Structure and Ultrastructure of the Tracheary Elements of Asplenium (Pteridophyta) from the “Yungas”, Argentina

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 microscopy (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 mature tracheary cells showed pit membranes with a mesh-like aspect and visible openings or pores. These characteristics are attributed to pit membrane hydrolysis, which facilitates water transport among tracheary cells. Key words: Asplenium, Pteridophyta, tracheids, secondary wall, pit membrane, 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]].

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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 species, 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.

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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).

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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 coating 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 and came into view as a random network of fibrils delimiting openings or pores (Fig. 14, 15).

Rhizome tracheary elements In transverse sections, the rhizomes exhibited a dictyostele with the meristeles disposed in a single circle. The number of meristeles varied according to the species and they displayed roundish to oval outlines (Fig. 16, 17). Each meristele was concentric and consisted of xylem enclosed by phloem. The tracheary elements were not intermixed with parenchyma cells (Fig. 18). As in roots, tracheid cells possessed various facets, thus numerous lateral wall con- tacts (Fig. 18, 19). LM of thin sections revealed continuous pit membranes between the tracheary cells (Fig. 19, 20). In longitudinal views, metaxylem cells showed rounded to tapered ends (Fig. 21–23) and scalariform pitting in most facets (Fig. 24). Circular to oval bordered pits were also present (Fig. 23, 25). Under SEM, pit membranes appeared intact in most cases, although porose or web- like to thread-like pit membranes were detected on the end walls of some tracheary elements (Fig. 26–28). As was observed in roots, the secondary walls of rhizome tracheary xylem elements appeared smooth and homogeneous under TEM (Fig. 29, 30, 33, 35). A thin electron- dense coating was frequently visible on the lumen surface of the secondary walls (Fig. 30, 33). In various samples the secondary walls appeared crenulated and showed globular electron-dense outgrowths (Fig 31, 32, 35). In some instances, a granular material was deposited on the pit membranes (Fig. 31). Signs of cytoplasm autolysis were evident in some tracheary cells where a thin electron-dense coating was deposited in the inner surface during cell death (Fig. 29).

← Figures 1, 3–5 & 10. LM micrographs of root tracheary elements. Figures 2 & 6–9. SEM micrographs of root tracheids. – 1: Root TS showing diarch actinostele with endarch xylem (x) surrounded by many layers of sclereids (s). – 2: Detail of tracheids with polygonal outline and scalariform pitting. – 3: Thin root section showing pit membranes between tracheids (arrows). – 4: Intertracheary pit membrane in detail (arrow). – 5: Variation in pit membrane thickness: relatively thin at left (arrowhead) and thicker at right (arrow). – 6: Longitudinal view of tracheids with scalariform pitting and smooth pit membranes on lateral walls (asterisk). – 7: Detail of lateral wall with scalariform pitting and disrupted pit membranes attributed to artifacts (asterisk). – 8: Portions of lateral wall tracheids with circular to oval bordered pits. – 9: Tip of tracheid with scalariform pits lacking pit membranes probably due to tearing off during sectioning. – 10: Tracheary elements with tapered ends and scalariform pitting.

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Figures 11–15. TEM micrographs of root tracheary elements. – 11: TS of tracheids showing relatively thick pit membrane between them (Pm). See also the thin electron-dense coating de- lineating the lumen surface (arrowhead). – 12: Detail of homogeneous compound middle lamella (Lm) and secondary walls (Sw). – 13: Detail of pit membrane with uniform fibrillar aspect. – 14: Pit membranes with loose appearance given by the disarranged disposition of fibrils. – 15: De- tail showing openings or pores (arrows).

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Figures 16–21. LM micrographs of rhizome tracheids. – 16: TS of roundish meristele with xylem (x) enclosed by phloem (ph). – 17: TS of meristele with oval outline. – 18: Detail of polygonal tracheary cells with various facets. – 19: Continuous pit membranes between the tracheids (arrow). – 20: LS of metaxylem cells with scalariform pitting and continuous pit membranes. – 21: Tracheids that show rounded to tapered ends and scalariform pitting.

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Figures 22–28. SEM micrographs of rhizome tracheary elements. – 22: Tracheids with tapered ends and scalariform pitting on most facets. – 23: Tracheid with rounded tip at left and intact pit membranes on lateral wall pits at right (asterisk). – 24: Metaxylem tracheids with scalariform pitting on lateral walls. Disrupted pit membranes are attributed to artifacts (asterisk). – 25: Detail of portions of tracheary cells with circular to oval bordered pits. — 26–28: Portions of end walls of tracheary elements. – 26: Porose pit membrane (arrow). – 27: Pit membrane with larger pores (arrow). – 28: Web-like to thread-like pit membrane.

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Figures 29–32. TEM micrographs of rhizome tracheary elements. – 29: Tracheid showing a thin electron-dense coating deposited in the inner surface during cell death (arrow). The adjacent cell is a parenchyma cell (p). – 30: A supposed more advanced condition of tracheid maturation where the electron-dense coating appears darker and not continuous (arrowheads). – 31: Tracheary cells with crenulated secondary walls and electron-dense deposits on the lumen surface (arrowhead) and pit membranes (arrow). – 32: Detail of secondary wall outgrowths (arrows).

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Figures 33–37. TEM micrographs of rhizome tracheary elements. – 33: Tracheary cells with thick and dense intertracheary pit membrane (Pm) with uniformly disposed microfibrillar material. Secondary walls (Sw) have a smooth and homogeneous appearance. – 34: Detail of less dense intertracheary pit membranes (Pm). – 35: Apparently more mature tracheary cells with thinner pit membranes. – 36: Detail of the network-like aspect of pit membranes with visible openings or pores (arrow). – 37: Thinner intertracheary pit membrane with pronounced openings (arrow).

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The pit membranes between tracheids varied in thickness (100–500 nm). In some instances, pit membranes were thicker and denser, composed of uniformly disposed microfibrillar material (Fig. 33, 34). In others, they showed a loose appearance and openings or pores were discernible (Fig. 35–37).

DISCUSSION AND CONCLUSION

Root and rhizome metaxylem of selected species of Asplenium consists of tracheids with various types of scalariform pitting. Under SEM, intact non-porose pit membranes were observed on lateral walls. The absence of pit membranes at the end walls of some tracheary elements was considered an artifact. Thus, no perforation plates and hence no vessel elements were noted in our study. However, porose to web-like or thread-like pit membranes were registered near the tips of some rhizome tracheary cells. Schneider and Carlquist (1999) also reported the presence of porose pit membranes in root and rhizome tracheary elements of Asplenium nidus. The occurrence of intertracheary pit membranes with a degree of porosity is in agreement with Carlquist and Schneider’s findings in other ferns (Carlquist & Schneider 2007). Recently, Luna et al. (2008) found tracheids with porose and web-like to thread-like pit membranes in the roots of Salpichlaena (Blechnaceae). Carlquist (1992) and Carlquist and Schneider (2001, 2007) interpreted pit membrane remnants as precursors to membrane disappearance and, as a consequence, a significant stage in vessel element evolution. In Asplenium, the presence of porose or web-like pit membranes in tracheids was not related to habitat or habit type (epiphytic, rupestral or terrestrial). As mentioned by Carlquist and Schneider (2007), no relationship seems to exist between the occurrences of porose or reticulate pit membranes and plant ecology or habit. In this manner, we agree that these types of pit membranes appear to be widespread among ferns, inde- pendently of plant habit. Various authors have studied the fine structure of the intervascular pit membranes in angiosperms (Meylan & Butterfield 1982; Dute et al. 1992; Sano 2004, 2005; Jansen et al. 2009, among others). According to their findings, a variation in the porosity of intervascular pit membranes exists, as well, in this group of plants. In the present work, we report new observations on tracheid wall fine structure in ferns. Under TEM, the secondary walls of Asplenium tracheary cells showed a smooth and homogeneous fibrillar appearance. Kenrick and Crane (1997) found that tracheid cell walls of most vascular plants typically are relatively thick, lignified and decay-resistant. However, Cook and Friedman (1998) and Friedman and Cook (2000) observed two distinct layers in the secondary walls of Huperzia and Equisetum tracheids: a “template layer” and a “resistant layer”. In Asplenium tracheids no distinct layers, like those characteristic of early tracheophytes, were distinguished. This is in agreement with Cook & Friedman (1998), who proposed that a trend toward reduction of the template layer and the augmentation of the resistant layer has produced the secondary cell wall thickenings characteristic of tracheids of extant seed plants. Another feature observed in Asplenium tracheid cells was the presence of a dark layer covering the secondary walls, as was mentioned for Huperzia and Botrychium

Downloaded from Brill.com10/02/2021 02:54:11AM via free access 238 IAWA Journal, Vol. 31 (2), 2010 tracheids (Cook & Friedman 1998; Morrow & Dute 1999). Morrow and Dute (1999) associated such coatings with wound response in Botrychium, whereas Cook and Friedman (1998) described them as products of cell autolysis. Although in the present study we did not include a developmental analysis, we observed in some Asplenium tracheids signs of cytoplasm autolysis. In this manner, we provisionally attribute the presence of coating material to remnants of lysed cytoplasm in our samples. Other characters reported here in Asplenium tracheids, such as crenulated secondary walls or electron-dense outgrowths, remain to be explained. With TEM, Asplenium intertracheary pit membranes displayed a different thickness in presumed association with their maturation stage. Intermediate thick and thinner pit membranes showed a loosened microfibrillar aspect apparently due to pit membrane hydrolysis. In some instances, small openings were also discernible. As tracheary cells mature, the pit membranes between tracheids lose some or all of their matrix material. According to O’Brien (1970), destruction of the protoplast in tracheary elements is accompanied by loss of the non-cellulosic polysaccharides (pectins and hemicelluloses) from the unlignified parts of the primary walls. Previous TEM studies in Huperzia and Botrychium tracheary cells showed the removal of matrix material from the pit membranes during their maturation (Cook & Friedman 1998; Morrow & Dute 1998). In addition, ultrastructural studies of intertracheary pit membranes in gymnosperms also illustrated wall matrix removal from pit membranes in association with cell autolysis (Dute 1994; Dute et al. 2008). In this manner, our work brings new information of the fine structure of the secondary walls and pit membranes in fern tracheary cells in different stages of maturation and gives more evidence of the occurrence of pit membrane hydrolysis. This process, together with the development of openings or pores in the pit membranes, are said to facilitate water transport between adjacent conductive cells.

ACKNOWLEDGEMENTS

The authors thank Rafael Urrejola of “Servicio de Microscopía Electrónica de Barrido”, Facultad de Ciencias Naturales y Museo and Susana Jurado, “Servicio de Microscopía Electrónica de Transmisión”, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, for their technical assistance. They also thank Dr. Sherwin Carlquist and Dr. Pieter Baas for their valuable comments and suggestions concerning the manuscript. This study was supported by the Research Projects of Consejo Nacional de Investigaciones Cientificas y Técnicas (PIP 5533) and Universidad Nacional de La Plata (11/ N465), Argentina.

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