P. Gardenal, M.A. Morbelli, and G.E. Giudice: Morphology and ultrastructure in Salvinia from southern South America 143 SPORE MORPHOLOGY AND ULTRASTRUCTURE IN SPECIES OF SALVINIA FROM SOUTHERN SOUTH AMERICA PAULA GARDENAL MARTA A. MORBELLI Cátedra de Palinología Facultad de Ciencias Naturales y Museo Universidad Nacional de La Plata Paseo del Bosque s/n. 1900 La Plata Argentina e-mail: paulagardenal@yahoo.com.ar GABRIELA E. GIUDICE Cátedra de Morfología Vegetal Facultad de Ciencias Naturales y Museo Universidad Nacional de La Plata Paseo del Bosque s/n. 1900 La Plata Argentina Abstract The morphology and ultrastructure of megaspores, microspores, and massulae of Salvinia Séguier 1785 species from Argentina, Bolivia, southern Brazil, Chile, Paraguay, and Uruguay have been analyzed. The analyses were performed using light microscopy, stereo microscopy, scanning electron microscopy, and transmission electron microscopy. The taxa studied were Salvinia auriculata Aublet 1775, Salvinia biloba Raddi 1825, and Salvinia minima Baker 1886. The spores of Salvinia biloba are described and illustrated here for the first time. The spores of Salvinia adnata Desvaux 1827were not described here because all the specimens analyzed had megasporangia and microsporangia which had not developed, or were aborted. The megaspores in all the species analyzed are trilete, 224–402 µm in polar diameter and 179–378 µm in equatorial diameter, with a circular outline, an irregular margin in polar view, and are ovoid in equatorial view. The surface is ridged and perforated and the sporoderm in cross section comprises a compact, two-layered exospore and a thick lacunose epispore which is projected proximally. The apertural area has unique characteristics in each species. The microspores are enclosed in spheroidal to elliptical massulae 145–240 µm in diameter. The individual microspores are trilete, rugulate, 15–36 µm in diameter, and spheroidal. In cross section, the exospore is two-layered. Both types of spores produced by the species analyzed exhibit little interspecific and intraspecific variability. Differences in general shape and proximal characteristics were found in megaspores at the species level. According to these, and previous, results Salvinia is a genus with stable palynological characteristics, all of them related to its adaptation to the aquatic environment. Key words: Salvinia; South America; megaspores; microspores; massulae; morphology; ultrastructure. INTRODUCTION This work is part of a project on the spore morphology of heterosporous Filicophyta from southern South America. The families that inhabit this area are the Azollaceae, the Marsileaceae, and the Salviniaceae. This contribution is on the spores of the Salviniaceae. The genus Salvinia Séguier 1785 is characterized by its adaptation to an aquatic environment and by the presence of heterospory. The species grow in nutrient-rich waters in temperate, subtropical, and Palynology, 32 (2008): 143–156 © 2008 by AASP Foundation ISSN 0191-6122 tropical regions and represent the food source of rich pleustonic communities. Due to their rapid vegetative reproduction some taxa are important aquatic weeds, producing obstructions, diminishing the water quality, and replacing native species where they were introduced anthropogenically (Mitchell and Thomas, 1972). These species have a similar vegetative morphology, and a high phenotypic plasticity, rendering their identification difficult. For these reasons the presence of Salvinia in an area makes it necessary to know which species are present, in 144 order to determine the level of environmental risk. Sexual reproduction is less frequent than vegetative reproduction in Salvinia. At maturation the plant decays and megasporangia and massulae with microspores become detached from the floating plants, are deposited at the water sediment interface, and germinate. The microspores are retained within the massulae; then surface apertures open, through which antherozoids are released (Bonnet 1957). There are ten species of Salvinia, eight of which grow in South America (de la Sota, 2001). The area of study constitutes the southern limit of distribution of the genus. This region comprises Argentina, Bolivia (southern part of Santa Cruz), southern Brazil (Parana, Rio Grande do Sul, and Santa Catarina states), Chile, Paraguay, and Uruguay, (de la Sota, 1973). The genus is present in the most humid part of this region, to the east, in the Rio de la Plata Basin, and in the Mata Atlántica of Brazil. As a consequence of being cultivated as ornamental plants, they can be found in other areas. Four taxa were reported to grow in the area by de la Sota (2001). They are, Salvinia adnata Desvaux 1827, Salvinia auriculata Aublet 1775, Salvinia biloba Raddi 1825, and Salvinia minima Baker 1885. Attempts were made to obtain palynological data on the spores of Salvinia adnata, but only empty, undeveloped, or sterile sporangia were found. The morphology of fossil and extant Salvinia spores has been studied more in the Old World than in the Americas, and knowledge of neotropical species is sparse. Most work on this genus in South America concerns floristics, morphology, or taxonomy. The contributions of de la Sota (1962a;b;c; 1963; 1976; 1995; 2001) mainly on morphology and taxonomy have been especially important. De la Sota (1962a) studied the morphology and structure of megaspores and microspores of Salvinia oblongifolia Martius 1834 from northern South America with the light microscope (LM). De la Sota and Cassá de Pazos (1992) described the morphology and structure of the spores of Salvinia martynii Kopp 1936 from northern Brazil. Sporogenesis and spores of Salvinia auriculata were observed with the LM by Bonnet (1955). Tryon and Tryon (1982) examined the vegetative and reproductive systems of Salvinia and the megaspore and microspore morphology and ultrastructure with the scanning electron microscope (SEM). Tryon and Lugardon (1991) analyzed the microspores of Salvinia auriculata and megaspores and microspores of Salvinia natans (L.) Allioni 1785 of the Old World with SEM and the transmission electron microscope (TEM). Gardenal et al. (2007) described megaspores, microspores, and massulae of Salvinia minima from Argentina. The spore morphology of Salvinia fossils has also been studied by Jain and Hall (1969), Martin (1976), Nasu and Seto (1976), Friis (1977), and Batten and Collinson (2001). The aim of the present study is to analyze the morphology PALYNOLOGY, VOLUME 32 — 2008 Text-Figure 1. The distribution of species of Salvinia that grow in southern South America (in gray). The distribution of the different species is marked with the following symbols: ▲ - Salvinia adnata Desvaux 1827; ■ - Salvinia auriculata Aublet 1775; ● - Salvinia biloba Raddi 1825; and + - Salvinia minima Baker 1886. and ultrastructure of megaspores, microspores, and massulae of the species that grow in Southern South America with the LM, SEM, and transmission electron microscope (TEM). The results are compared with those of other studies. The variability of the megaspores and microspores is also considered. MATERIAL AND METHODS This study is based on specimens from herbaria in the Instituto Darwinion (SI) and Museo de Ciencias Naturales de La Plata (LP), Argentina. The materials studied are listed below. Salvinia adnata Desvaux 1827 Brazil: Paraná, Paranaguá, Forno 29 (LP); Praia de Leste, River Guaraguazú, Forno 32 (LP); Rio Grande do Sul, Guaiba, Forno 23 (LP); Ídem, Forno 23b (LP); Santa Catarina, Florianópolis, Forno 18 (LP); Ponte sobre River Aracatuba, Forno 19ª (LP), Forno 28 (LP). P. Gardenal, M.A. Morbelli, and G.E. Giudice: Morphology and ultrastructure in Salvinia from southern South America Salvinia auriculata Aublet 1775 Argentina: Misiones, Posadas, Pérez and Guillén 172 (LP); Santa Fe, Burkart 9072 (SI). Bolivia: La Paz, Iturralde, west of River Beni, Beck and Haase 10177 (LP). Paraguay: Trinidad, Osten and Rojas 8441 (LP). Uruguay: Felippone s/n (SI), Montevideo, Arechavaleta s/n (LP). Salvinia biloba Raddi 1825 Argentina: Corrientes, Dto. San Cosme, Pellegrini, Solís Neffa and Berón 18 (LP); Santa Fe, Forno s/n (LP). Brazil: Rio Grande do Sul, Porto Alegre, Forno 25c (LP); Cabo Frio, Forno 33c (LP). Paraguay: Central, Hassler 124418 (LP). Uruguay: Maldonado, Gamerro s/n (LP). Salvinia minima Baker 1886 Argentina: Buenos Aires, Burkart 4554 (SI); Corrientes, Esquina, Krapovickas 27829 (LP); Formosa, Pilcomayo, Morel 1125 (LP); Ídem, Guaglianone, Sancho and Zuloaga 415 (SI); Santa Fe, Santa Fe, Tur 2053 (LP). Brazil: Rio Grande do Sul, Pelotas, Forno C 48 (LP). 145 acetate for 15 minutes, followed by treatment with lead citrate for three minutes. The LM slides, SEM stubs, and TEM grids have been given Herbaria sheet numbers and curated in the Cátedra de Palinología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Argentina. There is some disagreement regarding the terms used to describe some structures in Salvinia. Schneider and Pryer (2000) refer to the apical structures in megaspores of all heterosporous Filicophyta, including Salvinia, as gula. Nowak and Lupia (2004) used the term acrolamella. In the glossary by Punt et al. (1994), the term gula is applied mainly to fossil megaspores. According to Punt et al. (1994), acrolamella is a specific type of gula, formed of leaf-like segments. Due to the variety of terms used for the proximal features in Salvinia megaspores, these are described here as completely as possible, without adopting any of the aforementioned terms. In order to describe the male structures, the term massula was chosen; this was used by Bonnet (1955), Nasu and Seto (1976), and Schneller (1981). In general, the terms proposed by Tryon and Lugardon (1991) were used in the descriptions. RESULTS The general morphology of megaspores and massulae was studied with light, stereo, and scanning electron microscopes; the transmission electron microscope was used for Salvinia biloba. For light microscope study, the material was hydrated or acetolized using the method of Avetisian (1950), mounted in glycerine jelly, and examined with Olympus BH-B and BH-2 microscopes. The measurements given are based on 20 spores and massulae of each specimen examined. For study with the scanning electron microscope (JEOL JSM-6360LV), chemicallyuntreated spores were placed on double-sided sticky tape on bronze stubs and coated with gold. The megaspores were handled with fine dissection needles and separated from the sporangium wall, or gently rolled on doublesided sticky tape. Megaspores and massulae were sectioned into halves with a razor blade under the stereo microscope. For transmission electron microscopy, using a Zeiss EM 109-Turbo unit, dry material from herbarium specimens was rehydrated using the method of Rowley and Nilsson (1972). Then it was fixed with a mixture of 2% glutaraldehyde and 1% Alcian Blue in a phosphate buffer for 12 hours, and post-fixed with 1% osmium tetroxide in water and 1% Alcian Blue in a phosphate buffer. The spores were dehydrated in an acetone series and embedded in Spurr’s ‘soft mixture’ (Spurr, 1969). Semi-thin sections were stained with toluidine blue, and observed with the LM. Thin sections were stained with 1% uranyl No description of Salvinia adnata is given because all the specimens examined had not developed sporangia, or they had aborted. In the rest of the taxa analyzed, the production of megaspores and microspores was generally normal. Nevertheless, few small dark megasporangia were consistently present together with normal ones. Salvinia auriculata Aublet 1775 Plate 1, figs. 1–9 Megaspores (Plate 1, figs. 1–6). Spores trilete, 357.1– 418.9 µm in polar diameter and 268.3–310.8 µm in equatorial diameter, with a broadly circular but irregular outline in polar view (Plate 1, fig.1) and ovoid in equatorial view (Plate 1, figs. 2–4). The megasporangium wall is frequently strongly attached to the megaspore (Plate 1, figs. 2, 4). The megaspore surface is ridged and perforated. The perforations vary in size (Plate 1, figs. 1, 5). The sporoderm in section consists of an exospore and an epispore (Plate 1, fig. 5). Under the LM, the exospore is brown. The exospore has a verrucate surface; rodlets are also present between verrucae (Plate 1, fig. 6). In section, using the SEM, the exospore is 3.8–6.0 µm thick and homogeneous. Under LM, the epispore is white. It constitutes the main part of the wall and in some spores its thickness varies. It has a lacunose structure. Two zones can be distinguished according the size of the lacunae (Plate 1, fig. 5). The inner zone is 6.2– 146 PALYNOLOGY, VOLUME 32 — 2008 14.5 µm thick, with lacunae 200–600 nm in diameter. The outer zone is 27.8–70.0 µm thick with larger lacunae from 1.0–1.9 µm in diameter. Proximally the epispore forms a complex projection consisting of two parts, with intervening spaces. In surface view three subtriangular, foveolate, flap-like extensions of the epispore are evident; the areas between these flap-like extensions are thick (Plate 1, fig. 1). In section, the innermost part of the projection is a triradiate ridge 92.3–133.3 µm high (Plate 1, figs. 2, 4). The outer part is composed of the three triangular lacunose flap-like extensions of the epispore (Plate 1, figs. 2–4). Massulae (Plate 1, figs. 7, 8). Spheroidal to ellipsoidal bodies, the major axis is 154.7–240.0 µm long, covered by the microsporangium wall, with a reticulate surface. In section its structure is lacunose with one or two central spaces 110–140 µm in diameter (Plate 1, fig. 7). The microspores are located within some of the peripheral lacunae (Plate 1, figs. 7, 8). Microspores (Plate 1, fig. 9). Spores trilete, 18–36 µm in diameter, spheroidal, with a rugulate surface. Each laesura is 4–12 µm long. The sporoderm in section is composed of exospore 0.5–0.8 µm thick, increasing to 1.5 µm at the junction of the three laesurae (Plate 1, fig. 9). Salvia biloba Raddi 1825 Plate 2, figs. 1–11; Plate 3, figs. 1–6; Plate 4, figs. 1, 2. Megaspores (Plate 2, figs. 1–9; Plate 3, figs. 1–6). Spores trilete, 281.8–435.7 µm in polar diameter and 300.0– 339.3 µm in equatorial diameter, with a broadly circular but irregular outline in polar view (Plate 2, figs. 1, 2) and ovoid in equatorial view (Plate 2, figs. 4, 5; Plate 3, fig. 1). The megasporangium wall is frequently strongly attached to the megaspore (Plate 2, figs. 4, 5). The surface is ridged and perforate (Plate 2, fig. 3). The perforations vary in size. The sporoderm in section consists of exospore and epispore (Plate 2, figs. 6–9; Plate 3, fig. 4). Under the LM the exospore is brown. Under the SEM the exospore is verrucate and rodlets are also present between verrucae (Plate 2, figs. 6, 7). In section is 4.0–5.9 µm thick. Under the TEM, the exospore is two-layered (Plate 3, figs. 3, 4). The inner layer is 60–130 nm thick and electron-dense. The outer layer is thicker, and constitutes the main part of the exospore. In the inner zone, a three-dimensional network of connected channels filled with a dark material is evident. In the outer zone, the channels have mainly a radial orientation, their ends are open toward the exospore surface, and are connected to the alveolar structure of the epispore. Under the LM the epispore is white. It is 21.7–78.0 µm thick and has PLATE 1 Spores of Salvinia auriculata Aublet 1775 photographed using the SEM from Perez and Guillén 172 (LP). Figures 1–6 are megaspores, figures 7 and 8 are of a massula, and figure 9 is a microspore. 1 2 3 4 5 A megaspore in proximal view; the surface is ridged and perforated. An intermediate area between distal and proximal expansions is rather smooth. The three subtriangular flap-like structures can be seen (arrow). There are three thickenings at the three corners (arrowheads). Scale bar: 100 µm. The megasporangium wall (MeW) is adherent to the megaspore surface. In the proximal area, two thickenings (arrows), the inner triradiate ridge (asterisk), and two of the outer flap-like extensions of the epispore (arrowheads) are evident. Scale bar: 100 µm. A megaspore. In the proximal part, one of the flap-like structures (arrow) and two thickenings (arrowheads) are present. Scale bar: 100 µm. A megaspore within the reticulate megasporangium wall (MeW). Proximally there are two thickenings (arrowheads), the inner triradiate ridge (asterisk), and two of the flap-like extensions of the epispore (arrows). The globules in the inner part of the megaspore probably correspond to reserve products. Scale bar: 100 µm. Detail of the megaspore in figure 3; the sporoderm in section. The exospore (E) is verrucate, with a compact 6 7 8 9 structure, and is slightly detached. The epispore (Ep) has small lacunae in its basal zone (asterisk). At the top, the perforate-ridged surface of the megaspore is clear. Scale bar: 20 µm. Detail of section in figure 5 in the region of the exospore (E)/epispore (Ep) contact. The exospore surface has verrucae (arrows) and rodlets. Scale bar: 5 µm. A massula cut in half. On the top right corners, the reticulate surface of the microsporangium wall can be observed. A large central space lined by a continuous thin layer surrounded by a lacunose structure is evident. A spore is located in one of the lacunae (arrowhead). Scale bar: 50 µm. Detail of figure 7. The microspore surface is rugulate– verrucate. Scale bar: 5 µm. Proximal detail of a microspore in section; equatorial view. The surface is rugulate–verrucate. At the top, a laesura is evident (arrow). Exospore (E). The globules in the inner part of the microspore probably correspond to reserve products. Scale bar: 1 µm. P. Gardenal, M.A. Morbelli, and G.E. Giudice: Morphology and ultrastructure in Salvinia from southern South America Plate 1471 148 PALYNOLOGY, VOLUME 32 — 2008 a lacunose structure. The inner epispore surface has projections (Plate 2, fig. 8). In section, two zones can be distinguished according to the size of the lacunae (Plate 2, fig. 9). The inner zone is uniformly thick, 4.6–13.7 µm, with lacunae 100–400 nm in diameter. The outer zone is variable in thickness, 17–71 µm, with lacunae 2.1–3.2 µm in diameter (Plate 3, fig. 4). Proximally, there is a complex projection consisting of two parts. In surface view three subtriangular flap-like extensions of the epispore with lobed margins are present (Plate 2, fig. 1). In section, the innermost part of the projection forms a triradiate ridge 60– 90 µm high (Plate 2, figs. 2, 4). The outer part is composed of the three flap-like extensions of the epispore (Plate 2, figs. 1, 4, 5; Plate 3, fig. 1). With the TEM, both parts of the projection have a lacunose structure; the lacunae are smaller in the inner part (Plate 3, figs. 5, 6). In some specimens studied, the lateral flap-like structures are separated and the inner ridge is exposed. This phenomenon is followed by rupture of the megasporangium wall (Plate 2, figs. 1, 2). variable diameter. In some of these lacunae microspores are located (Plate 2, figs. 10, 11). Massulae (Plate 2, fig. 10). Ellipsoidal bodies, the major axis being 144.6–212.2 µm long, covered by the microsporangium wall with a reticulate surface. In section its structure is lacunose, there are one or two central spaces of 70.2– 96.9 µm in diameter and peripheral lacunae of smaller Salvinia minima Baker 1886 Plate 5, figs. 1–9 Microspores (Plate 2, fig. 11; Plate 4, figs. 1, 2). Spores trilete, 15–26 µm in diameter, spheroidal, with a rugulate surface. At the laesura level, scales or crests are observed (Plate 2, fig. 11). Each laesura is 7–11 µm long. Under the TEM, the exospore is 0.7–0.9 µm thick, increasing to 1.6 µm at the laesurae junctions (Plate 4, figs. 1, 2). In section this is two-layered; the inner layer is 45 nm thick and continuous and electron-dense (Plate 4, fig. 2). At the inner part of the laesura, there are nodules of material with a similar contrast to the inner exospore. The outer layer has a mesh of channels in its innermost part. The channels are fused forming arcs, and simple channels are present (Plate 4, fig. 2). On the exospore, a thin, dark layer of fibrilar material occurs. This layer covers the entire microspore surface and forms the scales or crests near the laesurae (Plate 4, fig. 2). Megaspores (Plate 5, figs. 1–6). Spores trilete, 178.6– 294.0 µm in polar diameter and 188.1–294.5 µm in PLATE 2 Spores of Salvinia biloba Raddi 1825 photographed using the SEM. Figures 1, 9: Forno 25 C (LP); figures 2–8, 10, 11: Forno s/n (LP). Figures 1–9 are megaspores, figure 10 is a massula, and figure 11 is a microspore. 1 2 3 4 5 6 The megaspore is covered by the sporangium wall. The three subtriangular flap-like structures (arrows) are partially open. Scale bar: 100 µm. Megaspore covered with the megasporangium wall; part of the subtriangular flap-like extensions of the epispore are partially broken. The perforated surface and the inner triradiate ridge (asterisk) are visible. Scale bar: 100 µm. Detail of the surface with ridges and perforations of variable diameter under the megasporangium wall (MeW). Scale bar: 10 µm. Section of megaspore. Two flap-like extensions of the epispore (arrows) and the inner ridge (asterisk) are visible. The reticulate megasporangium wall (MeW) is firmly attached to the megaspore surface. Scale bar: 100 µm. Longitudinal section of a megaspore with the exospore retracted, and the inner part of the proximal projection detached. The lateral flap-like extensions of the epispore (arrows) are visible. Scale bar: 100 µm. Detail of figure 3, the exospore (E) surface has verrucae and rodlets. The epispore (Ep) structure is lacunose. Scale bar: 20 µm. 7 8 9 10 11 Detail of figure 6. The outer surface of the exospore has verrucae and rodlets. Scale bar: 1 µm. Detail of figure 6. The inner epispore surface has projections. Scale bar: 2 µm. The sporoderm in section. The exospore (E) is homogeneous and compact; the epispore (Ep) is lacunose. The alveoli are smaller in the inner zone (asterisk). At the top of the epispore surface, the megasporangium wall (MeW) is attached. Scale bar: 10 µm. A massula; at the bottom of the figure the microsporangium wall (MiW) is reticulate. In the peripheral alveoli, the microspores are located (arrowheads). Scale bar: 50 µm. Detail of figure 10 showing a microspore within an alveolus. The microspore is surrounded by the lacunose structure of the massula. A laesura (arrow) and the rugulate surface are visible in detail. Along the surface next to each laesura, short crests are evident. Scale bar: 5 µm. P. Gardenal, M.A. Morbelli, and G.E. Giudice: Morphology and ultrastructure in Salvinia from southern South America MeW 3 1 2 Ep MeW MeW E 6 4 5 7 MeW Ep 9 E 10 MiW 11 8 Plate 2 149 150 PALYNOLOGY, VOLUME 32 — 2008 equatorial diameter, with a broadly circular but irregular outline, and rhomboidal to ovoid outline in equatorial view (Plate 5, figs. 1–3). The surface is ridged and perforate, with a background of minute perforations. The perforations are more dense in the equatorial area (Plate 5, figs. 1, 4). The sporoderm in section consists of exospore and epispore. Under the LM, the exospore is brown. The exospore is 3.2–6.0 µm thick and homogeneous under the SEM (Plate 5, figs. 5, 6). The epispore is 11.1–74.0 µm thick, lacunose, with two zones (Plate 5, fig. 6). The inner zone is 2.6–9.1 µm thick with lacunae 300–600 nm in diameter. The outer zone constitutes the main part of the wall, is variable in thickness in different areas, from 5.5– 65.0 µm, and has lacunae 2.0–7.2 µm in diameter. Proximally the megaspore has a complex projection consisting of two parts. In surface view three subtriangular flap-like extensions of the epispore are present; the areas between these flap-like extensions are thick (Plate 5, fig. 1). In section, the innermost part of the projection is a central ridge, with a channel open to the surface. The outer part is composed of the three flap-like structures, that can be banded toward the centre (Plate 5, figs. 2, 5) but in some cases they are reduced to lateral blunt projections (Plate 5, fig. 3). Massulae (Plate 5, figs. 7, 8). Spheroidal to elliptical bodies, the major axis being 150–197 µm long covered by the microsporangium wall with a reticulate surface (Plate 5, fig. 7). In section, its structure is lacunose and there are one or two central spaces of 44–123 µm in diameter, and smaller peripheriric lacunae of variable diameter. The microspores are included in some of these lacunae (Plate 5, figs. 8, 9). Microspores (Plate 5, fig. 9). Spores trilete, spheroidal, 16–36 µm in diameter. The surface is rugulate. Each laesura is 6–14 µm long. In section the sporoderm consists of exospore 0.5–0.9 µm thick, increasing to 2 µm at the laesurae. DISCUSSION All the specimens studied of Salvinia adnata (=Salvinia molesta Mitchell 1972) had small, dark, empty megasporangia and massulae. These observations are consistent with those of Mitchell and Thomas (1972), on material from Botswana, Rio de Janeiro (Brazil), and Sri Lanka, and those of de la Sota (2001) on specimens from southeast Brazil. Loyal and Grewal (1966) studied this species, as Salvinia auriculata (=synonym), and made an interpretation of the reasons for the interruption of the reproduction by means of spores. The morphology and sporoderm ultrastructure in megaspores, microspores, and massulae of Salvinia auriculata, are consistent with the descriptions of Bonnet (1955) and Tryon and Lugardon (1991). The latter authors considered the massula structure is lacunose and part of the microspore wall, and considered it to be epispore. The structure of the microspore exospore in Salvinia biloba is similar to the observations of Lugardon (1972; 1974) in homosporous Filicophyta, and those of Lugardon and Husson (1982) on the microspores of Salvinia natans. Nevertheless in the present work, an outermost thin electron-dense layer was observed under the TEM composed of fibrillar material. During chemical treatment, it was observed that megaspores and massulae remained on the surface of the liquid when centrifuged. It is considered that this is due to the PLATE 3 Megaspores of Salvinia biloba Raddi 1825 from Forno 25 C (LP). Figure 1 photographed using the LM; figures 2–6, photographed using the TEM. 1 2 3 Longitudinal section. Proximally, three flap-like extensions of the epispore are visible (arrows). Two on the sides are seen in transversal longitudinal section while the third (in the center) was cut obliquely. Inwards, two arms of the inner triradiate ridge (asterisks) can be seen. Scale bar: 100 µm. Section of the outermost part of the megaspore wall. Megasporangium wall (MeW) and underlying epispore (Ep). Scale bar: 10 µm. Detail of the two-layered exospore. The inner layer (Ei) is thin and electron-dense. The outer layer (Eo) has many spaces, all with a dark content. The spaces are evident both in longitudinal (white rectangle) and cross section (white circle). Scale bar: 1 µm. 4 5 6 Megaspore wall section that includes the inner and middle parts. Two-layered exospore (Ei and Eo) and a lacunose epispore (Ep). A channel (arrow) is visible. Scale bar: 10 µm. Transverse section of the proximal projection. The three flap-like extensions of the epispore with a lacunose structure are observed (arrowheads). In the center, part of the inner triradiate ridge (arrow) is visible. Scale bar: 10 µm. Transverse section of the proximal projection at a deeper level in the same material in figure 5. Two flaplike extensions of the epispore (arrowheads) and part of the inner central ridge, with a more compact structure (arrow) are evident. Scale bar: 10 µm. P. Gardenal, M.A. Morbelli, and G.E. Giudice: Morphology and ultrastructure in Salvinia from southern South America 1 2 MeW Ep 4 Ep 3 Eo Eo Ei Ei 5 6 Plate 3 151 152 alveolate structure of both the epispores of megaspores and the massulae. Undoubtedly this kind of structure increases bouyancy. Variations were observed in the orientation of the proximal megaspore flap-like extensions in Salvinia biloba megaspores, ranging from close to the center, to peripheral. Similar variations were reported by Nasu and Seto (1976) in megaspores of Salvinia natans from Japan, and were interpreted by these authors as steps in the process of apical opening during germination. The spores produced by austral species of Salvinia are similar to those produced by other species from South America. Thus, the proximal structures of the megaspores and microspores of Salvinia auriculata and Salvinia biloba are similar to those of Salvinia oblongifolia from Bahia, Brazil (de la Sota, 1962a). When comparing certain fertile characteristics of the Salvinia species studied here with those of other heterosporous Filicophyta such as Azolla Lamarck 1783, as described and illustrated in Di Fulvio (1961), Fowler and Stennett-Wilson (1978), Perkins et al. (1985), and Gardenal et al. (2007), some similarities were evident. Thus, the components of the proximal projection in Salvinia megaspores in section are similar in structure and location to those in Azolla. In addition, the microspores in both genera are also included within massulae with a relatively similar basic inner structure. It was noticed also that megaspores, microspores, and massulae of Salvinia from southern South America are similar to fossil material. Megaspores of extant Salvinia auriculata have a morphology and ultrastructure similar to Salvinia cobhamii Martin 1976 from lacustrine deposits from the Upper Paleocene of England (Martin, 1976). The megaspore wall ultrastructure of the three species analyzed here is indistinguishable from that of Salvinia cerebrata Nikitin ex Dorofeev 1974 studied by Friis (1977) from the Middle Miocene of Denmark, which differs in its ornamentation, and with those found with Salvinia reussii Ettingshausen 1886 from the Miocene of Bohemia (Collinson, 1991). Similarities were also found in the variability of ornamentation patterns, and the type and distribution of structural elements in megaspores between the extant species of Salvinia studied here and fossil species described by Friis (1977). The proximal projections of megaspores of Salvinia auriculata, Salvinia biloba, and Salvinia minima are similar to those observed by Jain and Hall (1969) in Salvinia aureovallis Jain and Hall 1969 from the Eocene of North Dakota, U.S.A. Furthermore, the massula morphology and ultrastructure of extant Salvinia species from southern South America are indistinguishable from those of Salvinia sp. described by Batten and Collinson (2001) from the PALYNOLOGY, VOLUME 32 — 2008 Paleocene/Eocene boundary of Belgium, Germany, and the Netherlands. CONCLUSIONS Species of Salvinia from southern South America are similar to other extant taxa from the Neotropical and the Old Worlds and fossil material. Southern South America represents the southern limit of distribution of these extant species. This area has large latitudinal differences, and this is reflected in the habitats. In spite of this, no intraspecific differences in specimens from the extremities of this region were noted. The megaspores of the three species studied share some characteristics such as, size, perforate outer surface, and ultrastructure. Both the megaspore size and the ovoid equatorial outline are similar in Salvinia auriculata and Salvinia biloba, whereas those of Salvinia minima are smaller, with an ovoid to rhomboidal equatorial outline. There are differences in the size and complexity of the proximal structures. The megaspores produced by Salvinia auriculata and Salvinia biloba also have a well-developed proximal inner ridge and outer flap-like extensions, while these elements in Salvinia minima are less prominent and compressed. These morphological differences contribute to the “Salvinia auriculata complex” of Mitchell and Thomas (1972), in which Salvinia auriculata and Salvinia biloba, are included. Based on this information, further research on the relationships within this complex in a broader phylogenetic context is needed. The massulae and microspores of the three species are indistinguishable at morphological and ultrastructural levels. The epispore in megaspores and massulae is similar and constitutes an interconnected system; both would probably accomplish the same functions. It would be necessary to investigate if both structures could be considered as homologous based on studies of sporogenesis. In spite of the fact that TEM study was made in only one of these species, the detailed knowledge obtained on the ultrastructure of megaspores, microspores and massulae raises questions and indicates that similar studies should be done on the other species. A three-dimensional network of channels with an osmiofilic content on the outer exospore of the megaspores is present, and there is a layer in the exospore of the microspores that is similar to a perispore. The inner ridge of the proximal projection and the thickenings between the outer flap-like extensions in Salvinia is comparable to the central column and the collar of Azolla respectively. Nevertheless the flap-like extensions of Salvinia are part of the megaspore wall, whereas in Azolla there is apparently no connection between the floats and the sporoderm, except for filaments which arise from the floats and the column and tangle together to secure the P. Gardenal, M.A. Morbelli, and G.E. Giudice: Morphology and ultrastructure in Salvinia from southern South America 153 floats to the column. The microspores included in massulae in Azolla and Salvinia are also similar. Studies on the development of both kinds of reproductive structures are needed in order to investigate the existence of homologies between these genera. By comparing the extant megaspores, microspores, and massulae in the representatives of the Salviniaceae studied here, with those from the Paleocene to Quaternary fossil record, a similar wall structure was found that indicates stasis. Nevertheless, minor differences were observed in wall stratification and ornamentation. This condition is a consequence of the success in competing with other taxa and reproduction in an aquatic environment. van Cittert, for valuable suggestions that have substantially improved the manuscript. Lic. Rafael Urrejola of the SEM Unit, Museo de La Plata, Ing. Luis Zimmermann, of the TEM Unit (LANAIS/CONICET) Facultad de Medicina, Universidad Nacional de Buenos Aires, and Mrs Isabel Farías are thanked for technical assistance. This work was supported by grants from the National Agency of Science and Technology Promotion (ANPCyT) for PICT 12758, and Universidad Nacional de La Plata for projects 363 and 451. ACKNOWLEDGMENTS AVETISIAN, N.M. 1950 Méthode simplifiée d’acetolyse dans la préparation de pollens. Botaniceskij Zurnal (Moscow and Leningrad), 35: 385–386. The authors would like to express their thanks to the reviewers, David Batten and Johanna van Konijnenburg- References Cited PLATE 4 A microspore of Salvinia biloba Raddi 1825 photographed using the TEM from Forno 25 C (LP). 1 2 Microspore within the lacunose structure of the massula. The exospore (E) is thicker at the laesura. Along the exospore surface, there is a thin electron-dense layer (arrows). Scale bar: 5 µm. Detail of the proximal area of the microspore. The inner layer of the exospore (Ei) is homogenous, thin and electron-dense. The outer layer (Eo) has a system of fused channels (black arrowhead), forming arcs in its innermost part and simple channels toward the outer surface (outlined arrowhead). At the laesura base, nodules of material are evident. A thin, fibrillar, electrondense layer is evident on the exospore surface (arrow). Scale bar:1 µm. 154 PALYNOLOGY, VOLUME 32 — 2008 BATTEN, D.J., and COLLINSON, M.E. 2001 Revision of species of Minerisporites, Azolla and associated plant microfossils from deposits of the Upper Palaeocene and Palaeocene/Eocene transition in the Netherlands, Belgium and the USA. Review of Palaeobotany and Paynology, 115: 1–32. BONNET, A.L.M. 1955 Contribution à l’étude des Hydroptéridées: recherches sur Salvinia auriculata Aublet. Annals de Sciences Naturelles Botanique, 16: 529–600. 1957 Contribution à l’étude des Hydroptéridées: recherches sur Azolla filiculoides Lamk. Revue de Citologie et de Biologie Végétales, 18(1): 1–89. COLLINSON, M.E. 1991 Diversification of modern heterosporous Pteridophytes. In: Blackmore, S., and Barnes, S.H. (eds.). Pollen and Spores. Systematics Association, 44: 119–150. Clarendon Press, Oxford. 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Scale bar: 100 µm. Megaspore sectioned in equatorial view. The wall is thickened laterally at the equator. Proximally the inner ridge (asterisk), and two flap-like expansions (see the detail of the proximal area in the longitudinal section of a megaspore in figure 5) of the epispore (arrows) can be seen. Scale bar: 50 µm. Megaspore sectioned in equatorial view. The wall in this megaspore is thickened distally. Two blunt lateral expansions are evident proximally (arrows). The globules in the inner part of the megaspore probably are reserve products. Scale bar: 50 µm. Detail of the surface with perforations of varying diameter. Scale bar: 10 µm. Detail of the proximal area of the spore in figure 2. There is a central projection (asterisk) with a channel open to the surface (double white arrow); at both sides 6 7 8 9 there are flattened expansions of the epispore (arrows). Scale bar: 20 µm. Sporoderm in section. The exospore (E) is homogeneous and compact. The epispore (Ep) is lacunose. 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