BLUMEA 36 (1992) 515-540
Sporogenesis in Polypodiaceae (Felicales). II.
The genera Microgramma Presl and Belvisia Mirbel
Gerda+A. van Uffelen
Rijksherbarium / Hortus Botanicus, Leiden, The Netherlands
Summary
Sporoderm developmenthas been studied in several species of polypodiaceous ferns belongingto
Presl and Belvisia Mirbel. Both transmission and electron micro- the genera Microgramma scanning
have been the latter This has shown different scopy applied, technique including freeze-fracturing. a succession of surface in the For each the ontogenetic spore patterns two genera. genus, ontogenetic series observed is also with surfaces in other compared mature spore congeneric species.
Introduction
of in This publication covers the study sporogenesis species belonging to the
Presl and Belvisia Mirbel. In the first polypodiaceous generaMicrogramma publica- tion in this series, sporogenesis in Drynaria sparsisora (Desv.) Moore is described
(Van Uffelen, 1990). In a third publication, sporogenesis in several other genera of
Polypodiaceae s.s. will be treated (Van Uffelen, in prep.). These publications are the result of of and the of in this a study spore morphology process sporogenesis family,
aimed at tracing possible new characters that may help to reconstruct relationships
This in this group of ferns (see Van Uffelen, 1990). may also help to explain the astounding variation in mature spore wall morphology as encountered in the family
Polypodiaceae s.s. as a whole (Van Uffelen & Hennipman, 1985; Hennipman,
1990).
MATERIAL AND METHODS
Material
15 and is sometimes The genus Microgramma Presl comprises about species treated of the The as part large genus Polypodium (Hennipman et al., 1990). species occur in the warmer areas of the American continent(Tryon & Tryon, 1982). Micro-
grammalycopodioides occurs also in Africa, Madagascar, and the Mascarenes.
of Presl The genus Belvisia, including part Lemmaphyllum (Hennipman et al.,
1990; Hovenkamp & Franken, in prep.), comprises 8-10 species and is confined to the tropics of the Old World.
Whenselecting species of Polypodiaceae for this study on sporogenesis, two cri-
should the selected all variation in teriawere applied. Not only species represent ma- 516 BLUMEA VOL. 36, No. 2, 1992
ture spores and sporogenesis as found in the family, they should also be regularly
different of time. and abundantly fertile, with many stages development present at a
Both Microgramma ciliata (Willd.) Alston and Belvisia mucronata (Fee) Copel. were
different tribes subfamiliesof the suitable as they represent or Polypodiaceae (Hennip-
and show man et al., 1990) quite different series of patterns during outer exospore formation. They were available in the botanic gardens ofthe universitiesof Leiden and
Utrecht, and were easy to sample.
Microgramma ciliata, recorded from the tropics of South America, has small di-
the fertile withround sori in Belvisia morphic fronds, ones arranged two rows. mu- cronata is known from the tropics of South East Asia and the Pacific. The sporangia
the midrib of the narrowed of the the are densely set along apical part frond, spike.
Mature spores of other species belonging to Microgramma orBelvisia have been studied with the SEM (see Table 1): Microgramma heterophylla (L.) Wherry, M. ly- copodioides (L.) Copel. and M. persicariifolia (Schrad.) Presl, and Belvisia novo- guineensis (Ros.) Copel., B. platyrhynchos (Kunze) Copel., and Lemmaphyllum abbreviatum (Fee) C. Chr., which is to be transferred to Belvisia (Hovenkamp &
Franken, in prep.). The spores were either taken from herbarium specimens or from
in the botanic of the universities of Leiden Utrecht plants grown gardens or (for methods, see Van Uffelen & Hennipman, 1985).
Methods
The of different found in different is difficult sequence exospore patterns species to discern and describe from two-dimensionalmicrographs as made with the TEM.
Therefore, as inDrynaria sparsisora, the freeze fracturing technique developed in
London by Blackmore & Barnes (1985) has been applied to sporogenesis in Micro- gramma ciliata and Belvisia mucronata. It involves the freeze fracturing of whole
followed with the SEM of the fractured in different sori, by study sporangia stages of development. In both species the development of the spore wall has also been studied with the TEM. Samples have been treated for study with the transmission electron microscope (TEM) and the scanning electron microscope (SEM) as describ- ed for Drynaria sparsisora (Van Uffelen, 1990), unless stated otherwise.
For SEM fixation, spikes ofBelvisia mucronata were cut into transverse slices of about2 mm thick (Plate V-l, 2). Slices from the base of the spike, where it is broadest, were cut in half longitudinally. The resulting pieces all have one transversal freeze
face fractured and one transversally cut face, and some also have a longitudinally cut
face. It is difficultto assess which one of the transversal faces is the result of freeze
fracturing, and which is not. Of one other species, Belvisia platyrhynchos, mature spores and immaturespores have with the fixed in been studied SEM Sporangia ofone specimen (see Table 1) were paraformaldehyde in the usual way (Van Uffelen, 1990), followed by critical point drying. Even without applying freeze fracturing, so many damaged sporangia with immature spores were found in the samples that a series of micrographs covering outer exospore development could be constructed and compared with the series as found in Belvisia mucronata. This surprising result shows that it is possible to study G.A. Uffelen: in Micro and Belvisia 517 van Sporogenesis gramma
wall without the time- and chem- series of surface patterns during spore development
ical-consuming procedure of freeze fracturing. The quality of the images is almost as
after the freeze this good as more complicated fracturing treatment. However, simpli-
fied technique can only be applied in species where the sporangia are exceptionally
after fragile, and at the same time retain enough of their contents for study breaking.
ciliata and Belvisia Germinating spores of both Microgramma mucronata were
in mediumuntil occurred, studied after cultivating mature spores a liquid germination
subsequent fixation in 4% paraformaldehyde, and critical point drying.
Table 1. Specimens studied.
Species Origin of material Sd Sm Sg Td Tm
M. ciliata LEI 19699 + + + + +
- - M. heterophylla Wright 799 (L) _- + _- _ _
M. lycopodioides LEI 22401 _- + _- _- _
M. persicariifolia Martius 384 (L)(L) _- + _- _ _-
L. abbreviation Cadire 97 (P) - + - - -
_— — - - L. abbreviationabbreviatum Gaudichaud s.n. (P) + _ _ _
B. mucronata LEI 21836 - + -— + +
B. mucronata LEI batchbatch 957 + + + - —-
B. novoguineensis Van Royen 11600 (L) _- + _- _- -_
B. platyrhynchos 86GR00027 U + + -— -— —
Sd = SEM of LEI Botanic of = study spore development LEI = Botanic garden of Leiden University
S = SEM of U = Botanic of Utrecht m study maturesporesspores garden University
= SEM of = Herbarium Leiden Sg study germinatingspores (L)
Td = TEM of Herbarium Paris Td = TEM study of spore development (P) = Herbarium Paris
Tm = TEM of study mature sporesspores
The temporal order of micrographs
The surface devel- micrographs representing spore patterns during outer exospore
opment in Microgramma ciliata, Belvisia mucronata, and B. platyrhynchos are pre-
sented as different series in their supposedly correct order, based on the author's
interpretation. Their order has been established using the following criteria (see also
Van Uffelen, 1990, on Drynaria sparsisora):
— spore length, which increases with the age of the spore; however, there is much
between overlap stages;
— of the of the laesural fold covered with height part not yet exospore material;
— exospore thickness, although it does not always seem to increase linearly with
time, and although exospore material may reduce in thickness by settling after its
deposition; 518 BLUMEA VOL. 36, No. 2, 1992
the author the in otherrelated — experience gained by while studying process species;
this lead in when the of however, may to some circularity argument using process
spore wall development in assessing relationships between species or other taxa.
As already mentionedwith respect to Drynaria sparsisora, sporangium size is no
criterion it is difficult if infer the from reliable as not impossible to ontogenetic stage it: sporangium size does not increase in a linear way with time, and sporangia may
be fractured their diameter. not through greatest
In the studied intoeach and species here, stages change gradually other, separate
difficult describe than in On the other stages are more to Drynaria sparsisora. hand, these gradual changes from one pattern into another have facilitated ordering the
into of the of wall formation. the micrographs a representation ontogeny spore In
of each series of is presentation results, micrographs thought to represent a true sequence in time, and is treated as such.
Terminology
In the spores included in this study, and in many other fern spores (Lugardon,
of the wall material is added from outside the 1978a, b), most spore spore by a
of material in series of different These superposition a usually patterns. patterns are
caused by local differences in wall thickness, be it on a large (e. g., verrucae) or on a small scale (e. g., in a scabrate surface), and have elements that are, in cross section,
continuous with the of usually rest the exospore. It makes sense to name a pattern by its most prominent features, which are usually protruding as well as eye-catching.
However, it is sometimes difficult to choose between depressions and elevations as the factor best characterizing a pattern. For instance, in mature spores of Belvisia, one may choose between a term based on depressions (grooves) and one based on protrusions (ridges).
In Belvisia, the patterns found during outer exospore formation are best described
in by depressions ('pits', see Van Uffelen, 1990) the surface, which is thus called foveolate. When of these form rows pits grooves, these grooves form the main ele-
of the the ment surface pattern. In mature spores ridges are the most prominent and
so the most easily described features. Therefore, in this genus the series of terms
describing subsequent patterns change from depression- to elevation-based.
The various patterns occurring during sporogenesis in Microgramma ciliata are best their described by most prominent features, the units or warts.
RESULTS
Microgramma Presl
Microgramma ciliata (Willd.) Alston
Fronds, sori, and sporangia
The sori are situated in two rows, one on each side of the main vein of the frond.
The sporangia of one sorus are implanted on a small semispherical cushion which is
to 0.5 in with hairs which up c. mm diameter, intermingled many are up to more than in Micro and Belvisia G.A. van Uffelen: Sporogenesis gramma 519
than the On freeze-fractured 1 mm long and are usually longer sporangia (Plate HI-6). lie pieces of frond this cushion is not always visible, as the plane of fracturing may found in beside it. More than 50 sporangia of different stages may be one sorus.
0.3 their stalk is about the Sporangia are up to c. mm long; as long as sporangium.
Sporogenesis formation consists of the In Microgramma ciliata the first part of outer exospore
smooth surface. After the thin deposition of small sporopollenin lumps over a this,
covered with which round to exospore layer gets contiguous warts ('units') are an- 2 in diame- gular in outline, irregularly sized (in all three dimensions), c. 0.4 to pm The wall is still rather thin, ter, and rounded and almost smooth at the top (Plate 1-1).
20 Some c. 0.2 pm in cross section (Plate 1-1). The spores are c. pm long. are partly
covered with tapetal material.
While the overall exospore thickness increases (Plate 1-3: c. 0.5 pm thick between
the remains the with units that ir- the warts), pattern same (Plate 1-2), contiguous are regular in size and oudine. However, they are more uniform in height. The laesural
material embeddedin the fold is not covered with much exospore or exospore layer,
3 The 28 and is at the most pm high (Plate 1-2). spores are now c. pm long.
Granular material is deposited over and between the warts, so that the warts grad-
ually become less prominent, with granular material in between, and with a slightly
in scabrate surface (Plate 1-4). The laesural fold is not yet completely embedded exo-
are now c. 32 spore material and is c. 2 pm high (Plate 1-5). The spores pm long.
Deposition of granular material continues (Plate 1-6, 7). This does not result in a conspicuous thickening of the exospore, which is about 0.4 pm thick between the units. The of the has become but the still top warts slightly more flattened, warts surface have the same outline, and have a similar fine structure. Small channel open-
The laesural fold still ings are present on and between warts (Plate 1-7). protrudes about 2 from the surface. The 36 pm exospore spores are c. pm long.
The deposition of finer exospore material causes the surface of the warts to lose its The become their granular character (Plate II-1). warts more prominent, and tops more irregular in shape. The laesura is also covered with a similar pattern (Plate II-2).
Small fragments of material are present, which are continuous with the spore sur- face. surface lies clear from the A few The spore spore coat or tapetum. spherical bodies of 2 in diameter found be attached the surface. The up to pm were to to spore spores are now c. 40 pm long.
49 When spore length has again increased to pm, the warts have become higher
The is 1-2 thick and their tops more irregular (Plate II-4). exospore now pm (Plate
II-3). By the end of exospore formation, the tops of the warts have become very irregular indeed (Plate II-6).
is In the mature exospore, the outer layer completely homogeneous, showing a few thin channels around the laesura and to the rest of the perpendicular exospore surface, sometimes ending in a fairly wide opening on the surface between warts
(Plate II-5).
formation with the the surface of Perispore starts appearanceon exospore a double membrane (Plate III-l) which is sometimes divided into overlapping fragments (Plate
In the 'blobs' of electron dense in III-2). tapetum many material, c. 0.1 pm diameter, 520 BLUMEA VOL. 36, No. 2, 1992
the membranes are present (Plate IH-3). They are subsequently deposited on (Plate ffl-1), thus forming the thin perispore layer. Part ofthis process can also be inferred fromPlate m-5. Each spine consists of a tapering bundle of thin rods (Plate EU-4).
It is not clear whether the spines are assembled lying in the tapetum, or in their final
of this position on the perispore surface. In some mature spores species the perispore is covered with a thin amorphous layer, although the perispore layer seems to be complete and finished.
Mature spores (Plate IV-1, 2)
Mature spores are monolete, concavo-convex to biconvex, rather shortly bean- shaped in lateral view (c. 49 pm long and 39 pm high, and c. 37 pm wide). The lae-
sura is c. 23 pm long.
The exospore surface is verrucate-colliculate (that is, with warts covering the whole surface, see Van Uffelen & Hennipman, 1985). The warts are c. 2-5 pm in
diameter, flattened, in outline irregular and round to oblong, their surface again ir-
bodies The that regularly verrucate. Spherical are rarely present. warts cover the area
around the laesura are sometimes less prominent (Plate IV-3). The inner exospore
layer is c. 0.05 pm thick, the outer layer is 0.5 pm thick between the warts, these
thickness. All adding up to 0.5 pm to its layers are slightly thicker near the laesura.
rather thin The perispore overlying this verrucate-colliculate pattern is and smooth,
which 0.5 The bearing minute spines (echinulae) are about pm long. laesura may also be covered with the verrucate exospore pattern and with perisporal spines.
Plate IV-4 shows a mature perispore. The perisporal surface of the warts and the
between them is smoother than the surface in the last of space exospore stages outer exospore formation. The outline of the mature exospore surface is still traceable through the thin perispore layer with its spines.
Plate IV-5 shows an opened spore from which the prothallium has fallenout, and of which the insideand section of the spore wall and laesura are well visible.
Other of species Microgramma: mature spores
Micrograrrum heterophylla (L.) Wherry (Plate IV-6)
The about spores are plano-convex, c. 49 pm long, 33 pm high, and 37 pm wide.
Their surface is the verrucate, warts usually round and c. 2 pm in diameter, some- times and 4 with sides and rounded flattened longish up to pm long, steep a or top, 1.5 lie c. pm high. The warts quite close together, but do not touch. The spore sur- face between the is warts smooth to slightly verrucate. The laesura is a prominent,
32 without c. pm long bar, any large warts. Around the laesura the larger warts are also The is thin and the lacking. perispore smooth, closely adhering to exospore sur- face, without any furtherornamentation.
Microgramma lycopodioides (L.) Copel. (Plate IV-7)
The spores are c. 50 pm long and 32 pm high. The exospore surface is entirely covered with or rounded warts. The are 3-5 low, slightly pointed warts round, c. pm in diameterand is 1 high. The laesura a c. 25 bar without pm prominent, pm long any ornamentation. The around area the laesura is less pronouncedly verrucate. The peri- G.A. Uffelen: in Micro and Belvisia van Sporogenesis gramma 521
the and bears minute echinae of spore is thin, closely adhering to exospore, scattered less than 1 pm long.
Microgramma persicariifolia (Schrad.) Presl (Plate IV-8).
43 34 31 The spores are c. pm long, c. pm high, and pm wide. The exospore sur- face consists of colliculate warts. The warts are round, up to 3 pm in diameter, and c. 1.5 pm high, with a rounded or slightly pointed top, smaller and lower in the area around the laesura. The laesura is a prominent, c. 25 pm long bar with only the slight- est ornamentation. The exospore is c. 1 pm thick, smooth on the inside. The peri-
spore is smooth and thin, adhering to the exospore surface and without any orna- mentation.
Belvisia Mirbel
Belvisia mucronata (Fée) Copel.
Fronds, sori, and sporangia
fertile of frond ofBelvisia consists of The part a mucronata an apical, long, thin,
folded with in different of structure ('spike') densely set sporangia many stages
development with paraphyses in between, which cover the sporangia when young.
become visible when almost Sporangia only they are mature; usually there are also
shorter stalked underneath. some younger, sporangia present
The spores of Belvisia, both young and old, are not always retained in the broken
sporangia as they are in Drynaria sparsisora and Microgramma ciliata. Thereforethey
are more difficult to study in situ. Furthermore, after having been observed for a first
time, they are more difficult to retrace for further study.
Sporogenesis
surface of The young spores that are already bean-shaped and have a distinct
laesural is almost smooth. low and small fold, entirely Very round lumps of exospore
material this surface. These 0.4 in are deposited on lumps are up to pm diameter. The short spore is then about 30 pm long, the laesural fold rather (c. 13 pm long) and c. 2.5 pm high (Plate V-3). TEM micrographs show that the outer exospore is much
thicker the side 0.4 than the distal side on proximal (c. pm thick) on (0.1-0.2 pm thick) (Plate V-4).
This is overlaid granulate pattern by a finely rugulate pattern. The rugulae are con- tiguous, covering all of the spore surface, separated only by tiny holes and short fine grooves; they are 0.2-0.3 pm broad, therefore on approximately the same scale as
that The the lumps cover slightly younger spores (Plate V-5, 6). spores are now over
30 The laesural fold is 1.3 that pm long. maximally pm high, so a considerablepart of the fold is already embedded in exospore material.
On this rather even surface more of the same rugulate surface is deposited. Grad-
shallow but surface become visible. ually, conspicuous depressions on the They are
less than 1 in and for the round, slightly pm diameter, are greaterpart scattered over the spore surface, while only a few of them touch each other (Plate V-7, 8).
As more exospore material is added, the depressions or 'pits' become deeper, but
Some hardly more numerous. start touching each other as they become wider (c. 0.4 pm in diameter). During this process the surface looses its finely rugulate character. 522 BLUMEA VOL. 36. No. 2, 1992
fine but the small holes that visiblebetween the The grooves disappear, were rugulae
there. These less than0.1 in diameterand are still clearly are pm probably correspond to small channels traversing the exospore layer. Such channels are also numerous in mature spores.
in number and become 1 in The pits increase slightly larger (c. pm diameter) and
deeper, so that the edges, which were fairly distinct at the beginning of pit formation,
The the fold are now beginning to slope. spores are now c. 40 pm long, laesural en- tirely embedded in exospore material. Some of the pits are fusing, giving a first in-
of the of and characteristic of the dication pattern grooves ridges mature exospore
(Plate VI-1, 2). On TEM, the pits and the smooth surface in between are well visible.
All pits show about the same depth, so they must have originated at the same time.
of the thick show The typical layering mature exospore begins to already (see Plate
The is about 1 thick the distal side and almost 2 thick Vm-1). exospore pm on pm
the laesura The become and in near (Plate VI-3). pits deeper start fusing greater num- bers. The laesura also bears a pattern resembling that of the rest of the spore surface
(Plate VI-4, 5). The spore surface between the fusing pits is rather smooth, apart
the still and visible small from numerous clearly holes.
Later on the pits are fusing to such an extent that it becomes difficultto distinguish individual pits. The pits, as far as they can still be counted, do not increase in num- ber any more. In this pattern of ridges and grooves the surface between the grooves is the small holes less becoming smoother, becoming numerous (Plate VI-6,7). The
of with pattern tortuous, irregular ridges grooves in between is now firmly estab-
lished.
The fairly smooth surface ofthe ridges and the laesura (see PlateVII-2) is becom-
ing more and more verrucate. The spore surface becomes distinctly knobby (Plate
At VII-1). first, the warts do not stand much apart from the rest of the surface, later
of them on some are recognizable as separate warts (Plate VII-3, 4). The spore sur- face is striate On the finely (Plate VII-4). mature exospore surface, the numerous warts deposited on the ridges are so conspicuous that in this species it has become
difficult to trace the underlying pattern of ridges and grooves (Plate VH-5).
As the consists of thin close the perispore a layer lying to exospore surface, on
SEM micrographs the perispore is not visibleexcept where it peels off. Therefore, it is best studied with the aid of TEM. On TEM the first indicationof perispore forma- tion is the appearance of thin double membranes (Plate VII-6) which develop into
membrane multilayered structures (Plate VII-6). They appear to be deposited by the
Blobs of material of about 0.1 in tapetum (Plate VII-8). tapetal pm diameterare also
found (Plate VII-7).
Mature spores (Plate VIII-1—3)
Mature spores are bean-shaped, plano-convex to slightly biconvex, c. 51 pm 35 and 33 wide. The long, pm high, pm irregularly shaped, tortuous ridges are c. 0.25 pm wide and more conspicuous than the grooves. Their surface is also irreg- ular, and set with of size densely warts varying that are around 0.25pm in diameter.
The 'grooves' often consist of a series of pits. The laesura is a prominent bar which is c. 21 pm long and covered with the same ornamentationas the ridges. The peri- is spore an extremely thin layer, closely adhering to the exospore surface. G.A. vanUffelen: Sporogenesis in Microgramma and Belvisia 523
Belvisia platyrhynchos (Kunze) Copel.
Sporogenesis
of have been found Of this species immature spores in different stages development after paraformaldehyde fixation and critical point drying withoutfreeze fracturing. In
the series of but there differ- most aspects patterns are similar, are some significant ences, despite the similarity between mature spores of B. mucronata and B. platy-
rhynchos.
surface with small After the stage in which the young spore has a smooth lumps
about 36 Plate the surface of c. 0.33 pm in diameter(spore length pm, IX-1), spore
does not show a finely rugulate pattern as in B. mucronata, but is almost entirely
smooth. The spore is already more than 40 pm long (Plate IX-2), but the laesural
in material: than 1 is still fold is not yet entirely embedded exospore more pm un- covered.
shallow visible During a further deposition of exospore material, pits become in a
smooth surface instead of in a rugulate surface as in B. mucronata. At first only a
few the distal and in pits are present on a spore (Plate IX-3), mainly on side, varying
size from0.5 to 2 pm in diameter.However, as they get larger and deeper, they also
get more numerous (Plate IX-4). The first pits occur when the spore is already quite
50 but when the laesural fold is covered with long (about pm), not yet entirely exo-
spore material.
2 in As in B. mucronata, the pits get larger (about pm diameter) and deeper, and
start fusing into the usual pattern of grooves, separated by ridges (Plate IX-5). Grad-
ually, the surface develops into that of the mature spore, as described for B. mucro-
nata (Plate IX-6, 7). It differs from that of B. mucronata in that the grooves and
ridges are on a slightly smaller scale, and are eventually covered with even more
warts.
Mature spores (Plate IX—8)
The spores are bean-shaped, concavo-convex to plano-convex, c. 60 pm long.
Their surface consists of small, rounded c. 1-3 pattern irregular, warts, pm in diam-
eter, arranged along a pattern of ridges. The laesura is small, c. 14 pm long, incon-
spicuous, and also bears some warts.
Other species in Belvisia
All other species studied in the genus have basically the same exospore pattern.
all have thin which is visible SEM They a perispore, not on micrographs.
Lemmaphyllum abbreviatum (Fée) C. Chr. (Plate VIII-5)
The spores are slightly concavo-convex to plano-convex, about 48 pm long, 36 pm wide. The ridges on the surface are more prominent than the grooves. The ridges
0.25 wide 0.5 are around pm (sometimes up to pm) and irregular as to surface and
with and between them. The laesura is about 14 form, grooves deeper pits pm long, distinct, devoid of much surface ornamentation. 524 BLUMEA VOL. 36, No. 2, 1992
Belvisia novoguineensis (Ros.) Copel. (Plate VIII-4)
The spores are approximately plano-convex, about 53 pm long, 40 pm high, and
wide. The surface 44 pm exospore pattern is smaller in all dimensions than in the
that the almost 0.1-0.2 other species studied, so spores are smooth inoutline. The
wide stand from a smoother surface, albeit with smallish than in pm ridges out pits, the other species. The ridge surface is sparsely set with small warts of up to 0.1 pm in diameter. The laesura is a prominent rounded bar of about 16 pm long, without much ornamentation.
DISCUSSION
Van Uffelen (1986) distinguished six main stages during sporogenesis inDrynaria sparsisora, mainly based on the study ofmicrographs made with the TEM:
1) presence of spore mother cells (smc's) 2) meiosis
3) formationof the spore plasmalemma
4) formationof the inner exospore (ie)
5) formationof the outer exospore (oe)
6) formation ofthe perispore
In Van Uffelen (1990), where sporogenesis in Drynaria sparsisora is described in
detail, 5 - the formationofthe - has been stage outer exospore divided into different
found the of substages, based on the different surface patterns during development this layer in this fem as seen on SEM micrographs.
In the species of which is described in the the sporogenesis present paper, stages
1-4 are similar to those of Drynaria sparsisora, therefore their description has not been included here. the first of During part outer exospore formation (stage 5) the differences in spore wall formation between these species begin to show after the deposition of small sporopollenin lumps on a smooth surface. As already mentioned in the material and section on methods, the change from one pattern into another
in the treated here that occurs so gradually species a subdivision of stage 5 is not
In the three the of the formationof the practicable. species studied, onset stage 6,
is the of double followed perispore, indicated by appearance membranes, by a multi- membrane layer of sometimes overlapping lamellae. In all these species, similarblobs of material involved in formation. tapetal are perispore
The genus Microgramma
Although in both Drynaria sparsisora (Van Uffelen, 1990) and Microgramma cili- ata outer exospore formationstarts with the deposition of small lumps of sporopollenin on a smooth surface, followed by the deposition of neatly defined units of roughly similar size and shape, the formationof the rest of the outer exospore layer is rather different. While in Drynaria sparsisora outer exospore formationis characterized by a succession of dramatically different patterns, on which a clear subdivision of this
be in ciliata the that succeed each stage can based, Microgramma patterns other are
and from into another. not strikingly different, change gradually one in and Belvisia 525 G.A. van Uffelen: Sporogenesis Microgramma
of In Microgramma ciliata, increase in spore length seems to occur by stretching There the developing spore wall rather than by intercalary growth. are two arguments for this supposition:
in the first of outer — The considerable increase spore length during stages exospore
- - - Plate 1-4 Plate formation, from 20 to 36 pm (see series Plate 1-1 Plate 1-2
of units the 1-6), occurs without any increase in the number covering spore sur-
from the face. Therefore, the increase in spore length apparently results stretching
of existing elements.
material Plate 1-4 and Plate exo- — Despite the additionof new granular (see 1-6,7)
this is in with the of the spore thickness does not increase; contrast length spore,
which increases from 32 to 36 pm. However, one part of this effect may be due
to the setting of the sporopollenin after its deposition, and another part to a not
unusual variation in spore length between different spores.
is also in Increase in spore length by stretching supposed to occur Drynaria sparsi-
unit size attributed sora (Van Uffelen, 1990), where the increase in was partly to
a in surface This be a uni- spore wall stretching (and partly to change pattern). may
of in ferns, that the materialof versal trend in the process exospore growth indicating which the exospore consists remains quite elastic for a long time.
An amorphous layer covering the perispore has also been foundin some spores of Pyrrosia and other Polypodiaceae. It is not clear whether it consists of remnants
which the when the of tapetum, are deposited on mature perispore sporangium opens transition the is rather early, before the from the liquid to gaseous phase completed.
If the sporangium opens only when its contents have reached the gaseous phase, these tapetal remnants are deposited either as a fine granulate layer, or as a much thinner one, and mainly on the inside of the sporangial wall.
the Assuming that the perispore is deposited centrifugally, i.e., spines are only formed after the basal layer of the perispore has been finished, then the occasionally observed amorphous layer covering the perispore can only be explained by an abnor- mal, later deposition of tapetal residues.
the the In most recent publications concerning family Polypodiaceae s.s., genera
Drynaria and Microgramma are placed in different tribes of the subfamily Polypo- dioideae (Drynarieae and Polypodieae, respectively: Hennipman et al., 1990), or even in different subfamilies(Drynarioideae and Polypodioideae: Hennipman, 1990).
The differences in outer exospore formation support a not very close relationship between these genera.
Within the genus Microgramma (Tryon & Tryon, 1982), or, alternatively, the
Microgramma-group in Polypodium, there is some, but not a striking degree of vari-
ation in mature spore surfaces (see above, and Tryon & Lugardon, 1991): all c. 14
species have a thin perispore adhering closely to the exospore surface, which is al- ways discernible in mature spores; in some species the perispore bears minute
of echinae; the mature exospore surface bears similarwarts more or less irregular ap-
for M. has with pearance, except megalophylla (Desv.) Sota which large spores a
shallowly rugate surface. 526 BLUMEA VOL. 36, No. 2, 1992
The genus Belvisia
As is the case in Microgramma ciliata, the first differences in wall deposition be- tween Drynaria and Belvisia became visible during outer exospore formation.In both
Belvisia Drynaria sparsisora and mucronata the granulate stage (smooth-with-small-
is overlaid After in lumps) by a finely rugulate pattern. this, Drynaria sparsisora the
is covered with while in Belvisia shallow wide surface units, depressions appear, a
development not found in eitherDrynaria sparsisora or Microgramma ciliata. As in
Microgramma ciliata, clear-cut substages cannot be recognized during further outer exospore development because of the gradual change in pattern.
The mechanism underlying the formationof the pits that are so characteristic for
formation in Belvisia is still the first outer exospore unclear. During very appearance of shallow depressions (Plate V-7), nothing that may have guided their formationis
either in the the surface. this is apparent, tapetum or on spore Unfortunately, the case with featuresof wall for the laesural of which most spore ornamentation, except fold, the position is fixed by the position of the young spore in the tetrad. However, the mechanism of formation of these depressions can be speculated upon. I prefer to
the easiest the surface is in unknown postulate process: spore marked an as yet way, which indicates places where further exospore material will not be deposited. In this
and located of the wall where in way depressions, pits, grooves are on parts spore a
certainphase of the deposition of exospore material no more material is deposited.
left The way in which these places are open must be governed by the spore surface at the onset of pit formation, and probably not by some attribute ofthe material depos- itedin between the depressions (see Van Uffelen, 1991). A mechanism needing both a marker system and a complicated enzyme system governing the local dissolving of
material in order exospore to 'scoop out' depressions in an already existing layer, is far more complicated.
The lack of a rugulate stage between the smooth-with-small-lumps-stage and the smooth-with-deepening-pits-stage in the series reconstructed for outer exospore for-
in Belvisia result from maton platyrhynchos may the fact that it was not present in the broken I that it does sporangia studied, or not occur at all in this species. In both possible cases it indicates that there are two independent processes at work: pit for- mation (found in both from the from smooth-with- species) and, apart that, change a
via small-lumps-stage a rugulate surface to a smooth surface as found in at least one of the species, Belvisia mucronata.
In B. with platyrhynchos, spore lengthening occurs early respect to exospore de- position processes, such as pit formation and the covering of the laesural fold. All this timed of of suggests a fairly loosely occurrence independent processes spore and growth exospore deposition, which may vary betweenrelated species and still result in similar mature spore surfaces. One has also to take into account that mature
B. platyrhynchos spores are usually larger than those of B. mucronata (c. 60 pm and c. 51 pm, respectively).
the In genusBelvisia, which comprises 8-10 species (Hovenkamp & Franken, in
all have the and all prep.), species basically same exospore surface, they have a sim- ilar thin perispore without any ornament (Tryon & Lugardon, 1991). Sporangia in several species of Belvisia tend to shed their spores before they are ripe; in all of G.A. Uffelen: in Micro and Belvisia van Sporogenesis gramma 527
these species (e.g., B. platyrhynchos) immature spores with similar pitted patterns
found. This has led the conclusion that in Belvisia similar series of were to patterns
have led to similar mature exospore surfaces. It also shows the necessity to check
whether even spontaneously shed spores are truly mature.
When observing immature spores as if they were mature, the conclusion that neo-
be of is obvious. this is teny may common among species Belvisia However, not
necessarily so. Neoteny may only have occurred affecting a very late stage of outer
exospore formation, as the ridges in some species carry many warts (e. g., B. mucro-
while in found the nata), other species not so many warts are on mature spore sur-
face (e.g., B. novoguineensis). The occurrence of neoteny depends on whether the
situation found in B. is with 'non- 'warty' as mucronata plesiomorphic respect to the in B. where in warty' or reverse. Evidently, platyrhynchos, mature spores the
ridges are densely set with irregular warts (Plate IX-8), this warty pattern is formed
and be addition the the later, may seen as an to ontogenetic series, leading to mature
spore surface as found in some other species of Belvisia, e.g., B. novoguineensis.
In the related generaDrymotaenium, Lemmaphyllum, and Lepisorus (including
which with the Belvisia form Paragramma), together genus the tribe Lepisoreae (Hen-
similar surfaces nipman et al., 1990), rugate mature exospore are found (Tryon &
Lugardon, 1991). However, in some species of Lemmaphyllum and Lepisorus the
mature exospore surface is verrucate or tuberculate, reminiscentof mature surfaces in
Microgramma - to make things more complicated, the spores of the one deviating spe-
cies in the genus Microgramma, M. megalophylla, are shallowly rugate. However,
in the series of surface establishing relationships, not only patterns during outer exo-
which rather different in ciliata the spore development, are Microgramma on one
hand and several species of Belvisia on the other, are of value, but also mature exo-
spore ultrastructure as seen on TEM is important. Hennipman (1990) described the
Belvisia type of exospore ultrastructureand characterized it by its extreme thickness,
its verrucate, fissured or cerebriform surface, and by its abundant microchannels and
bands section. He indicated that this of is tangential on cross type exospore typical for and confined to the tribe Lepisoreae. In his Table2.2, however, the Belvisia type of is also listed for perispore the Microsorinae and Polypodiinae, to which last one
the Microgramma group belongs. However, the exospore in the species of Micro- gramma studied here with the TEM indicate no similarity in ultrastructure with the
Belvisia type.
CONCLUSIONS
all of In species Polypodiaceae s. s. studied up to now, the process of sporogene- sis with the series of in Van Uffelen concurs stages described (1986). But there are
differences in the of formation the level of large way outer exospore on genera or
of is groups genera. Therefore, it impossible to divide outer exospore formation in
sub-stages that may be generally applied to all species in this family.
In the species where increase in spore length could be related to changes in other
spore features, such as exospore thickness, embedding of the laesural fold in exo-
in spore material, and changes spore surface patterning, spore length apparently in- creases by stretching ofthe existing spore wall material, and not by intercalary growth. 528 BLUMEA VOL. 36, No. 2, 1992
Comparison of outer exospore formation in Belvisia mucronata with the same
has shown that differ- process in B. platyrhynchos during outer exospore ontogeny, ent sequences of processes are at work at the same time, but more or less indepen-
dentof each other.
Despite differencesin perispore morphology, membranes and characteristic blobs of material of tapetal origin are involved in the process of perispore formationin all species of the genera studied so far. formation Similarities in the process of outer exospore correspond with a similar mature exospore ultrastructure and surface, as is the case in the tribeLepisorinae.
ACKNOWLEDGEMENTS
Dr. B. Lugardon of the Universite P. Sabatier (Toulouse, France) and Dr. S. Blackmore and Dr.
S. Barnes of the Natural History Museum (London) have helped in makingmicrographs, and also in
freely discussing sporogenesis. Many of the TEM pictures have been made in Toulouse, and several
SEM pictures have been made in London. Prof. Dr. E. Hennipman, Prof. Dr. C. Kalkman, and Dr.
G.M. Lokhorst critically read this manuscript, and their comments are much appreciated. I want to thank the staff of the botanic gardens in Leiden and Utrecht for the good care they have taken of the plants I studied. The investigations were supported by the Foundation for Fundamental Biologi- cal Research (BION), which is subsidized by the Netherlands Organization for Scientific Research
(NWO).
REFERENCES
BLACKMORE, S., & S.H. BARNES. 1985. Cosmos pollen ontogeny: a Scanning Electron Microscope
study. Protoplasma 126: 91-99.
E. 1990. The of the SEM for character of of HENNIPMAN, significance analysis spores Polypodiaceae
In: D. Electron in and functional (Filicales). Claugher (ed.), Scanning Microscopy taxonomy
morphology. Syst. Ass. Special Vol. 41: 23-44. Clarendon Press, Oxford.
HENNIPMAN, E„ K.U. KRAMER & P. VELDHOEN. 1990. Polypodiaceae. In: K.U. Kramer & P.S.
Green I. and In: K. Kubitzki Families and (eds.), Pteridophytes gymnosperms: 203-230. (ed.),
ofvascular Berlin 404 genera plants. Springer Verlag, etc. pp.
HOVENKAMP, & N.A.P. FRANKEN A of the Belvisia Mirbel. P.H., (in prep.). revision genus
Blumea.
LUGARDON, B. 1978a. Isospore and microspore walls of living pteridophytes: identificationpossi-
bilities with different observation instruments. Proc. Fourth Int. Palyn. Conf. I: 152-163 +
2 pi. Lucknow.
B. 1978b. between and walls. Proc. Fourth Int. LUGARDON, Comparison pollen pteridophyte spore
Palyn. Conf. I: 199-206 + 1 pi. Lucknow.
& TRYON, A.F., B. LUGARDON. 1991. Spores of the Pteridophyta. Surface, wall structure, and di-
based electron studies. versity on microscope Springer Verlag, New York etc. 648 pp.
TRYON, R.M., & A.F. TRYON. 1982. Ferns and allied plants, with special reference to tropical
New York 857 America. Springer Verlag, etc. pp.
UFFELEN, G. A. VAN. 1986. Sporogenesis in Drynaria sparsisora (Desv.) Moore (Polypodiaceae) (ab-
stract). Acta BoL Neerl. 35: 116-117.
UFFELEN, G. A. VAN. 1990. Sporogenesis in Polypodiaceae (Filicales). I. Drynaria sparsisora (Desv.)
Moore. Blumea 35: 177-215. G.A. vanUffelen: Sporogenesis in Microgramma and Belvisia 529
G.A. 1991. The control of wall formation. In: S. Blackmore & S.H. Barnes, UFFELEN, VAN. spore
Pollen and Spores, Patterns of diversification. SysL Ass. Special Vol. 44: 89-102. Clarendon
Press, Oxford.
UFFELEN, G. A. VAN. (in prep.). Sporogenesis in Polypodiaceae (Filicales). III. Several species. Dis-
cussion. Blumea.
G. E. HENNIPMAN. 1985. The of Mirbel UFFELEN, A. VAN, & spores Pyrrosia (Polypodiaceae), a
SEM study. Pollen et Spores 27: 155-197.
LEGENDS OF PLATES I—IX
(pages 532—540)
Plate I: Microgramma ciliata, sporogenesis (1)
SEM - covered with which are rounded and almost smooth 1: young spores contiguous warts, at
the top; x 3150.
- similar to found in 2: SEM lateral view of a spore with a surface pattern that younger spores
(Plate 1-1); x 1850.
- section of from the with thicker 3: SEM cross a spore same sporangium, a substantially exo-
than the Plate the laesural fold is covered with spore spores on 1-1; (arrow) not yet exospore ma-
terial; x 7000.
SEM - detail of which material found and between the 4: proximal a spore on granular is on warts;
x 7000.
5: SEM - detail laesural from the material has of a fold, same sporangium; some exospore (arrow)
been depositednear the base ofthe laesural fold; x 8000.
6: SEM - lateral view of in later approximate a spore a slightly stage, showing further deposition
of granular material, and a large number of small holes on and between the warts; x 1350.
7: SEM - detail of Plate 1-6; x 5000.
Plate II:Microgramma ciliata, sporogenesis (2)
SEM - with ofwhich the surface has lost its 1600. 1: a spore warts granular character, x
2: SEM - detail of from the the laesura is also covered with similar a spore same sporangium; a
pattern; x 8000.
- a of which 3: SEM cross section of spore the warts have become higher, and their tops more
irregular; the exospore is 1-2 pm thick; x 4500.
SEM - from the 8000. 4: surface view of a spore same sporangium; x
5: TEM - cross section showing a few narrow channels around the laesura, perpendicular to the
sometimes in wide the surface exospore surface, ending fairly openings on (arrow); x 10,000;
oe = outer exospore.
6: SEM - surface view of the end of the of the a spore at exospore formation; tops warts have
become quite irregular; x 3300. 530 BLUMEA VOL. 36, No. 2, 1992
Plate HI: Microgramma ciliata, sporogenesis (3)
TEM - of formation: double membrane the 1: start perispore a (arrow) appears over exospore sur-
face; x 40,000.
- 10,000. 2: TEM the double membrane is sometimes divided into overlapping fragments (arrow); x
TEM - the contains 'blobs' of electron dense = 3: tapetum many material; x 10,000; w sporan-
gium wall.
4: TEM - a spine, attached to the basal perispore layer; each spine consists of a tapering bundle of
thin rods; x 40,000; oe = outer exospore.
as is also visible on this SEM micro- 5: SEM - part of perispore formation shown with TEM
lateral view of 1500. graph, a a spore; x
6: SEM - cross section of a freeze-fractured sorus; x 19.
Mature of different of Plate IV: spores species Microgramma
1: SEM-M. ciliata: proximal view; x 1000.
2: SEM - M. ciliata: lateral view; x 1000.
- ciliata: with thickness and 3: SEM M. mature spore a peeling perispore, showing perispore struc-
1200. ture; x
4: SEM - M. ciliata: detail of the perispore surface; x 6500.
5: SEM - M. ciliata: the inside and section of the wall and laesura a germinated spore; spore are
clearly visible; x 1000.
6: SEM - M. heterophylla: lateral view; x 1050.
7: SEM - M. lycopodioides: proximal/lateral view; x 950.
8: SEM - M. persicariifolia: lateral view; x 1100.
Plate V: Belvisia mucronata, sporogenesis (1)
1: SEM - cross section of a sorus; x 17.
2: SEM - detail of the cross section ofthis sorus; x 90.
SEM - surface with low and small round 3: young bean-shaped spore; lumps of exospore material;
x 1800.
TEM - section of the laesural and the which is 4: a young spore, showing fold, outer exospore,
much thicker on the proximal side (c. 0.4 pm) than on the distal side (0.1-0.2 pm); x 4000.
SEM - the shown Plate V-3 is overlaid 5: pattern as on by a finely rugulatepattern; x 1650.
6: SEM - detail of Plate V-5; x 3900.
7: SEM - surface shallow but rugulate spore showing conspicuous depressions; x 2300.
8: SEM - detail of Plate V-8; x 5300.
Plate VI: Belvisia mucronata, sporogenesis (2)
SEM - the of the 1: spore showing a first indication of grooves plus ridges pattern mature exo-
the have in and have become and 1700. spore; pits increased number, larger deeper,x
2: SEM - detail of Plate VI-1; note the numerous small holes (arrow); x 6000.
TEM - section of which the about thick 3: cross a spore of exospore is 1 pm on the lateral side,
and almost 2 thick 4500. pm near the laesura; x
SEM - 4: lateral view ofa spore with deeperpits, which start fusing in greaternumbers; x 1400.
SEM - detail of Plate VI-4: the laesura shows similar 5000. 5: a pattern; x
6; SEM - which it has become difficult individual the spore on to distinguish pits; pattern of
ridges and grooves is evident; x 1300.
7: SEM - detail of Plate VI-6; x 5000. Uffelen: in Micro and Belvisia 531 G.A. van Sporogenesis gramma
Plate VII: Belvisia mucronata, sporogenesis (3)
SHM - lateral view of of the Plate with 1: a spore approximately same stage as VII-1, a knobby
1250. looking spore surface; x
2: TEM - cross section of the laesural area; the surface of the ridges, as well as that of the laesura
has become more distinctly verrucate; x 6000; oe = outer exospore.
SEM - surface which 1450. 3: spore on some warts are recognizable as separate warts; x
- detail ofPlate the surface is 6500. 4: SEM VII-3; spore finely striate; x
on the are SEM - lateral of the 5; view a mature exospore; numerouswarts deposited ridges so con-
of and 2800. spicuous that it has become difficult to trace the underlying pattern ridges grooves; x
6: TEM - section of with double membranes and membrane cross a spore multi-layered structures
(arrow) on its surface; x 40,000.
involved in x w = 7: TEM - blobs of tapetal material (arrow) perispore formation; 29,000; sporan-
gium wall.
- section of where the membranes be the 8: TEM cross a spore appear to deposited by tapetum (t);
x 40,000.
Plate Vm: Mature of different ofBelvisia spores species
of a x in 1: TEM - B. mucronata: cross section mature spore; 2300; note layers outer exospore
(arrow).
2: SEM - B. mucronata: proximal view; x 950.
which has the 1100. 3: SEM - B. mucronata: germinatedspore, opened along laesura; x
4; SEM - B. novoguineensis: lateral view; x 1000.
5: SEM - Lemmaphyllum abbreviatum: lateral/proximal view; x 1000.
Plate IX; Belvisia platyrhynchos - sporogenesis and mature spore
SEM - lateral view of surface smooth with small 1500. 1: a young spore; lumps; x
2: - almost SEM proximal/lateral view; spore surface entirely smooth; x 1250.
SEM - lateral view of with few 1000. 3: a spore only a pits; x
SEM - with that and 4: spores pits are larger, deeper, more numerous; x 900.
SEM - lateral view of where the into the of and 5: a spore pits start fusing pattern grooves ridges;
x 1000.
SEM - lateral immature 6: view of a slightly older, spore; x 1150.
SEM - view of 800. 7: proximal spore; exospore not yet finished; x
8: SEM - lateral/proximal view of a mature spore; x 850. 532\I BLUMEA VOL. 36, No. 2, 1992
Plate I (legend on page 529) in Micro and Belvisia 533\II G.A. van Uffelen: Sporogenesis gramma
Plate II (legend on page 529) 534\III BLUMEA VOL. 36, No. 2, 1992
Plate III (legend on page 530) G.A. Uffelen: in Micro and Belvisia 535\IV van Sporogenesis gramma
Plate IV (legend on page 530) 536\V BLUMEA VOL. 36, No. 2, 1992
Plate V (legend on page 530) G.A. Uffelen: in Micro and Belvisia van Sporogenesis gramma 537\VI
Plate VI (legend on page 530) 538\VII BLUMEA VOL. 36, No. 2, 1992
Plate VII (legend on page 531) 539\VIII G.A. van Uffelen: Sporogenesis in Microgramma and Belvisia
Plate VIII (legend on page 531) 540\IX BLUMEA VOL. 36, No. 2, 1992
Plate IX (legend on page 531)