Morphology and ultrastructure of megaspores and microspores of Isoetes sehnemii Fuchs ( Lycophyta )

The morphology and wall ultrastructure of megaspores and microspores of Isoetes sehnemii that grows in Brazil were analyzed as part of the study of the Isoetaceae present in Southern South America. The observations were performed with light, scanning and transmission electron microscopes. The megaspores are trilete, 350-450μm in equatorial diameter. The surface is reticulate. In section, the sporoderm is 100μm thick including the ornamentation. The wall is composed of a siliceous perispore, which consists of short fused flatten, elements forming a three-dimensional mesh. The exospore has two zones of different structure. The endospore is fibrillar. The microspores are monolete, 21-27μm in equatorial diameter. The sporoderm is composed of a sporopollinic rugulate perispore. A space between the paraexospore and the exospore is evident. The exospore is compact. The endospore is fibrillar. The ultrastructural analysis makes homologies evident concerning structure and organization of the layers below the perispore in both spore types. A possible similarity and stability in the ultrustructure of the present spores and fossils could be also inferred. In addition, there would be a correlation among the plant habitat, the spore ornamentation and the geographic distribution.


INTRODUCTION
The Isoetaceae constitute a family with a wide distribution.They live from mild to warm regions of all the continents, from sea level to near 4200 m.In southern South America, they are present in Argentina, Brazil, Bolivia, Chile, Paraguay and Uruguay.As part of the palynological study of the Isoetes that grow in this region, the morphology and ultrastructure of the spores of Isoetes sehnemii that grows in the state of Rio Grande do Sul, Brazil, are analyzed.Fuchs-Eckert (1982) carried out an updated study of the South American species of Isoetes and found a list of 64 taxa, among which the I. sehnemi (nomen nudum) is included.Then, Fuchs-Eckert described and typified the Isoetes sehnemii Fuchs in the Flora Ilustrada Catarinense (1986).
Systematic (Pastore 1936, Capurro 1968) and floristic (de la Sota et al. 1998) aspects of Isoetes in southern South America have received some attention, but little information can be found in those contributions regarding palynological characteristics.In 2003, Musselman mainly studied the microspores of North America species with scanning electron microscopy (SEM).The microspores of some species of southern South America have been analyzed with SEM and transmission electron microscopy (TEM) by Macluf et al. (2004).In 2006a, b, Macluf et al. analyzed with SEM the microspores of all the species of Isoetes that grow in southern South America.In this analysis, a description of the adapted Isoetes sehnemii spores of Fuchs-Eckert (1986) is included.The joint study of microspores and megaspores in taxa of Isoetes with electronic microscopy has only been carried out for Isoetes savatieri Franchet by Morbelli (1980), Hickey (1985Hickey ( , 1986)), Hickey et al. (2003) and Macluf et al. (2003b).The spores of other species of Isoetes were examined with TEM by Lugardon (1973Lugardon ( , 1986)), Robert et al. (1973), Prada Moral and Saenz de Rivas (1978), Tryon and Lugardon (1991), Taylor (1992Taylor ( , 1993) ) and Uehara et al. (1991).
In megaspores, a siliceous cover, a sporopolleninous exospore and a well-developed endospore can be distinguished in the sections of mature megaspores, from the outside to the inside, respectively.In microspores, the sporoderm comprises the perispore forming the ornamentation, the para-exospore, a compact exospore and a fibrillar endospore.The term para-exospore (Lugardon 1972) is used to refer to the group of several large superimposed elements located between the exospore and the perispore (Macluf et al. 2006b).
The aim of this work was to study the morphological characteristics of megaspores and microspores of Isoetes sehnemii using light (LM), scanning electron (SEM) and transmission electron (TEM) microscopy, in order to determine whether those sculptural and structural characteristics are reliable enough to be used for systematic purposes, for being regarded as microspore and megaspore characters, whether they are coherent or not, and also to determine if the organization of the sporoderm, the structure and the surface of both types of spores respond to the general characteristics of Isoetes.

MATERIALS AND METHODS
Spores were obtained from herbaria specimens housed at the Herbario Anchieta, Instituto Anchietano de Pesquisas, São Leopoldo, Rio Grande do Sul, Brazil (PACA).
For LM, the material was mounted in glycerinejelly without any chemical treatment.Dimensions were estimated for 25 spores per specimen.The minimum and maximum values in micrometers are given in the text.
For studies with SEM, the spores were handled with moist brushes without any chemical treatment and placed on double-stick tape on bronze stubs.The samples were coated with gold and examined under a Jeol JSM-35 CF microscope.
For studies with TEM, the dry material was hy-drated by following the technique of Rowley and Nilsson (1972)

MEGASPORES
The megaspores are triletes (Plate I, 1-8), 350-450μm in equatorial diameter and subtriangular in polar view.The proximal face is convex and the distal one is hemispheric in equatorial view (Plate I, 2).Each laesure is fused to the equatorial zone, which is low, slightly differentiated from the ornamentation.Adjacent to the area, in the distal face, the girdle (cf.Tryon andLugardon 1991, Taylor et al. 1993) shows the same ornamentation as the rest of the spore (Plate I, 2).The surface is reticulate heterobrochate in both polar faces.The proximal muri are lower compared to those of the distal face (Plate I, 5).In the lumens, the background consists of anastomosed bars that form a three-dimensional mesh with irregular spaces (Plate I, 7).The terminal ends of some elements are joined and form echinulae.In the muri, these echinulae are arranged on both sides of the major axis of each wall, with its ends oriented towards the lumens (Plate I, 8).

Megaspore wall structure and ultrastructure
The megaspore wall in section (Plate II, Plate III, Plate IV) from the outside to the inside is composed of a An Acad Bras Cienc (2010) 82 (2) siliceous cover (Si), a sporopolleninous exospore and an endospore; (Plate III, 12).The sporoderm is 100μm thick, including the muri and 40μm among muri (Plate II,9,10,11).The perispore and the outer part of the exospore are involved in the apertural differentiation.A siliceous cover is 16 to 40μm thick, formed by short fused flattened elements forming a three-dimensional mesh and leaving their free ends at the surface.This layer is thicker in the equator and distally, and most of its thickness forms the ornamentation (Plate II, 11).The distal ornamentation is formed by higher structural elements.The silica cover (Si) is strongly contrasted when fixed and stained for TEM.It is formed by structural elements that are rectangular to lenticulate, flattened and fused by their ends.(Plate III,12,13,14).The chains formed by the link of these elements take articulated forms (Plate III, 13) and also form an open network that communicates with the outside.A connection with the exospore is also observed in some points (Plate III, 14).The exospore is 6 to 10μm thick, and less contrasted than the siliceous cover.The structural elements show a more contrasted margin.They are tangentially elongated, arranged in several levels and fused with others of different levels, forming elongated spaces and lacunae.Due to the presence of a great amount of spaces, the thickness is variable.Silica is present in a variable amount in the outermost exospore spaces.The presence of silica decreases towards the inside and is absent in the internal area near the endospore (Plate IV, 15, 16).The nature and contrast to TEM of silica in the exospore spaces is seen identical to that of the perispore.The exospore area with silica impregnation is 1.5 to 4μm thick (Plate IV, 16).Among the exospore elements, a space or "gap" is observed, which varies in amplitude distally and equatorially in different spores (Plate IV, 15).The endospore is 1 to 2μm thick, has a fibrillar type structure (Plate IV, 15) and can be deposited in two stages probably marked by a discontinuity.

MICROSPORES
The microspores are monolete (Plate V, 17-22), 21-27μm long and 18-20μm wide, elliptic in polar view, and biconvex in equatorial view.A swelling (cf.Musselman 2003) perpendicular to the laesura was observed (Plate V, 17).A supra-laesural expansion is present.An equatorial 4.5μm high projection is evident (Plate V, 20).The perispore is rugulate on the whole surface.The background is composed of perforations and granules.

Microspore wall structure and ultrastructure
The microspore wall in section from the outside to the inside is composed of perispore, para-exospore, exospore and endospore (Plate V, 23); (Plate VII, 28).The sporopollinic perispore, 0.4 to 3.5μm thick, shows an uniform thickness in all the contour of the spore.Two layers can be seen in the cross sections, both with lacunar structure but with different characteristics of their elements (Plate VII, 26).The outer stratum (Po) has thin, fused, laxly distributed bars (Plate VII, 26).The outside of this stratum forms the ornamentation.The inner perispore stratum (Pi) consists of thicker bars, circular in section, fused in different directions.This stratum is in contact with the elements of the para-exospore (Plate VII,26,27).
The para-exospore is 0.3 to 0.6μm thick and formed by large structural elements tangentially arranged, which are fused to others at different levels (Plate VI, 23); (Plate VII,26,27).The center of each element is seen with less contrast than the margins (Plate VII, 26, 27).The elements of the internal para-exospore are generally thinner.An equatorial-distal separation or gap between the para-exospore and the exospore is present (Plate VI, 23, 24).
In a transverse section of a microspore along its major polar axis, a supra-laesural expansion is observed, which forms a kind of vestibule of variable height that is higher at the central part and diminishes towards both ends of the laesurae (Plate V, 23, 24).All the thickness of the perispore and the more external structural elements of the para-exospore participate in the expansion (Plate VI, 25).The structure and arrangement of the external para-exospore in the supra-laesural expansion takes the form of a zip (Plate VI, 25).

DISCUSSION AND CONCLUSIONS
The organization of the sporoderm, the number and ultrustructure of the layers respond to the general characteristics of both types of Isoetes spores.Homologies have been found as regards the organization and structure of the internal layer to the perispore, i.e. exospore of megaspores and para-exospore of microspores.In both, the structural elements are long, tangentially arranged and fused in different levels.They also determine spaces that may or may not be filled with silica.Macluf et al. (2003bMacluf et al. ( , 2006a) ) have found in different studies characters such as: type of ornamentation, morphology and distribution of the structural elements, and the characteristics of the equatorial area of the mi- crospores, which may be used with systematic purposes.Likewise, the ornamentation of megaspores is a very useful diagnostic for the infrageneric classification.In virtue of this, megaspores and microspores of Isoetes sehnemii show superficial characters that, in an isolated or combined ways could be applied to the systematics.
Even if ornamentation is determined by the perispore in both types of spores, in megaspores almost all the thickness forms the structural elements, whereas in the microspores only the outside stratum participates.
In megaspores, the sporoderm shows spaces in all of its thickness.From the exospore towards the surface of the perispore, a system of interconnected spaces is observed.This is a characteristic that is repeated in the microspores from the para-exospore towards the perispore surface.Due to the way in which the structural CECILIA MACLUF, MARTA MORBELLI and GABRIELA GIUDICE Plate III -Megaspores of I. sehnemii as seem with TEM -Figure 12 -Section through the sporoderm.The silica cover (Si) consists of elements fused forming bars that form a lacunose structure.The exospore (Ex) shows silica in the lacunae of the most external layers.There is a space (G) between the elements of the outer and the inner exospore.Below the exospore, the endospore (En) is in contact with the cytoplasm (Cy).Scale bar: 1μm. Figure 13 -Detail of the silica cover structure.It consists of structural elements that are rectangular to lenticulate and fuse with one another.Scale bar: 1μm. Figure 14 -Section that shows the sporoderm.The silica cover (Si) consists of short rectangular to lenticulate elements and forms a closed or dense structure.A connection with the exospore (Ex) is observed.Silica impregnates the exospore lacunae.The exospore elements have a less contrasted center and a dark edge.Scale bar: 1μm.elements of the siliceous layer of the megaspores fuse forming chains, they are likely to show a certain mobility or flexibility when living.The siliceous wall of the megaspores of I. pedersenii (Macluf et al. 2003a) shows a similar structure.
Based on the dominant sculptural element on the microspore surface, the microspores would be located within the rugulate pattern, among the scultural patterns of microspores proposed by Macluf et al. (2006a) for the microspores of the genus of the species of the area.A swelling associated with the laesure described by Musselman (2003) and later by Macluf et al. (2006a) is also found in the microspores of the studied species.It has been observed that the perispore, which has a lacunar structure, is similar to that one observed in the microspores of I. pedersenii (Macluf et al. 2006b), where it is seen as camerate.In I. sehnemii it consists of bars, whereas in I. pedersenii it is formed by short rodlets, An Acad Bras Cienc (2010) 82 (2) It has a fibrillar structure.Scale bar: 1μm. Figure 16 -The silica cover (Si) is in contact with the exospore, which is composed of anastomosed elements.Silica impregnates the most external layers of the exospore (Eo).Scale bar: 1μm.but of the same structural type.The perispore thickness increases towards the equatorial-distal area in both species.Personal observations carried out in microsporangium sections with different degree of development in other species of the area, such as I. ekmanii and I. savatieri (C.Macluf, unpublished data), would indicate that the amplitude of the "gap" or space between the paraexospore and the exospore in the microspores ranges depending on the stage of development of the spore, and it is observed that it decreases in mature spores.These characteristics were described by Uehara (1991) in his study about the development of the wall in microspores of I. japonica.
The "pluristrata areas" of the apertural area in microspores, described by Lugardon (1972Lugardon ( , 1973) ) and Tryon and Lugardon (1991) for Isoetes brochoni, Isoetes echinospora, I. setaceum, I.durieui and for Selaginella selaginoides, were not observed in the studied material.This could be attributed to the place where the transverse section has been made.
Isoetes sehnemii grows in the south of Brazil, in low and floodable areas of subtropical climate.I. pedersenii, grows in the northeast of Argentina, in similar habitat and climate.The coincidence in the ecological preferences could be linked to the similarities found at the ultrastructural level of microspores.Both species produce microspores with open structure sporoderm with wide spaces.On the other hand, it was compared to I. escondidensis, which grows in the NW of Argentina, in a mild climate, and is submerged aquatic, with differences in the wall ultrastructure being observed since it shows a closed structure without the spaces among the elements.Based on these data, a probable correlation between the sporal ultrastructure and the plant habitat could be inferred.Likewise, Macluf et al. (2006a, b) related the increase of the ploidy level with the increase in the size of the microspores and with the habitat in microspores of Isoetes of the South, and no particular reference was made to I. sehnemii.
If organization, ornamentation and structure of the sporoderm of the studied spores were compared with those of other studies, particularly of fossil material of the Cretaceous (Gamerro 1977, Archangelsky 1965), a general stability or similarity could be inferred in the morphology and surface of spores.This would show a non-change or "stasis" condition according to Tryon's concept (1986) when he refers to the conservation of structures in the sporoderm of Pteridophyta.Likewise, Kovach (1994) suggests that there is an ultrastructural affinity between mesozoic megaspores and present Lycophyta megaspores, which would also indicate a probable conservation of the sporoderm structure.Lugardon et al. (1999Lugardon et al. ( , 2000) ) and Grauvogel-stamm and Lugardon (2004) compared megaspores and microspores of living species of Isoetes with spores of the Triassic period, assigned to Isoetes, and came to similar conclusions in the sense that there is a similarity in the ultrastructure and organization of the fossil and living spores.
An Acad Bras Cienc (2010) 82 (2) Plate VII -Microspores of I. sehnemii as seem with TEM -Figure 26 -Section through the apertural area following the polar axis.The perispore (P) and the para-exospore (Pex) participate in the formation of the supra-laesural expansion.The perispore is lacunar with two strata.The inner stratum (Pi) is attached to the para-exospore (Pex).The structural elements of the para-spore show a center that is less contrasted than the margins.
The exospore (Ex) is homogeneous and has a dark-contrasted thick cover with low, blunt elements.The endospore (En), in contact with the cytoplasm (Cy), has a strongly contrasted central zone and shows a fibrillar structure.Scale bar: 1μm.
Plate I -Megaspores of I. sehnemii as seen with SEM -Figure 1 -Proximal view.The laesurae are low and are fused to the equatorial area, which is slightly developed.The sculpture of the silica cover is reticulate in both faces.Scale bar: 100μm.Figure 2 -Equatorial view.The distal face is at the top.The zone (arrow) marks the equator.The surface is reticulate in both polar faces.The girdle (arrowhead) has the same sculpture as the rest of the distal face.Scale bar: 100μm.Figure 3 -Detail of the silica surface proximally among the laesurae.The background is composed of a three-dimensional network of fused rodlets forming irregular spaces.Scale bar: 10μm.Figure 4 -Detail of the proximal surface showing the background with heterogeneous spaces.Some of the bars join and form echinulae.Scale bar: 2μm. Figure 5 -Distal view.The sculpture of the silica cover (Si) is reticulate.The muri are higher in this face than in the proximal face.Scale bar: 100μm.Figure 6 -Detail of the distal sculpture showing an irregular reticule.The muri are high.Scale bar: 20μm.Figure 7 -Detail of the distal surface.Background of a lumen consisting of a three-dimensional mesh of bars.Some of them are fused and form echinulae.Scale bar: 2μm. Figure 8 -Detail of the distal surface showing echinulae on both sides of the muri, with their ends towards the lumens.Scale bar: 5μm.
Plate II -Megaspores of I. sehnemii as seen with LM -Figure9-Section of the megaspore.The distal ornamentation (lower part of the photograph) consists of the highest structural elements of the silica cover (Si), which are ramified and with different shapes.Below, the sporopollinic exospore (Ex) and the fibrillar endospore (En).Scale bar: 40μm.Figure 10 -Detail of the sporoderm in section.The silica cover (Si) consists of silica flattened and fused elements.Low contrasted with toloudine blue.The exospore (Ex) consists of tangentially elongated and anastomosed elements, with an external area impregnated with silica.The endospore (En) is shown as fibrillar.Scale bar: 40μm.Figure 11 -Detail of the sporoderm in section.Most of the thickness of the silica cover (Si) forms the ornamentation.Below, the exospore (Ex), endospore (En) and cytoplasm (Cy).Scale bar: 20μm.
MORPHOLOGY AND ULTRASTRUCTURE OF Isoetes sehenemii SPORES347Plate IV -Megaspores of I. sehnemii as seem with TEM -Figure15-Section that shows a space (G) that divides the exospore into two areas.The exospore (Ex) elements show a more contrasted margin and are arranged in different directions.The endospore (En) is visible under the exospore.
CECILIA MACLUF, MARTA MORBELLI and GABRIELA GIUDICE Plate V -Microspores of I. sehnemii as seen with SEM -Figure 17 -Proximal view showing rugulate sculpture.The supra-laesural expansion is evident.Swelling perpendicular to the laesura (arrowhead).Scale bar: 5μm. Figure 18 -Detail of the proximal face.The background is composed of perforations and granules (arrowhead).Scale bar: 1μm. Figure 19 -Distal view.The ornamentation consists of rugulae.Scale bar: 5μm. Figure 20 -Lower equatorial view showing the proximal expansion of the laesura (arrow) and the equatorial projection (arrowhead).Scale bar: 5μm. Figure 21 -Major equatorial view showing a supra-laesural expansion.Scale bar: 5μm. Figure 22 -Fracture that exposes the two strata of the perispore.The internal lacunar stratum is evident (arrow).Scale bar: 1μm.