Structure and Ultrastructure of the Tracheary Elements of Asplenium (Pteridophyta) from the “Yungas”, Argentina
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IAWA Journal, Vol. 31 (2), 2010: 227–240 STRUCTURE AND ULTRASTRUCTURE OF THE TRACHEARY ELEMENTS OF ASPLENIUM (PTERIDOPHYTA) FROM THE “YUNGAS”, ARGENTINA María Luján Luna1, 3,*, Gabriela Elena Giudice1, María Alejandra Ganem2 and Elías Ramón de la Sota1, 4 SUMMARY The structure of root and rhizome tracheary cells of Asplenium spp. (Fili- cales, Pteridophyta) growing in NW Argentina was studied using light mi- croscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In all cases, tracheary cells consisted of tracheids with various facets, mainly with scalariform pitting. With SEM, intertracheary pit membranes appeared smooth and non porose in most cases. In some instances, porose or web-like to thread-like pit membranes were noticed in rhizome tracheids. Under TEM secondary walls displayed a smooth and uniform appearance. Pit membranes showed a variation in thickness in presumed association with their maturation stage. More ma- ture tracheary cells showed pit membranes with a mesh-like aspect and visible openings or pores. These characteristics are attributed to pit mem- brane hydrolysis, which facilitates water transport among tracheary cells. Key words: Asplenium, Pteridophyta, tracheids, secondary wall, pit mem- brane, ultrastructure. INTRODUCTION The water-transport system (xylem) of Pteridophytes consists mainly of tracheids with tapered ends and scalariform or circular to oval bordered pits (Bierhorst 1960; Ogura 1972; Gifford & Foster 1989). Certain ferns possess vessels. Bliss (1939) and Bierhorst (1960) documented the presence of vessel elements with scalariform perforation plates in rhizomes, petioles and roots of Pteridium aquilinum. Vessel elements with simple perforation plates were observed in roots and rhizomes of Marsilea sp. (White 1961; Bhardwaja & Baijal 1977) and in rhizomes of Actiniopteris radiata (Singh et al. 1978). Reports of vessels in Astrolepis (Carlquist & Schneider 1997) and Woodsia (e.g., Carlquist & Schneider 1998) are considered valid by Carlquist and Schneider (2007). 1) Cátedra Morfología Vegetal, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata. Paseo del Bosque s/n (1900), La Plata, Argentina. 2) Cátedra Botánica General, Facultad de Ciencias Agrarias, Universidad Nacional de Jujuy, Argentina. 3) Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. 4) Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina. *) Author for correspondence [E-mail: [email protected]]. Downloaded from Brill.com10/02/2021 02:54:11AM via free access 228 IAWA Journal, Vol. 31 (2), 2010 Various SEM studies on macerated material reported absence of pit membranes in some tracheid pits of various species of Pteridophyta (i.e. Carlquist & Schneider 1997, 1998, 1999, 2000a, 2000b; Carlquist et al. 2000; Schneider & Carlquist 1997, 1998, 1999). In a revision of their studies, and employing other preparative techniques, the authors reinterpreted their data and concluded that fern xylem consists mainly of tracheids, with different ranges of pit membrane porosity in end walls (Carlquist & Schneider 2007). Carlquist & Schneider (2007) found that in their earlier studies, use of macerations (which involve oxidative techniques and high temperatures) may ac- count for excessive removal of pit membranes on some tracheids. However, use of thick sections, prepared with razor blades of ethanol-fixed material, reveals porose patterns in end wall pits of tracheids, patterns that do not appear to be artifacts (Carlquist & Schneider 2007). They recommended against the use of macerations in studies on pit membrane presence in fern tracheids. The same was observed by Luna et al. (2008) in the root and rhizome tracheary cells of Salpichlaena. Most studies of tracheary element fine structure have been conducted on conifers and flowering plants (Friedman & Cook 2000; Dute et al. 2008, 2010). Morrow and Dute (1998) studied with TEM the development of the torus-bearing pit membranes of Botrychium. Cook and Friedman (1998) and Friedman and Cook (2000) described the development of the secondary cell walls of Huperzia (Lycophytina, Lycopodiaceae) and Equisetum (Euphyllophytina, Equisetaceae) tracheids. In both cases, they found that secondary walls were composed of a first-formed layer, the degradation-prone or “template layer”, and a later-formed degradation-resistant layer. Recently, Choat et al. (2008) have emphasized the importance of pit membrane studies in general, especially within ferns and basal angioperms. The aim of the present work was to analyze the structure and ultrastructure of the tracheids of Asplenium (Euphyllophytina, Filicales), in order to contribute to the knowledge of xylem morphology and evolution in ferns. Asplenium (Aspleniaceae, Pteridophyta) is a cosmopolitan genus of nearly 650 spe- cies, about 150 of which occur in tropical America (Tryon & Tryon 1982). Asplenium species are terrestrial, rupestral or epiphytic. The stems (rhizomes) are erect or decum- bent, rarely long-creeping; the roots are usually long and fibrous. In Argentina, near 38 species of Asplenium grow from the NW–NE to Patagonia (Sylvestre & Ponce 2008). MATERIALS AND METHODS For microscopic study fresh material of different species of Asplenium was collected at the “Yungas”, NW Argentina: A. argentinum (on cliffs), A. gillesii (rupestral, among rocks), A. praemorsum (epiphyte), A. serra (epiphyte or rupestral) and A. squamosum (terrestrial). The Yungas Phytogeographic Province is one of the most diverse ecosystems of Ar- gentina, with Sub-Andean humid Sierras, mountain forests, fertile valleys, canyons and “Altiplano” or “Puna”. In the NW of Argentina this region occurs between 400–3000 m, in areas that receive 1500–3000 mm of rain during the summer. Downloaded from Brill.com10/02/2021 02:54:11AM via free access Luján Luna et al. — Tracheary elements of Asplenium 229 Portions of roots and rhizomes were prepared for light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Specimens for LM were fixed in formaldehyde-acetic acid-alcohol, dehydrated through an ethanol series and embedded in Paraplast. Sections (8–12 µm thick) were double stained with safranin-fast green (Johansen 1940). Part of the material was macerated according to Jeffrey’s technique. Samples were placed in Jeffrey’s solution for 12 hours at room temperature, and then washed with distilled water. Preparations were stained with safranin and observed under a Nikon Photolab 2 light microscope. For SEM study, material was treated according to the methods of Dute et al. (1992) and Jansen et al. (2008). Transverse and longitudinal sections of roots and rhizomes were split using a razor blade. Sections were placed in 80% ethanol, then in 90% ethanol, followed by absolute ethanol and finally allowed to air dry. Samples were attached to aluminum stubs using double sticky tape, air dried and sputter-coated with gold-palladium. Observations were made in a JEOL, JSM-35 CF scanning electron microscope. For TEM analysis, samples were fixed in a 2% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.5) and vacuum infiltrated for 2 hours, then rinsed three times in the same buffer and post-fixed for 2 hours in 1% osmium-tetroxide. Specimens were then dehydrated in an ethanol-acetone series and embedded in Epon 812. Sections were mounted on grids, stained with uranyl acetate followed by lead citrate and examined with a JEOL, JEM 1200 EX II transmission electron microscope. RESULTS In all analyzed species, metaxylem consisted of tracheary cells with various facets, mainly with scalariform pitting and intact pit membranes. Root tracheary elements As in most ferns, roots of Asplenium are slender, thus only a few tracheary elements could be observed in each sample. In transverse sections, roots showed a diarch actinostele with exarch protoxylem and central metaxylem (Fig. 1). The stele was surrounded by many layers of thick-walled sclereids which made difficult microtome sectioning. The tracheary cells appeared polygonal in shape revealing the existence of various facets (Fig. 2). Pit membranes between xylem cells were easily observed in transverse thin sections (c. 1 µm) (Fig. 3–5). In longitudinal views, metaxylem tracheary elements showed tapered ends and scalariform pitting on most facets (Fig. 6, 7, 10). Facets with circular to oval bordered pits were also noted (Fig. 8). In most instances pairs of bordered pits on lateral walls showed intact pit membranes with smooth appearance; thus when they were disrupted this was attributed to artifacts during sectioning (Fig. 6, 7). In a few cases pit membranes were lacking in pits near the tips of tracheary cells, apparently due to the fact that they were torn off during sectioning (Fig. 9). Downloaded from Brill.com10/02/2021 02:54:11AM via free access 230 IAWA Journal, Vol. 31 (2), 2010 Downloaded from Brill.com10/02/2021 02:54:11AM via free access Luján Luna et al. — Tracheary elements of Asplenium 231 With TEM the compound middle lamellae and the secondary walls presented a smooth and homogeneous aspect (Fig. 11, 12). The middle lamellae appeared grey whereas the secondary walls appeared light grey (Fig. 12). A thin electron-dense coat- ing was visible on the lumen surface of the tracheary elements (Fig. 11). In some cases, pit membranes between tracheids were relatively thick and dense (250 nm) and showed a uniform microfibrillar appearance (Fig. 11, 13). In others, microfibrils were loosely packed