366. Taylor, T.N. and Brack-Hanes, S.D., 1976. The structure and reproductive significance of the exine in fossil lycopod megaspores. Scanning Electron Microsc. 1976 (IITRI): 513- 518. Taylor, T.N. and Brauer, D.F., 1983. Ultrastructural studies of in situ Devonian spores: Barinophyton citrulliforme. Am. J. Bot., 70: 106-12. Vitt, D.H., 1968. Sex determination in mosses. Mich. Bot., 7: 195-203. Zimmerman, R.P. and Taylor, T.N., 1970. The ultrastructure of Paleozoic megaspore membranes. Pollen Spores, 12: 451-68.