Silica bodies and their systematic implications at the subfamily level in Podostemaceae

Podostemaceae is a family of eudicotyledonous rheophytes with unique morphology, sometimes resembling algae, lichens or bryophytes. This study evaluates the taxonomic value of silica-body morphology (presence, location, size, shape, ornamentation) in species representing the three subfamilies: Tristicha trifaria (Bory ex Willd.) Spreng. (Tristichoideae), Weddellina squamulosa Tul. (Weddellinoideae) and Diamantina lombardii Novelo, C.T.Philbrick & Irgang (Podostemoideae). T. trifaria and D. lombardii have silica bodies in stems and leaves. In contrast, W. squamulosa exhibits silica bodies only in the scale-like leaves. The morphology of these bodies is unique to each species. The results denote the taxonomic usefulness of silica characteristics and a need for a more complete study of silica bodies throughout the family.


Introduction
Podostemaceae Rich.ex Kunth is a eudicotyledonous family comprising approximately 50 genera and 300 species.These plants grow in rapidly flowing water, attached to rocks or other solid substrate.Species occur in the tropics with a few species reaching temperate regions (Cook & Rutishauser 2007).The family is divided into three subfamilies (Engler 1930;Kita & Kato 2001).Tristichoideae (three genera), which is sister to the rest of the Podostemaceae, is represented by a single species (Tristicha trifaria (Bory ex Willd.)Spreng.) in the neotropics.The monotypic Weddellinoideae is endemic to the neotropics.The largest (47 genera) and most widespread subfamily is Podostemoideae, which occurs in both paleo-and neotropics (Ruhfel et al. 2011).
Many authors have reported on the unusual vegetative body of Podostemaceae.For example, Rutishauser (1997) and Rutishauser et al. (2008) have discussed the difficulty of applying the classical root-stem-leaf model to these plants.The unusual morphology of riverweeds is linked to their environment, although specific cause-effect relationships remain unclear (e.g.Pfeifer et al. 2009).
Podostemaceae are also notable in terms of their anatomy.Plants are characterized by the lack of aerenchyma (otherwise common in aquatic plants), lack of stomata, lack of an endodermis and pericycle, inconspicuous vascular tissue, faint distinction between xylem and phloem, abundant starch grains, presence of laticiferous and/or secretory channels and presence of silica bodies (e.g.Schnell 1967, Cusset & Cusset 1988, Cook & Rutishauser 2007).Although the presence of silica bodies in Podostemaceae has been noted by numerous authors in many genera, such as Apinagia, Diamantina, Podostemum and Weddellina (e.g.Schnell 1967, Rutishauser et al. 2005, Koi & Kato 2007), morphological descriptions and comparative studies involving taxonomic or phylogenetic perspectives are lacking.
Plants take up silicon (Si) as silicic acid (Si (OH) 4 ) and accumulate it as hydrated silica (SiO 2 .nH 2 O) in the cytoplasm (Currie & Perry 2007), vacuoles or cell walls of roots, stems and leaves, or reproductive structures (Kealhofer & Piperno 1998;Neethirajan et al. 2009).Silica particles can be present in roots in many shapes, such as spherical, dumb-bells, saddles, bowls, and boats (Cooke & Leishman 2010).These patterns, as well as metric data, have been used as important taxonomic characteristics, even at the infrageneric level, in monocots, like Poaceae, Zingiberaceae, and Orchidaceae, as well as in Pteridophyta (e.g.Pteridaceae) (Kealhofer & Piperno 1998;Prychid et al. 2003;Sundae 2009).In Podostemaceae the silica can be found occupying the cytoplasm or only in the vacuoles (Jäger-Zürn 2011).Although the presence of silica bodies is well documented in Podostemaceae, they have been poorly investigated in terms of morphology and not utilized taxonomically.Moreover, recent phylogenetic studies of neotropical Podostemaceae (Tippery et al. 2011) reveal a lack of apparent morphological synapomorphies for some major clades.Clearly further examination of morphological features of neotropical Podostemaceae is needed.
The aim of this study was to evaluate the potential application of silica bodies in the taxonomy of neotropical Podostemaceae at the subfamily level.In this sense, the presence, location and morphology of the silica bodies were determined for three representative species, one from each of three subfamilies.Tristichoideae, which is largely paleotropical, was represented by Tristicha trifaria (Bory ex Willd.)Spreng.Weddellina squamulosa Tul. was included as the sole member of subfamily Weddellinoideae.Podostemoideae, the largest subfamily, was represented by Diamantina lombardii Novelo, C.T. Philbrick & Irgang, the most basal member of this subfamily (Ruhfel et al. 2011).

Material and Methods
Mature plants were fixed in FAA 70 (Johansen 1940) and preserved in 70% ethanol.Longitudinal and transversal hand-cut sections were cleared in 50% commercial sodium hypochlorite solution and rinsed in 1% acetic acid and distilled water.Samples were then stained with alcian blue and fuchsin 0.1% (Luque et al. 1996).A portion of the fixed samples were dehydrated in an ethanol series, subjected to pre-infiltration and subsequently infiltrated in hydroxy methacrylate resin following standard procedures.Transverse and longitudinal sections (5µm thick) were made using a rotary microtome.
Sections were stained with 0.05% toluidine blue at pH 4.7 (O'Brien et al. 1965).Histochemical tests were performed on freehand-sections to detect starch, using Lugol's solution, and clove oil and heated phenol to detect silica bodies (Johansen 1940).For morphological studies of silica bodies in light microscopy, fragments of vegetative material were softened in a solution proposed by Franklin (1945), soaked with clove oil and stained with phenol.All samples were mounted in 50% glycerin on slides with cover slips.Observations were carried out with an Olympus ® (CX-31) light microscope.To observe the shape and ornamentation of silica bodies with scanning electron microscopy, fragments of specimens were carbonized in a muffle furnace for 6 hr at 600°C, soaked with 70% ethyl alcohol (Wattiez & Sternon 1942), and transferred to aluminum stubs.Stubs were sputter coated with gold platinum (Denton Vacuum Desk IV) and examined using a Jeol JSM 6390 LV scanning electron microscope.
In size descriptions, the lower and upper ranges were listed parenthetically.Arithmetic mean and standard deviation values were based on 20 measurements of one specimen of each species.At least one more voucher of each species was analyzed and 10 measurements were taken for comparison.Selected specimens for morphometric data shown in

Results and Discussion
This study confirmed the presence of starch grains in parenchyma cells and silica bodies occupying the cell cytoplasm in each of the species examined.The morphology (shape and ornamentation) of the silica bodies was different in each species analyzed.In Tristicha trifaria they were rectangular with many crests oriented perpendicular to the main axis (Fig. 1a-b).Mean length and width were 62.4 µm and 17.6 µm, respectively (Tab.1).In Weddellina squamulosa the silica bodies were triangular, ellipsoidal, ovate or oblong, with many rounded depressions and projections distributed somewhat uniformly along the surface (Fig. 1c-d).Average size was 44.1 µm in the longest dimension (Tab.1).Lastly, in Diamantina lombardii the silica bodies were rectangular or oblong with many holes of variable diameter (Fig. 1e-f).These holes were distributed only on the surface of the body oriented to the exterior of the plant (Fig. 2e).Average length and width were 32 µm and 16.6 µm, respectively (Tab.1).Thecomparison specimens analyzed showed uniformity of morphological and morphometric data.
In Podostemaceae, the aggregation of silica bodies in peripheral tissues can be referred to as forming a "carapace," which may provide protection against mechanical damage and drought stress  (Metcalfe & Chalk 1979).Our results indicated that Tristicha trifaria (Fig. 2a) and Diamantina lombardii (Fig. 2b) have silica bodies in epidermal and in some hypodermal cells of stem and leaves.In contrast, Rutishauser et al. (2005) reported that silica bodies occur in only a few epidermal and hypodermal cells in stem and leaves of D. lombardii; a difference that may have been induced by methodological bias.The silica bodies forming a carapace were present here in sections made with a Ranvier microtome.In contrast, sections made with a rotary microtome showed fewer silica bodies in all specimens.Methodological bias provided by rotary microtome (thinner sections) causes removal of the silica bodies in tissues (Fig. 2d).
In Weddellina squamulosa these structures were present only in the epidermis of the scale-like leaves (Fig. 2c), corroborating the report of Koi & Kato (2007).Interestingly, this species is often used as fish-bait (C.P. Bove, personal communication).Consequently, if silica bodies serve an antiherbivory function, it likely relates to non-fish herbivores (e.g.invertebrates).It is also notable that leaves of Podostemaceae are present in stomach content analyses of fish species (e.g.Astyanax, Characidae) indicating it as a potential food source (Villela et al. 2002).
The ornamentation and shape of the silica bodies were notably different among the subfamilies investigated.Moreover, whether such variation is useful at lower taxonomic levels (e.g.among genera or species) remains to be determined as more species are analyzed.The potential applicability of silica body morphology and Silica bodies in Podostemaceae systematics occurrence in Podostemaceae to systematic purposes, based on broad taxonomic and phylogenetic sampling, is presently underway.Due to the high capacity of fossilization, the morphology of silica bodies can also be applied to paleobotanical/paleoecological examination (see "opal phytoliths" in Alexandre et al. 1997).It is notable that silica bodies may not migrate through strata to the same extent as do other microfossils, such as pollen grains and diatoms (Prychid et al. 2003).Perhaps better understanding of the form and diversity of silica bodies in Podostemaceae can contribute to new insights into the history of these fascinating plants, for which a fossil record is largely absent, and help on the reconstruction of ancient aquatic ecosystems.

Figure 1 -
Figure 1 -a-b.Isolated silica bodies of Tristicha trifaria -a.rectangular silica bodies on light microscopy; b. silica body surface with crests perpendicular to main axis on scanning electron microscopy.c-d.Isolated silica bodies of Weddellina squamulosa -c.triangular, ellipsoidal, ovate and oblong silica bodies on light microscopy; d. silica body surface with circular depressions on scanning electron microscopy.e-f.Isolated silica bodies of Diamantina lombardiie.rectangular and oblong silica bodies on light microscopy; f. silica body surface with holes of irregular form (arrows) on scanning electron microscopy.

Figure 2 -
Figure 2 -a-e.Transversal (a-c) and longitudinal (d-e) sections of the vegetative body on light microscopy.a. Tristicha trifaria.Arrow shows a group of silica bodies (dark content) in epidermal cells of stem forming a "carapace.b.Section made with Ranvier microtome of Diamantina lombardii.Arrow shows many silica bodies (dark content) in epidermal cells.c.Weddellina squamulosa.Arrows shows silica bodies in epidermal cells of scale-like leaf attached to stem.d.Section made with rotary microtome of D. lombardii.Arrow shows a removed silica body from epidermal cell.e. D. lombardii.Arrow showing a silica body with the holes oriented toward the external surface of plant.

Table 1 -
Shape, ornamentation, occurrence and size of silica bodies from three species of Podostemaceae, one representing each subfamily.Data are mean ± standard deviation; minimum and maximum values in parentheses (n=20).