Structural and histochemical aspects in leaves of six species of Anemia (Anemiaceae) occurring in rocky outcrops

: Rocky outcrops are known for low humidity, rainfall and high solar radiation, factors that limit the development of some vegetables. However, some species of the genus Anemia occurring in these environments. Thus, understanding the anatomical characters present in these vegetables are important for botanical and biodiversity knowledge in rock fi elds. We described the leaf anatomy of six species of Anemia to identify characters adapted to rocky outcrops for ferns. Herbarium samples were rehydrated. Field-collected leaves, were also sampled, the material was subjected to standard anatomical study by light microscopy, and secretions were evaluated by histochemical of the secondary compounds, with ruthenium red, tannic acid, ferric chloride, lugol, Sudan black B, vanillin/hydrochloric acid, Dragendorff’s reagent and ponceau xylidine. Histochemical tests were positive for phenolic compounds, alkaloids, polysaccharides, and proteins in A. buniifolia , A. oblongifolia , A. presliana , and A. trichorhiza . Our fi ndings revealed that several structural and histochemical characters of Anemia with trichomes, conical stegmatas, phenolic compounds, mucilages and alkaloids are related to reducing water loss, providing an adaptive value to species in extreme environments, such as rocky outcrops, in addition to new data relevant to the group taxonomy, such as the presence of amphistomatic leaves in A. trichorhiza .


INTRODUCTION
A nemiaceae is a pantropical family of ferns comprising ca.115 species in a single genus, Anemia Sw. (Mickel 2016).Most of its species are confi ned to the Neotropical region, with few reaching Africa, Madagascar, southern India, and the Indian Ocean Islands (Mickel 2016).Species of Anemia frequently occupying xeric environments such as dry tropical forests and savannas (Hietz 2010), from open and dry areas, to the terry environment to rocky outcrops (Mickel 2016).This habitat is characterized by exposure to severe environmental conditions determined by limiting abiotic factors, such as intense sunlight incidence, high temperatures, hydric stress, and low nutrient availability (Biedinger et al. 2000).In addition, rocky outcrops represent refuges for endemic, threatened, and geographically disjointed species, commonly being the source of new species discovery (Oliveira & Godoy 2007, Silva 2016).
O ver the last two decades, few studies have focused on rocky outcrops ferns (Xavier & Barros 2003, Santos & Sylvestre 2006, Santos et al. 2006, Ribeiro et al. 2007, 2011, Silva 2016).Ribeiro et al. (2007) analyzed the leaf anatomy of A. tomentosa var.anthriscifolia and A. villosa Humb.& Bonpl.ex Willd., evaluating the species' adaptive strategies to hydric stress in rocky outcrops.Despite the importance demonstrated by these studies' results, additional studies on the leaf anatomy of Anemia might reveal different characters adapted to hostile environmental conditions (Oliveira & Godoy 2007).Anatomical traits related to such conditions have been poorly studied for ferns (Ribeiro et al. 2007), with rare reports for plasticity on ecologically adaptative characters (Bradshaw 1965).Another few investigated field in Anemia's anatomy is the diversity of secretory structures and its compounds' chemical nature, which may present an untapped potential as new sources of natural products of economic importance (Santos et al. 2006, Ribeiro et al. 2007).
Thus, in order to identify anatomical structures of Anemia and interpret its presence in the environment of occurrence, we intend to comparatively analyze the leaf structure of six species and the histochemistry of four species of Anemia occurring in rocky outcrops.

MATERIALS AND METHODS
To contemplate the largest number of Anemia specimens, selection considered the availability of living specimens from field expeditions and herborized specimens deposited at the MG herbaria.Fully expanded leaves of A. buniifolia, A. oblongifolia, A. presliana, and A. trichorhiza were collected from natural populations at Serra of the Martírios/Andorinhas State Park (PESAM).This integral protection area is located in the municipality of São Geraldo do Araguaia, southeastern State of Pará (06°03'00" to 06°23'00" S, 48°22'30" to 48°36'30" W).In this location, the average monthly rainfall is less than 60 mm (Martorano et al. 1993).We additionally complemented our sampling with herborized specimens of A. elegans and A. phyllitidis from the MG herbarium.All information, including species authorship and voucher specimens, is summarized in Table I.
Herbarium samples from 18 specimens (fragments from the: 1. median portion of the leaf-blade's interveinal region, 2. midrib, 3. margin, 4. petiole, and 5. rachis) of all six samples were rehydrated according to the methodology of Smith & Smith (1942) which consists of boiling the material in water for 5 min or until submerged, then placing 2% potassium hydroxide for 30 minutes, washing several times with distilled water at intervals of 30 min, dehydrate in ethylic series from 20% -60% and at the end stored in 70% ethanol.They were then dehydrated in a decreasing ethanol series and embedded in 2-hydroxyethyl methacrylate resin for sectioning (Historesin Leica®, solutions were prepared according to manufacturer's instructions) following Meira & Martins (2003).These samples were transversely and longitudinally sectioned from 3 to 7 μm thick.Sections were stained with toluidine blue pH 4.6 (O'Brien et al. 1965), and slides were mounted in resin (Permount®, Fisher Scientific, New Jersey, USA).Some samples were also cleared with 5% sodium hydroxide and 20% hypochlorite solutions, stained with 50% ethanol-diluted fuchsin (Shobe & Lersten 1967), and mounted in glycerinated gelatin (Only samples collected Kaiser 1880).
In the field were used for histochemical tests Table III.Three fixative treatments were used for three different sets of samples.The first set was fixed in FAA formalin: acetic acid: 50% ethanol, 1:1:18 by volume, Johansen 1940) for 24 h for both structural characterizations in light microscopy and histochemical hydrophilic substances.The second set was fixed in neutral buffered formalin (NBF) for 48 h (Lillie 1965) to preserve lipophilic substances.The third was fixed in FSF ferrous sulfate in formalin (Johansen 1940) to detect total phenolic compounds.Part  (2022) 94(3) e20210392 3 | 15 of each sample set was also dehydrated in a tert-butanol series (Johansen 1940), embedded in histological paraffin with DMSO (Paraplast® Embedding Medium, Oxford Lab., USA), and serially sectioned at 10-12 μm thick (Kraus & Arduin 1997).
The histochemical tests were only performed on field-collected samples of A. buniifolia, A. oblongifolia, A. presliana, and A. trichorhiza.All species have idioblasts dispersed in their leaf tissues, besides secretory structures and glandular trichomes (Fig. 3a-3l).
For standard control, a sample without reagent was made to compare the histochemical test in each reaction.Slides were mounted in glycerinated gelatin (Kaiser 1880).Sections were obtained with a rotary microtome (model RM 2245, Leica® Biosystems, Heidelberg, Germany) using tungsten knives (Leica® Biosystems).Observations and photographic documentation were performed with a light microscope (Axio Scope, A1, ©Carl Zeiss, Göttingen, Germany) equipped with a digital camera (AxioCam HRc, ©Carl Zeiss).Macro images were obtained using a stereomicroscope (SteREO Discovery, V8, ©Carl Zeiss) coupled to a digital camera (AxioCam ICc5, ©Carl Zeiss).Anatomical descriptions were made using the terms adopted by Brasil ( 2009) contour of the Petiole and leaf, Mickel & Lersten (1967) for stomata typology, by Metcalfe & Chalk (1950), Ogura (1972), and Fahn (1990) for trichomes, and by Ogura (1972) for the vascular system.

Petiole
The petiole contour is convex plane (Fig. 1a) in transversal section in A. elegans and A. trichorhiza, slightly square (Fig. 1b) in A. oblongifolia and A. presliana, and cordate (Fig. 1c) in A. buniifolia and A. phyllitidis.All species showed cuticle (Fig. 1f ) and unistratified epidermis (Fig. 1h), with stomata low frequency observed above the epidermic cells in A. buniifolia, A. oblongifolia, A. phyllitidis and A. presliana forming the forming aerophore (Fig. 1d).Glandular trichomes A. elegans, A. phyllitidis and A. trichorhiza (Fig. 1e) were identified, but non-glandular trichome was observed in all species (Fig. 1f).Conic stegmata (Fig. 1g) on periclinal walls of the epidermis were only identified in A. oblongifolia, A. presliana, and A. trichorhiza.All species have he cortex with 1-5 layers of sclerenchyma (Fig. 1l), 5-13 layers of parenchyma (Fig. 1b), except for A. elegans, in which the cortex only shows parenchymatous cells.Pericycle is formed by 1-2 layers of parenchymatous cells in all species, except for A. phyllitidis, in which it may have up to 3 cell layers.The vascular system is amphicribal (Fig. 1h), arranged in an arch in all species.

Rachis
The leaves of A. buniifolia and A. elegans lack a rachis.In contrast, the rachis of A. phyllitidis was cordate (Fig. 1i) in transversal section, cylindrical (Fig. 1j) in A. oblongifolia and A. trichorhiza, and slightly square (Fig. 1k) in A. presliana.All species present slender cuticle (Fig. 1k), unistratified epidermis (Fig. 1j), and glandular trichome (Fig. 1e), except for A. trichorhiza that trichomes.Additionally, all species have a cortex of 1-7 layers of sclerenchyma (Fig. 1l), followed by 3-17 layers of parenchyma (Fig. 1m) (Table II).The pericycle has 1-2 cell layers in A. oblongifolia and A. trichorhiza, while in A. phyllitidis and A. presliana it may have up to 3 layers (Table II).The vascular system was amphicribal and arranged in an arch, except for A. trichorhiza, which was arranged in a "V".

Leaf-blade
All species have both surfaces of the epidermis (Fig. 2a-b) with sinuous anticlinal walls in frontal view.Non-glandular (Fig. 2c) and glandular (Fig. 2d) trichomes occur in all species, except for A. buniifolia that lacks glandular trichomes.Floating stomata (in surface view, they appear surrounded by an annular subsidiary cell, without coming into contact with any of its anticlinal walls (Mickel 1962)) (Fig. 2e) are present only on the abaxial surface of leaves of A. buniifolia, A. oblongifolia, A. phyllitidis, and A. presliana.Alternatively, in A. elegans, they only occur on the adaxial surface, and in A. trichorhiza, they appear on both surfaces (Fig. 2l) (amphistomatic leaves).Suspended stomata (presents intruded wall of surrounding epidermal cell (Mickel & Lersten 1967)) (Fig. 2f) were also observed on the abaxial surface only of A. buniifolia.
The leaf-blade of all species have epidermal characters similar to petioles, with conical stegmata observed only in A. oblongifolia, A. phyllitidis, A. presliana, and A. trichorhiza.The mesophyll of A. elegans is homogeneous with a lacunose parenchyma.The other species, dorsiventral mesophyll.The midvein is concaveconvex (Fig. 2k) with the vascular system comprising amphicribal bundles in A. buniifolia, A. phyllitidis, and A. trichorhiza.Even though a midvein was absent in A. elegans, A. oblongifolia, and A. presliana, their vascular bundles are also amphicribal (Fig. 2l).A sheath extension made of parenchymatous cells with thickened walls was only recorded in A. elegans.Alternatively, A. phyllitidis and A. trichorhiza have a sheath extension made of parenchymatous cells with thickened walls plus sclerenchymatous cells near the epidermis (Fig. 2m).

Secretory structures
During the collection expedition, no secretion was observed macroscopically on the leaf surfaces of the species.Histochemical tests were performed on petioles, rachis, and leaf-blades, with positive results for phenolic compounds (Fig. 3a-c.),alkaloids (Fig. 3d-e), acidic mucilage (Fig. 3f-h), starch, and total proteins (Fig. 3k-l) in A. buniifolia, A. oblongifolia, A. presliana, and A. trichorhiza.The same species showed negative results for total lipids, tannins, and neutral mucilage.

DISCUSSION
Ferns may exhibit morphological patterns that indicate ecological plasticity in response to heterogeneous environments (Arens 1997).This plasticity might explain their adaptive success to diverse conditions (Bradshaw 2006), such as the limited supply of water and nutrients, high irradiation of sunlight, and wide temperature variations, as observed in rocky outcrops (Burke et al. 2002).
All six studied species of Anemia showed common leaf epidermal cells with sinuous anticlinal walls, corroborating a pattern already reported to rocky outcrop species (Pant & Khare 1972, Ribeiro et al. 2007, 2011).The sinuosity of anticlinal epidermal cell walls may be related not only to a high incidence of solar radiation (Wilkinson 1979, Graçano et al. 2001) but also to low water availability.This sinuosity is associated with leaf expansion/contraction due to water inflow/outflow, which may lead to the development of mechanical adaptations that help prevent the collapse of the organ (Krauss 1949), such as rocky outcrops species of Anemia.The presence of sinuous walls confirms that environmental factors influence this character's plasticity, the primary way plants react to their habitat's heterogeneity (Bradshaw 1965, Barboza et al. 2006, Valladares et al. 2007).
The amphistomatic leaf-blade found in A. trichorhiza is considered a rare trait among ferns (Kramer 1990).This character state is recorded in this study for the first time for Anemia.Stomata occurrence on both leaf surfaces is considered a xeromorphic feature (Parkhurst 1978) wich allows for higher carbon dioxide conduction and increased photosynthetic capacity, both of which provide adaptive advantages to plants living in environments subjected to high sunlight incidence, establishing itself as pioneers in the process of ecological succession (Mott et al. 1982).Amphistomatic leaves are also observed in Angiosperms, being common mainly in Asteraceae that occur in open areas, of high light intensity (Silva et al. 2019, Vieira-Neto et al. 2020), so this is an important feature for kind of open environments (Liesenfeld et al. 2019).However, studies suggest that photosynthetic efficiency is Figure 3a-3l.Anemia oblongifolia (g, j), Anemia presliana (a, h) and Anemia trichorhiza (b, c, d, e, f, i, k, l).Petiole (b, d, h, i, j, l) and leaf-blade (a, c, e, f, g, k).Results of histochemical tests applied to the secretory product of the foliar secretory structures of Anemia species, a-c) Formalin with ferrous sulphate: Total phenolic compounds, notice the secretory product in the glandular trichomes (arrow), d-e) Dragendorff's reagent: Alkaloids (arrow), f-h) Ruthenium red: Acid mucilage, notice the secretory product in the glandular trichomes (arrow), i-j) Lugol: Starch grains, k-l) Ponceau Xylidine: Total proteins, notice the secretory product in the glandular trichomes.Bars: 30 µm (a, d, g, j, k), 50 µm (b, c, h, i, l).related to a regular gradient of carbon dioxide in the leaf and the presence of stomata on both leaf surfaces is not favorable to this gradient.Thus, stomata on both leaf surfaces do not contribute to the efficiency of photoassimilate production in arid environments (Mcelwain et al. 2005).
The epistomatic leaf-blade, which we found in A. elegans, is an uncommon trait in Anemia.Hypostomatic leaves, on the other hand, such as those found in A. buniifolia, A. oblongifolia, A. phyllitidis, andA. presliana, are more common (Ogura 1972, Ribeiro et al. 2007).
Although floating stomata have been initially described based on A. phyllitidis (Link 1841), they are not widely distributed in Anemia, being absent in A. adiantifolia (L.) Sw. and A. villosa Humb.& Bonpl.ex Willd.(Mickel & Lersten 1967).On the other hand, floating stomata are not exclusive to Anemia, having also been reported to genera of Polypodiaceae and Salviniaceae, such as Lemmaphyllum C.Presl (Kondo 1962), Pyrrosia Mirb.(Mickel & Lersten 1967), Pleopeltis Humb., and Azolla Lam.(Inamdar et al. 1971).From an evolutionary perspective, floating stomata are possibly specialized structures (Mickel 1962), yet no specific functional or ecological role has ever been attributed to them (Mickel & Lestern 1967, Inamdar et al. 1971, Sen & De 1992).In xerophyte plants, stomata are typically sunk in epidermal depressions, which protect the plant against excessive water loss (Beck 2010), in the opposite hand, the Anemia analyzed presents stomata exposed to edaphyte factors, making them more susceptible to water stress, on the other hand, it can be assumed that different biochemical and physiological mechanisms are found in plants that undergo water scarcity (Bohnert et al. 1995), as observed in Drymoglossum piloselloides (L.) Presl and Pyrrosia longifolia (Burm.)Morton, in which biochemical studies confirmed the presence of CAM metabolism (Wong & Hew 1976), reinforcing the presence of this type of metabolism in some ferns (Evert & Eichhorn 2014), responsible for increasing water use efficiency (Bohnert et al. 1992).Therefore, we infer a probable relationship that ferns with floating stomata, among them Anemia, present CAM metabolism, as an adaptive strategy to water stress, since no morphological adaptations were observed strong enough to withstand long periods of drought.Further in-depth analyses of their ecophysiological role, especially in extreme environments, are needed.
Glandular and non-glandular trichomes are common among species of Anemia (Roux et al. 1992, Ribeiro et al. 2007, 2011).Both are considered xeromorphic characters, as they play a significant role in preventing water loss (Gibson 1996) by regulating the temperature and reflecting the excess radiation (Larcher 2000).It also optimizes the absorption of atmospheric humidity by improving water retention on the leaf surface (Hietz & Briones 1998).Additionally, Manetas (2003) proved that trichome can protect tissues from uv-b damage.These characteristics reinforce the probable role of reducing the sweating played by trichome (Barros & Soares 2013).
In terms of plant-animal interactions, trichomes also contribute to reducing herbivory (Dickison 2000).Calo et al. (2006) cited that trichomes or other external leaf structures act as a physical barrier against herbivore attacks.In contrast to this statement, Eisner et al. (1998) state that this type of protection depends on this structure's morphology, such as the hooked trichomes of Mentzelia pumila var.pumila (Nutt.)Torr.& Gray, capable of trapping and killing some arthropod species that come into contact with the leaf.In that sense, it should be noted that no visitor was seen during field collections.A more extensive study on the existence of possible plant-animal interactions would be worthwhile.
Stegmata are characterized by a thickened wall adjacent to the underlying sclerenchyma cells, with progressively thinner lateral walls and thin outer walls with silica bodies inside,are observed in families of monocotyledons Orchidaceae, Arecaceae, Bromeliaceae and Cyperaceae (Prychid et al. 2004), and apparently typical in Trichomanes L. (Mettenius 1865).
Conical stegmata, found on petioles and external periclinal walls of common epidermal cells in Anemia, are variably shaped (i.e., conical, elliptical, or spherical) and genetically regulated, being little influenced by environmental factors (Møller & Rasmussen 1984).These structures are restricted to some plant groups, such as Anemia, in which its occurrence on the leaf-blade epidermis might be related to the reduction of water loss (Campos & Labouriau 1969, Ribeiro et al. 2007), representing a xeromorphic trait associated with the efficiency of water intake (Zanenga-Godoy & Costa 2003).In animal-plant interactions, stegmata also contribute to the defense against microorganisms and smaller herbivores, blocking the latter's urinary trait (Vicari & Bazely 1993).
Some types of anatomical characters in the mesophyll can optimize the photosynthetic process due to environmental variations (Erbano & Duarte 2010).The most accepted hypothesis is that mesophyll architecture is determined by light intensity, being a highly plastic feature (Arens 1997), hypothesis corroborated by our findings, where the type of mesophyll varied according to light intensity.The homogenous-type mesophyll with spongy parenchyma found in A. elegans commonly occurs in species growing in shaded environments (Graçano et al. 2001), as it allows for optimized use of solar radiation (Larcher 2000), increasing photosynthetic efficiency (DeLucia et al. 1996).On the other hand, the dorsiventral mesophyll usually occurs in species of Anemia from rocky outcrops (Ribeiro et al. 2007(Ribeiro et al. , 2011)).
The occurrence of the dorsiventral mesophyll in ferns is closely related to environments subjected to high sunlight incidence (Arens 1997), with the plicate parenchyma favoring increased photosynthetic rates due to its high concentration of chloroplasts (Queiroz-Voltan et al. 2011).
The synthesis of secondary metabolites in plants represents a necessary physiological process that might their effect intensified when these compounds interact, hindering development and resistance in the event of simultaneous attacks by herbivores and microbes (Wink 2010).These properties make secondary metabolites more efficient in biologically defending plants (Wink 2008).It is noteworthy that intrinsically correlated abiotic factors may jointly affect the secondary metabolism of the plant.Examples of such factors may include seasonality, rainfall, temperature, and altitude (Gobbo-Neto & Lopes 2007).
Phenolic compounds are usually related to different adaptive strategies like protection against drying and attack by animals (Feio et al. 2013), being commonly found in ferns (Ogura 1972) and xerophytes from other plant groups (Pyykkö 1966).In both groups, their production increases under unfavorable conditions, therefore acting as a stress bioindicator (Siddiqui & Arif-uz-Zama 2004, Achakzai et al. 2009).Phenols are essential for the adaptation of plants to terrestrial environments, acting in the defense not only against fungal proliferation (Croteau et al. 2000, Taiz & Zeiger 2006) but also against herbivory by providing plants with unpleasant tastes and smells that prevent them from being attacked (Strack 1997).Furthermore, phenols have also been reported to play a significant role in protecting plants against drought and high sunlight intensity (Waterman & Mole 1994).
The presence of alkaloids, which we found in all species analyzed, has been under studied among seedless plants.However, such a rarity could be due to the scarcity of fern studies that include histochemical analyses on this metabolic class (Evans 1996, Watson et al. 2001, Feio et al. 2013).Aside from reducing palatability, alkaloids are also related to the defense against herbivores and parasites (Vicari & Bazely 1993, Facchini 2001).They also play an allelopathic role (Robinson 1974), frequently found in increased amounts on plants under some kind of stress (Vicari & Bazely 1993).They represent a more economical strategy than defoliation in nutrient-poor environments (Gershezon 1983), such as rocky outcrops.
Mucilages are natural constituents of the plant body, being more common in organs with water-retention function (Simões et al. 2000).Due to their physical characteristics, they might aid in leaf extension, increasing the water supply during hydric stress (Thadeo et al. 2009).These mixed-nature compounds are pivotal for protecting developing organs, acting in the defense against herbivores and tolerance against desiccation (Gregory & Baas 1989).Furthermore, proteins, in addition to being crucial components in the metabolism and structure of plants, (Thadeo et al. 2009) can also act together with tannins against agents in protection against herbivores and pathogens ( Klein et al. 2004, Markham et al. 2006, Miguel et al. 2006).Its presence in these structures can (Thadeo et al. 2009).
Although naturally found in the plant cell protoplast, starch has been suggested to have a storage function in the leaves of A. villosa (Ribeiro et al. 2007).Additionally, starch may also be related to the prevention of mechanical damage to the cell membrane during hydric stress (Vicré et al. 1999) since it occurs in tissues of desiccation-tolerant seeds.Feio et al. (2013) indicated that in species of Elaphoglossum Schott ex Smith, the starch grains found on the leaf-blade might be transiently stored in chloroplasts during the day and degraded at night to maintain the metabolism in plants under stress.The stage of development, hydric stress, and leaf loss may alter the amount and regions where starch is found in plant structures.This polysaccharide was observed in the rhizome of the morphological and histochemical study of Adiantum latifolium Lam.(Pteridaceae) (Da Cruz et al. 2019).
Histochemistry in representatives of Anemiaceae or closely-related groups is still incipient.The absence of studies focusing on these groups means that the discussions are new and often based on unrelated groups.However, taxonomic studies can be useful for a preliminary analysis of the identification of secretions in secretory structures (Vizzotto et al. 2010).
Despite the environmental restrictions imposed by the rocky outcrop habitat, anemia species are well adapted to this type of environment, adaptive success is related to the presence of anatomical characters with trichomes and conical stegmatas and histochemical characters such as phenolic, mucilages and alkaloids, which in addition to reducing water loss, protect against herbivory, which suggests the development of adaptive strategies for this type of environment.The present study also provides new data relevant to the taxonomy of the group, such as the presence of ampyrtomatic leaves in A. trichorhiza that increase knowledge about the leaf anatomy of Anemia.
PEDRO G. DE MORAES NETO et al.LEAF ANATOMY OF Anemia (ANEMIACEAE)An Acad Bras Cienc TableII.Number of layers of parenchyma cells, sclerenchyma cells, endoderm and pericycle in the petiole and rachis, (-) inapplicable.

Table III .
Results of histochemical tests applied to the secretion of leaf structures present in Anemia species.Notes: (+) positive result; (-) negative result.