Elaphoglossum ( Dryopteridaceae-Fern ) of Amazon Rainforest in Brazil : Anatomic Characterization and Adaptative Strategies *

This study describes the anatomy of sterile leaves of Elaphoglossum discolor (Kuhn) C. Chr., E. flaccidum (Fée) T. Moore and E. laminarioides (Bory ex Fée) T. Moore, the most representative species of the genus in the Ecological Park of Gunma in Pará State. It reports the main diagnostic characters and provides new systematic data for the group. In addition, it locates the production and accumulation sites of bioactive compounds to determine possible adaptive strategies of these species in the Amazon rainforest environment. Diagnostic structural features include stoma typology, central veins and margin forms, type of mesophyll, and the presence of schlerenchymatous sheaths in the cortex, among others. Among the bioactive compounds related to defense adaptation are phenolic compounds, which occur in all three species, and alkaloids and mucilage, which are exclusive to E. laminarioides. Of the three species studied, E. laminarioides has features that make it the best suited to the rainforest environment.


Introduction
Because many of the features of Elaphoglossum Schott ex Smith do not allow for the diagnosis of sterile individuals, it is one of the fern genera with the greatest need for further studies [1,2].In fact, most infrageneric classifications are based on morphological characters such as plant size, blade form and size, and scale color and type [2][3][4][5][6].However, most of these characters are only relevant when plants are in their reproductive stage [3].
Elaphoglossum grows in primary formations and is sensitive to environmental changes.Some species may be resistant for some time in human modified habitats, but different light and moisture conditions have considerable influence on their morphology [7].
Since the 1970s, these species have been mentioned in floristic studies in the Amazon [8][9][10][11][12][13][14][15].Their constant presence in inventories is due to different adaptation strategies developed during their evolution, especially in the Amazon rainforest environment.Such strategies in-clude various phytochemical defenses, high resistance to diseases in moist environments, high tolerance to acute nutrient imbalance in substrates, plasticity to adapt to different ecological opportunities, mycotrophic interacttions [16] and the epiphytic habitat, in which Elaphoglossum prevails [17].
The scarcity of studies on fern anatomy hinders the understanding of their structural organization.Among the main anatomical studies carried out on this genus are [18], who analyzed rhizomes and leaves of Jamaican species; [19] who described the leaf anatomy of Elaphoglossum in Tucumán, Argentina and [20,21], who addressed leaf architecture of Argentinean species by focusing on their induments.None of these studies examined Brazilian species, indicating the lack of the knowledge about their anatomy and possible relationships to the environments where they grow.
The aim of this study is to characterize the anatomy of sterile leaves of E. discolor, E. flaccidum and E. laminarioides, listing their main diagnostic characters and providing new data on the systematics of this group, in addition to identifying production and accumulation sites of bioactive compounds, in order to determine possible adaptation strategies of these species in the Amazon rainforest environment.

Plant Materials
Botanical material was collected in the Ecological Park of Gunma (PEG) located in Santa Bárbara do Pará (01˚13'25''S, 48˚17'40''W), Pará State, Brazil.The area is covered by lowland Amazon forest with a maximum altitude of 100 m above sea level.Adult individuals with totally unwound leaf blades were collected.The vouchers of the fertile material deposited in the herbarium MG are: E. discolor (MG 195547;MG 193856), E. flaccidum (MG 195545;MG 193858), and E. laminarioides (MG 195548;MG 195551;MG 195552;MG 193852).

Field Observations
Elaphoglossum discolor and E. flaccidum were always found and collected in shady areas within the forest, while E. laminarioides was found at the forest edges, more exposed to the sunlight.Although the individuals of the latter presented many leaves that seemed to have been chewed, no visitors were observed during our ten excursions to the field over two years.

Light Microscopy and Histochemical Tests
Leaves were fixed in FAA 70 for 24 h [22], and in neutral buffered formalin [23] and formalin-ferrous sulphate [22] for 48 h.Leaf samples (n = 7) were dehydrated in a tertiary butanol series [22] and embedded in histological paraffin.Leaves were cut into sections with a Leica RM 2245 microtome and stained in safranin and Astra blue [24].

Scanning Electron Microscopy (SEM)
The samples fixed in FAA 70 were dehydrated in an ethanol series [22], critical point dried with CO 2 [35] and metallized with gold for SEM analyses.Images were cap-tured with an LEO 1450 VP electron microscope.Frontal views of epidermis were observed in a scanning electron microscope because chemical and mechanical dissociation techniques were unsuccessful.

Leaf Blade
In frontal view, the epidermis of each of the three species presented sinuous, thin anticlinal walls on both faces (Figures 1(a) and (b)), with subtle cuticular striations (Figure 1(c)), as well as fungal hyphae along the surface.All species were hypostomatic (Figures 1(a) and (b)).Stomata are on the same level as the other epidermal cells (Figures 3(a) and (e)).In E. discolor and E. flaccidum, stomata were exclusively polocytic (Figures 1(d) and (e)), while E. laminarioides presented anomocytic (Figure 1(g)) and copolocytic stomata (Figure 1(h)) associated to polocytic (Figure 1(f)) stomata.Cross sections of the epidermis of each species showed that it consists of one layer of cells covered with a thin cuticle (Figures 2(b), (f) and (j)).However, in E. laminarioides, the outer periclinal walls are prominent on both faces (Figure 2

(j)).
In cross sections, the midrib differed according to species: in E. discolor, both the adaxial and abaxial faces (Figure 2(a)) are similarly concave; in E. flaccidum both faces were concave, but the adaxial one was more pronounced (Figure 2(e)) and in E. laminarioides, the adaxial face is flat (Figure 2(i)).The midrib also revealed a pattern of the structural organization of the studied species: schlerenchymatous cells subjacent to the epidermis of both faces formed an arch comprised of 2 -6 layers (Figures 2(b), (f) and (j)) with the rest filled with parenchymatous tissue.The midrib enclosed two large and one small vascular bundles, each of which was surrounded by a cortical schlerenchymatous sheath (Figures 2(c), (g) and (k)), which was thicker in E. laminarioides.Adjacent to this sheath was the endodermis, followed by a 2-layered pericycle (Figures 2(d) and (h)).Vascular bundles were bicollateral, with curved xylem at their tips.

Petiole
Cross sections of the petiole were cylindrical (Figures 4(a), (e) and (i)), but concave on the adaxial surface of E. laminarioides (Figure 4(i)).The epidermis of all species was formed by thin-walled rounded cells (Figures 4(b),  (f) and (j)).The cortical region, which comprised 6 -10 strata of schlerenchymatous cells, is more evident in E. discolor and E. flaccidum.Below this strata, the rest of the cortex was filled with parenchymatous tissue.Unlike E. laminarioides (Figure 4(k)), the cortex sheaths of E. discolor and E. flaccidum were inconspicuous (Figures 4(c) and (g)) in both the medial and basal regions of the petiole.The petiole vascular system was similar to that described for the midrib of the taxa, with the only difference being that it presented five vascular bundles at its basal portion (Figures 4(d), (h) and (l)), which merged to three bundles as they approached the midrib median region.

Secretory Tissues
In all the species analyzed, the secretory tissues were epi-Copyright © 2013 SciRes.AJPS

Discussion
The species analyzed showed significant variation in their structural characters, especially leaf blades and histochemical features.Based on our results, E. laminarioides was the most different in terms of structural variation and the highest number of bioactive compounds.Some of the leaf structural features studied, such as sinuous epidermis and hypostomatic leaf blades, concur with the descriptions by [18] for E. latifolium, E. muscosum, E. pallidum and E. villosum and by [20,21]  While polocytic stomata predominated in all species, they were associated to copolocytic and anomocytic stomata in Elaphoglossum laminarioides.Although polocytic and copolocytic stomata are among the five types that can be found in Elaphoglossum [36], anomocytic stomata are not described for this genus.[37,38] did not correlate this stoma type with those found in E. laminarioides, but Sen and De (1992) asserted that polocytic stomata are never associated to anomocytic ones.Nevertheless, latter studies carried out by [20,21] identified anomocytic stomata in Argentinean species of Elaphoglossum.This variation in stomata typology is relevant since it can be used as an infrageneric diagnostic feature.
As only E. flaccidum presented branched cells and rostriform margins, mesophyll and leaf margins are also useful infrageneric features.[39] stated that homogeneous mesophyll with branched cells occurs only in Pteridaceae species that grow in shady habitats.Since this is the case of E. flaccidum, the present study corroborates their assertion.
One of the most common features mentioned in anatomical studies on ferns is their lignified schlerenchymatous sheath in the cortex [40][41][42][43][44][45][46], which only encloses the petiole vascular bundles in E. laminarioides.It is considered a specialized character found in specimens of most of the derived families, such as Dryopteridaceae [45].
Although no visitors were observed in loco, many leaves were chewed and pathogens such as fungal hyphae were observed on leaf epidermis.This is likely due to the presence of lipids, which are considered nutritionally important for different classes of visitors and pathogens [47].
The starch grains observed on the leaf blade of the studied species are believed to be transitorily stocked in the chloroplast during the day and degraded at night to maintain plant metabolism [48].
Although we know that epiphytes are more vulnerable to water stress [49], E. laminarioides has always been collected in areas more exposed to the sun.The muci-  laginous compounds found exclusively in this species thus serve to retain water and protect the plant against herbivory [50], since some chewed leaves were observed.When [51] studied fungus-insect-plant interactions, they observed that insects feed on the fungal hyphae present on the plants (mycophagy), which could explain the oc-currence of both chewed leaves and fungal hyphae on leaf surfaces.Phenolic compounds are known components of defensive adaptations.According to [52], they present pharmacological and antinutritional properties that inhibit lipid oxidation and the proliferation of fungi.[53] asserted that they are involved in adaptation processes as a defense against radiation and pathogenic aggressions.
Likewise, alkaloids, found only in E. laminarioides, are toxic and act as a defense against herbivores and parasites, in addition to playing an allelopathic role [54].Although no Elaphoglossum monospecific formations were observed in the field (which could lead us to induce some kind of dominance), the potential allelopathic activity of many fern genera has been proved, e.g. by [55] for Pteridium Gled.ex Scop., and [56] for Dicranopteris Bernh., Gleichenia Sm. and Sticherus C. Presl.
Thus, the structural data reported constitute new characters of the representatives of Elaphoglossum in vegetative stage.The bioactive compounds identified in situ also provide important unpublished data, adding information on the species and suggesting the development of adaptive strategies.This could be observed especially on E. laminarioides, which in addition to the compounds of communal occurrence on the species studied, was the only species presenting alkaloids and mucilage.These compounds possess peculiar properties related to herbivore and pathogen avoidance, demonstrating that E. laminarioides is possibly better adapted to its environment, producing secondary metabolites that are more varied and directly related to adaptive success.