121 Comparative Morphology and Anatomy of the Leaf and Stem Of

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Núm. 42, pp. 121-136, México, 2016 DOI: 10.18387/polibotanica.42.6

Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum (Rutaceae) from central Argentina

Morfología y anatomía comparada de la hoja y el tallo de las especies de Zanthoxylum (Rutaceae) del centro de Argentina

Antonia J. Oggero1; Marcelo D. Arana1, y Herminda E. Reinoso2 1Orientación Plantas Vasculares, 2Orientación Morfología Vegetal, Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico Químicas y Naturales, Universidad Nacional de Río Cuarto, X5804ZAB, Río Cuarto, Córdoba, Argentina. Correo electrónico: [email protected]; [email protected]; [email protected]

Abstract whereas Z. coco is usually a deciduous tree with terminal inflorescences. Both species Zanthoxylum is one of the major genera of also share the presence of secretory cavi- Rutaceae with at least 200 species in tro- ties, glandular trichomes and hypostomatic pical and temperate regions worldwide. In leaves. However each species presents par- Argentina is represented by 10 species, two ticularities of the leaves structure that could of them inhabiting in the central region, the be important and related to the environment native Zanthoxylum coco and the naturalised that the plants develop. Zanthoxylum armatum var. armatum. These species are found in xerophytic deciduous Key words: Zanthoxylum armatum, Zan- forests of Chaco biogeographic province. thoxylum coco, xeric habitats, flora. Besides the floristic and ethnobotanical importance of these species, their leaves RESUMEN and stems have been scarcely studied. The objective of this contribution was to present Zanthoxylum es uno de los principales a comparative morphological and anatomi- géneros de Rutaceae con al menos 200 cal study of the leaves and young stems of especies de las regiones tropicales y temp- the species of Zanthoxylum from central ladas de todo el mundo. En Argentina está Argentina. The analysis was done using her- representado por 10 especies, dos de ellas baria vouchers, freshly collected and fixed habitan en la región central, Zanthoxylum material. The species share morphological coco, nativo, y el naturalizado, Zanthoxy- features, as armed stems, leaves alternate, lum armatum var. armatum. Estas especies odd-pinnately, 3- to many foliolate. The se encuentran en los bosques caducifolios main differences are that Z. armatum is an xerofíticos de la provincia biogeográfica evergreen shrub with axillar inflorescences del Chaco. A pesar de la importancia flo-

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Núm.42: 121-136 Julio 2016 rística y etnobotánica de estas especies, They are shrubs, trees, or sometimes herbs, sus hojas y tallos han sido escasamente sometimes scrambling or scandent, some- estudiados. El objetivo de esta contribu- times armed, with aromatic volatile oils ción es presentar un estudio morfológico y contained in glands visible at surface of, anatómico comparativo de las hojas y tallos at least leaves, young branchlets, inflores- jóvenes de las especies de Zanthoxylum del cences, flower parts, fruit, or cotyledons in centro de Argentina. El análisis se realizó seed. The genus Zanthoxylum L. is one of the utilizando ejemplares de herbario, material major genera of Rutaceae with at least 200 fijado y fresco. Las especies comparten species in tropical (mainly) and temperate rasgos morfológicos, como tallos armados, regions of the world (Figueiredo Melo & hojas alternas, imparipinnadas, 3- multifo- Zickel, 2004; Zhang & Hartley, 2008). It lioladas. Las principales diferencias son que includes shrubs sometimes scrambling, Z. armatum es un arbusto de hojas perennes trees, or woody climbers, evergreen or con inflorescencias axilares mientras que deciduous, usually armed. The leaves are Z. coco suele ser un árbol de hojas caducas alternate, odd-pinnately 3- to many foliolate con inflorescencias terminales. Ambas or sometimes digitately 3-foliolate. Zan- especies también comparten la presencia thoxylum is represented in Argentina by 10 de cavidades secretoras, tricomas glandu- species, two of them inhabiting in central lares y hojas hipostomáticas. Sin embargo, region of the country, Zanthoxylum coco cada especie presenta particularidades de Gillies ex Hook. f. & Arn. and Zanthoxylum la estructura de las hojas que pueden ser armatum DC var. armatum. These taxa are importantes y relacionadas al ambiente en the only species of Zanthoxylum found in el que las plantas se desarrollan. Pampean hills of central Argentina, which belong to the biogeographic province of Palabras clave: Zanthoxylum armatum, Chaco (Morrone, 2001; 2004). This province Zanthoxylum coco, ambientes xéricos, flora. is characterized by xerophytic deciduous forests of Vachellia caven (Molina) Seigler & Introduction Ebinger, Aspidosperma quebracho-blanco Schltdl., Celtis ehrembergiana (Klotzsch) The family Rutaceae Juss. is placed in Liebm., Lithraea molleoides (Vell.) Engl. order Sapindales primarily because of the and Zanthoxylum coco as predominant pinnately compound leaves, absence of resin species of trees. Although these species are ducts in the bark, wood rays, leaf veins, the studied in floristic, taxonomic and ethno- presence of triterpenoid compounds and flo- botanic treatments, there are very scarce wers with a distinct nectar disk (Cronquist, morphological and anatomical studies of 1992; AGP III, 2003; Judd et al., 2008) and vegetative organs of these species. includes species of great economic importance as ornamental plants, medicinal or Anatomy describes the internal structure culinary uses or perfume industry such as the of plants and is considered as a source of orange, lemon, lime, kumquat, mandarine correct identification of taxa. Anatomy and grapefruit. The Rutaceae are represented centres on the spatial arrangement of the in Argentina by seven genera and approxi- dermal, ground, and vascular tissue systems mately 18 species (Seo and Xifreda, 2008). (Nancy & Dengler, 2002). Similarly foliar

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Oggero, A.J. et al.: Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum, Central Argentina epidermal microscopic features like shape southwestern part of Cordoba province, of epidermal cell, type of stomata, presence Argentina (fig. 1). or absence of pubescence and cell wall thickness are also considered as useful The comparative morphological description tools for correct taxa identification and their of the leaves and stem was carried out using phylogenetic relationship with other taxa fresh material and herbaria specimens and (Stace, 1965; Babalola & Victoria, 2009). it is based in Escalante (1961) for Zan- thoxylum coco and Arana & Oggero (2009) Although Zanthoxylum coco is one of the for Zanthoxylum armatum. The herbaria most common species of trees in forests consulted were CORD, LIL, LP, SI, RCVC in central Argentina Hills and it is used as and RIOC, and digital images of the types medicinal plant (Battista et al., 2007), their (provided by G and K) were also seen. The leaves and stems have not previously been selected samples of the specimens collected studied. The shrub Zanthoxylum armatum were deposited in the Herbarium of Depar- is an important medicinal plant extensively tamento de Ciencias Naturales, Facultad de used, in the countries which is native, for Ciencias Exactas, Físico-Químicas y Natu- curing Pneumonia, tick infestation and rales, Universidad Nacional de Río Cuarto gum diseases (Iqbal & Hamayun, 2005), (RCVC) and registered as follows: fruit is used for toothache, dyspepsia, and seeds are used as condiment and flavoring Zanthoxylum armatum: agent (Arshad & Ahmad, 2005; Abbasi et ARGENTINA. Córdoba. Depto. Calamu- al., 2010). Recently the leaves and fruits chita, Embalse Cerro Pelado, A. J. Oggero were investigated for various pharmacolo- & M .D. Arana s.n. (RCVC); Villa Amancay, gical activities and has showed good results 14-III- 2009, M. D. Arana & A. J. Oggero including cytotoxic, phytotoxic, antipyretic s.n (RCVC); Depto. Río Cuarto, Alpa Co- activities and rationalise its local uses (Suks- rral, 30- III-2000, C. A. Bianco, s.n. (RCVC athan et al., 2009; Barkatullah et al., 2011) 3625). and in Argentina was found as naturalized species (Arana & Oggero, 2009). Keeping Zanthoxylum coco: in view the significance of morphological ARGENTINA. Córdoba. Depto. Calamuchi- and anatomical studies for taxonomy and ta, Embalse Cerro Pelado, 14-III- 2009, A.J. ecology, a comparative morphological Oggero & M.D. Arana s.n (RCVC); Depto. and anatomical study of the leaves and Río Cuarto, Sierra de Los Cóndores, 10- X- young stems of the species of Zanthoxylum 1997, M. Ceballos, s.n. (RCVC 3948). from central Argentina is carried out. Morphological studies Material and methods The analysis of the stem and leaf structure Plant material was done using freshly collected material or fixed material. The leaves located in The collections of the botanical material the nodes of the middle part of the newest and the field observations were carried out branchlets (there is one leaf per node in Sierra de Comechingones, located in the along the stem) were used to carry out the

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Fig.1. Location of Sierra de Comechingones (central Argentina) where the study was carried out.

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Oggero, A.J. et al.: Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum, Central Argentina anatomical study. The samples were taken The stomatal density, trichomes and (just from the central zone of the middle leaflet in case of Zanthoxylum coco) glands were and the rachis. To anatomical studies of counted using ten microscope fields (0.25 stem, the internodes (a portion of a plant mm2) on each epidermis of six leaflets. stem between nodes) of the middle region of the branchlets were collected. A standard Zeiss Model 16 microscope was used to assess the histological preparations The samples were placed in FAA (95% and photomicrographs were taken with a ethanol: glacial acetic acid: 37-40% for- Zeiss Axiophot microscope with an equip- maldehyde: water; 50:5:10:35, v/v). The ment of image capture and digitalization dehydration of samples was done according AxioVision 4.3, with camera AxioCam HRc. to the procedures outlined in Johansen (1940) using graduated solutions of ethanol Results and xylene. Fully infiltrated tissues were embedded in Histowax (highly purified Zanthoxylum armatum var. armatum paraffin wax blended with polymer additi- ves). A series of transverse sections 12 μm Young stem and leaves morphology thick were obtained from the sample blocks using a Minot rotary microtome. The sec- The plants of Zanthoxylum armatum are tions were triple-stained with hematoxylin, shrubs to 5 m tall, deciduous, with dark safranin O and fast green FCF as described brown cilindrycal stems, alternate phyllo- by Johansen (1940). To verify the presen- taxis and compound, imparipinnate leaves ce of starch, semi-permanent slides with (fig. 2b). Stems, branchlets and leaflet blades Iodine dissolved in an aqueous solution of abaxially on midvein with dark brown, stout Potassium iodide (Lugol´s Iodini solution) prickles with a broad flattened base. Young colored were made. branchlets and inflorescence rachises are glabrous or rust-colored pubescent. The Epidermal studies were carried out using leaves are 3-5(7) foliolate with rachis and leaflet samples fixed in FAA which were petioles glabrous or rust-colored pubescent, submerged in a solution 40% of sodium broadly winged, wings to 6 mm on each side. hypochlorite and heated to epidermal sepa- The leaflet blades are dark green coloured, ration. The epidermal samples were washed subsessile, opposite, narrowly elliptic (5-15 with water and safranin coloured and the × 1-3 cm), base attenuate to broadly cuneate, semipermanent slides for microscope using the terminal leaflet is the largest. The mar- water-glycerine (1:1) solution were made, gin is crenate, finely serrulate or entire and according to the usual techniques. revolute when dry, apex acute to acuminate, with the midribs impressed above, elevated The stomatal index (SI) of each taxon was beneath. The lateral veins are 6-7 on each calculated from five fields in the middle side of the midrib with pellucid gland on region on lower surface of the blade of six margins. leaves taken from different plants, using the formula proposed by Cutter (1969).

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Fig. 2. Newest branchlets of Zanthoxylum (a) Z. coco and (b) Z. armatum.

Anatomy of leaves and young stems The rachis is winged (fig. 3d). The epidermis of the wings is similar to the leaflets but it The leaflets are dorsiventral. In transverse is observed a difference in the proportion section the epidermis of adaxial surface pre- of the palisade tissue, which represent less sent isodiametric cells bigger than the abaxial than the 50% of the mesophyll (fig. 3d). The surface. The stomata are present in abaxial central zone of the rachis presents a pith surface and are at the same level of the surroun- surrounded by xylem and phloem, where se- ding cells. The palisade tissue is unilayered, condary growth is observed. The secondary represents the 50% of the mesophyll and it phloem is in contact with chlorenchyma in is continuous, even in the midrib region (fig. the abaxial surface, where a layer of collen- 3a, b). The secretory cavities are present in chyma is also found (fig. 3e). the margins of the leaflets only. The vascular bundles are collateral, small and are proximal The epidermis of both, the leaflets and each other and have bundle sheaths (fig. 3c). rachis, present peltate trichomes, which are glandular and multicellular. The trichomes The midrib is surrounded by parenchyma are sunken in the epidermis (fig. 3e). and it is very prominent in the abaxial surface (fig. 3a).

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Oggero, A.J. et al.: Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum, Central Argentina

Fig. 3. Micrographs in transverse section of leaves and young stems of Zanthoxylum armatum (A) midrib region of leaflet (B) detail of mesophyll (C) margin of the leaflets with secretory cavities, (D) midrib region of rachis, (E) detail of wing of rachis with peltate tricome, (F) young stem (G) and (H) detail the different zone of showing the starch in cells. co, cortex; ep, epidermis; p, pith; pa, parenchyma; ph, phloem; pp, palisade parenchyma; pt, peltate trichome; s starch; sc, secretory cavity; sp, spongy parenchyma; xi xylem; ue, upper epidermis. Scale bars = 50 µm (a), (c), (e); 40 µm (b), (f);100 µm (d), (h), and 20 µm (g), (i), (j).

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Fig. 4. Micrographs of leaf epidermis of Zanthoxylum armatum (A) adaxial epidermis, (B) abaxial epidermis withpeltate trichome; and Zanthoxylum coco (C) adaxial epidermis, (D) abaxial epidermis, (E) gland on adaxial epidermis, (F) peltate trichome. gl, gland; pt, tricome peltate; st, stoma. Scale bar = 50 µm.

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Oggero, A.J. et al.: Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum, Central Argentina

The epidermis of leaves is unilayered in stout conic prickles when young and armed both, the adaxial and abaxial surfaces and branchlets with straight pale brown prickles. present a slight striated cuticle (fig. 4a, c). The phyllotaxis is alternate with compound, The epidermal cells of the adaxial surface imparipinnate or rarely paripinnate leaves have straight walls and are pentagonal or (fig. 2a). Young branchlets and inflorescence quadrangular of different sizes, while the rachises are glabrous or minutely pilose, cells of the abaxial surface are irregular with sulcate. The leaves are 3-7 foliolate with sinuous anticlinal walls. There are multice- rachis and petiole glabrous, without wings. llular peltate hairs in both epidermical faces. The rachis is sulcate, with two prominent These trichomes present a multicellular glands in each node. The leaflet blades are peduncle and a head with four central cells membranaceous, glabrous, pale green colo- with strong walls and numerous peripheric ured, with transparent oil glands dispersed cells (fig. 4b). The average density of the on all surface. The leaflets are subsessile trichomes was 3 per mm2 in abaxial surface or pedicellate, opposite, narrowly obovate, and lower than 1 per mm2 in adaxial surface. base attenuate and obtuse apex, the terminal The leaves are hypostomatic, the stomata leaflet is similar to the lateral ones. The mar- are anomocytic and present different sizes gin is crenate or serrulate and not revolute and a heterogeneous distributional patron, when dry. Rachises and petioles are armed some of them are so closed that the occlu- with pale brown, straight or slightly curved sive cells of the neighbouring stomata are prickles. in contact (fig. 4d). The average SI was 22.4 whereas the stomatal density was Anatomy of leaves and young stems 252.3 mm2. In transverse section of the leaflets blade, The young stems present a dermal tissue the cells of both epidermal faces tissue are that consists in a unilayered epidermis and similar in form and size and are protected a cortex formed by storage parenchyma by a thick cuticle. The stomata in abaxial subepidermic strata with abundant starch face of the leaflet are at the same level of granules (fig. 3g). The central zone of the the surrounding epidermal cells. In the same stem presents a parenchymatous pith is face of the blade there is an important vas- surrounded by parenchyma with abundant cular bundle that shows a secondary growth, starch granules (fig. 3h) and secondary whereas to the adaxial face there is one little xylem forming a continuous cylinder with collateral vascular bundle that shows an rays. A cambial zone separated the secon- inverted position. The vascular tissue is su- dary xylem from the secondary phloem rrounded by parenchyma and it is prominent (fig. 3f). in both surfaces of the leaflet. The mecha- nical tissue is absent in this area (fig. 5a). Zanthoxylum coco In the mesophyll the vascular bundles are collateral and surrounded by a bundle sheath. Young stem and leaves morphology The palisade tissue constitutes the major part of the mesophyll and occurred on either The plant is a tree to 8 m tall, deciduous, side of the spongy mesophyll. The cells of with grey armed stems with short grey the palisade tissue in the adaxial surface

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Fig. 5. Micrographs in transverse section of leaves and young stems of Zanthoxylum coco (a) midrib region of leaflet (b) detail of mesophyll (c) margin of the leaflets with secretory cavity, (d) rachis, (e) detail of (d) showing peltate trichome, (f) young stem, (g) detail the (f) showing secretory cavity. c, carenae; ch, chlorenchyma; co, cortex; ep, epiderms; p, pith; pa, parenchyma; ph, phloem; pp, palisade parenchyma; pt, peltate trichome; sc, secretory cavity; sp, spongy parenchyma; xi xylem; ue, upper epidermis. Scale bars = 50 µm (a), (b), (c), (e) and 100 µm (d), (f),(g).

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Oggero, A.J. et al.: Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum, Central Argentina are long and narrow, while the cells of the unilayered with stomata in the same level that abaxial surface are short and wide (fig. 5b). the surrounding cells. The epidermis presents In certain zones of the mesophyll the pali- multicellular trichomes, which are peltate and sade parenchyma tends to dissapear and is glandular, located in cavities of the surface. replaced by the spongy one. Under the epidermis there are chlorenchyma interrupted by secretory cavities. There is a The secretory cavities are present in all continuous cylinder of xylem and phloem su- parts of the leaflets, even in the margins rrounding a parenchymatous pith (fig. 5d, e). (fig. 5c). These secretory cavities are con- The young stems (fig. 5f) also present a sidered lysigenous because cellular remains unilayered epidermis covered by a thick in the borders of the cavities are observed. cuticle protecting the parenchymatous The secretory cavities are also found in all cortex, which presents secretory cavities analyzed parts of the plant. of relatively big size near to epidermis (fig. 5g). The epidermis is interrupted by schle- The Zanthoxylum coco epidermis of lea- renchyma, with cells highly lignified, in the ves is unilayered in both, the adaxial and areas where the p rickles are present. The abaxial surfaces and the cuticle is striated. vascular tissue has the same organization The epidermal cells are similar in both as the described for the rachis. surfaces, they are polygonal and present different sizes, with straight or slightly cur- Discussions and conclusions ved anticlinal cell walls (fig. 4d, e). There are notorious multicellular peltate trichomes The studied Zanthoxylum species share in both surfaces. These trichomes present a morphological features, as armed stems, multicellular peduncle and a head with many leaves alternate, odd-pinnately, 3- to many central and peripheral cells (fig. 4f). Also foliolate. The main differences are that Z. there are multicellular glands in both epi- armatum is an evergreen (or sometimes dermis, the average density of these glands deciduous) shrub with axillar inflorescences was 5 per mm2 in the adaxial surface and 3 whereas Z. coco is usually a deciduous tree per mm2 in the abaxial surface (fig. 4e). The with terminal inflorescences. density of the trichomes was 4.5 per mm2 in abaxial surface and 1.5 per mm2 in adaxial Study of different types of tissues and other surface. The leaves are hypostomatic, the microscopic techniques like linear measure- stomata in the abaxial surface are separated ments, determination of leaf constants and by similar distances among them but they quantitative microscopy are indispensable in not showed a specific distributional pattern the initial identification of plants materials (fig. 4e). The predominant type of stomata is (Jarald & Jarald, 2007). Some observed anomocytic and there are some cyclocytic, features in both species are characteristic of both types of stomata are surrounded by 4- 6 plants that inhabit places with xeric conditions epidermic cells. The average SI was 8.03, as occurred in central hills of Argentina. and the stomatal density was 189.5 mm2. Both species share the presence of secretory cavities, glandular trichomes and hyposto- The rachis is bicarenated and the carenae matic leaves. However each species presents are in the adaxial surface. The epidermis is particularities that could be important and

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Núm.42: 121-136 Julio 2016 related to the environment that the plants was also observed in an analysis of twelve develop. These features are basically related species of the genus Zanthoxylum (Xochi- to the leaves structure, owing to the fact that cale et al., 2010). Stomatal arrangement the leaf is the most sensitive organ and the and types are considered best taxonomic first in showing responses to environmental criteria and provide efficient basis for rela- conditions. The leaves reflect the morpho- tionship in taxonomic hierarchy (Hameed logical alterations as consequences of im- et al., 2008). Some workers like Sen & portant environmental changes (Trewavas, Hennipman (1981) had the idea that they 2003). Leaf epidermal studies are of im- may not be so an effective tool in taxonomy mense significance in finding taxonomy of because of their inconsistent arrangement in closely related species. Taxonomists have epidermises, however stomatal values like given prime importance to leaf epidermal stomatal number and stomatal index are of features to resolve taxonomic conflicts (Taia, great value in the evaluation of leaf origin 2005). Both species present thick epidermis crude drugs (Evans, 2002; Barkatullah et al., being really striate in Z. coco. As well it has 2014). Also, according to Wilkinson (1979) been informed that cuticle strias are under the values of SI are diagnostic features of a strong genetic control (Ahmad, 1962). It importance in Systematics, because they is also considered that its development as not present alterations. However in this the increase in thick of the epidermis, it can work, this affirmation is valid just for Z. be influenced by environmental factors. In coco, where the found value of SI (8.03) is most of xerophytes, the epidermis presents similar to the SI (7.65) found in plants of Z. different kind of strias while in species of coco inhabiting the Yungas in northwestern mesophytes and hygrophytes the epidermis Argentina (Arambarri et al., 2009). In Z. ar- is plain (Dunn et al., 1965; Wilkinson, matum we found a SI= 22.4, which is higher 1988). As well, it has been indicated that than the value of SI= 15, found in plants of a thick epidermis contributes a protection Z. armatum from Thailand by Suksathan of the photosynthetic tissues of a high sun et al. (2009). Kürschner et al. (1998) and radiation by an increase of the reflectance Salas et al. (2001) express that the value and striations can interfere in the absorption of SI is a parameter which is affected by of the light by the mesophyll chloroplasts stress conditions, specially nutritional and (Roth, 1990). In both species the walls of environmental ones. This allows us to de- epidermic cells tend to be straight and thick, termine that Z. armatum, which is exotic this characteristic could be associated to in Chaco biogeographical province, could aridity conditions and/or to a bigger sunlight be suffered remarked epidermal changes as exposition (Wilkinson, 1988). By other hand result to adaptation to the xeric conditions the anticlinal walls of the cell of the abaxial of chacoan environment. Also we found that epidermis always present more undulations Z. armatum presents higher stomata density, than in the adaxial surface, this is coincident that it would be possible, under hydric con- with what observed by Stace (1965) and ditions, a higher gaseous exchange than Z. agree with Roth (1990) who expresses that coco. Adaptation skills of plants depend on this characteristic is due to high shadow stomatal arrangement on the epidermises, as and humidity conditions. Both species pre- transpiration and photosynthesis are closely sent predominantly anomocytic stomata, this related to stomata. These are also useful in

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Oggero, A.J. et al.: Comparative morphology and anatomy of the leaf and stem of species of Zanthoxylum, Central Argentina taxonomic categorization and detection of balidis & Kofidis, 2002; Fahn, 1964). It future clues for observing environmental is necessary to highlight that Zanthoxylum factors. Micro and macro elements in plants coco is native of the northern and central are also closely related to stomatal density areas of Argentina while Zanthoxylum arma- (Nabin et al., 2000; Brownlee, 2001). Very tum has recently been reported as naturalised little work has been done on the stomatal (Arana and Oggero, 2009) and both species study of family Rutaceae, Ogunkunle & Ola- are found in the same habitat. Although the dele (1997) reported paracytic, hemiparacy- tissues organization in the stem is similar in tic, brachy paracytic, brachy paratetracytic both species, the stem of Zanthoxylum arma- and anomocytic stomatal complexes with tum shows a great capability to store starch uniform size from abaxial epidermises of in cells of the medulla, xylem and cortex as various Citrus species (Rutaceae) and also showed by the stained method used in this proved that in spite of high stomatal density, work. This species presents a small number they have relatively low transpiration rate of glandular trichomes and secretory cavities as compared to species with low stomatal that are observed only on the margin of the density. During a dry season, the margin leaves and the alate rachis, while in Z. coco of the leaves of Z. armatum are bended in the secretory conducts were very numerous direction to the abaxial area; this curving can in every analyzed organs and besides in this also be suffered by the wings of the rachis. species it has been found glands in both Therefore the appearance of the leaves chan- foliar epidermis. ges, the stomata are protected, an attenuate diffusion gradient between chlorenchyma All these structures are common of ob- and the environment is generated and per- serving in many species of Rutaceae (Judd mits the reduction of stomatal transpiration. et al., 2008) and it has considered that This strategy allows the plants can use water can contribute to the general regulation of efficiently so they are tolerant when this transpiratory rate moderating any hydric resource is limited (Fahn & Cutler, 1992). unbalance. Kakic et al. (2009) studied Z. acanthopodium DC., and informed that the As well the curving of the leaves is an espe- type of secretory structure and the secreted cial answer to the hydric’s stress conditions substances by this species and the members that are observed in many species (Stevano- of this genus must be considered such as vic, 1986; Stevanovic et al., 1992, Zivkovic ecological meaningful systems of protection et al., 2005), it is also related with a strategy and defence that has a plant, since they are for reducing the foliar area to an important important for the taxonomic diagnostic (Me- sun radiation (Bocher, 1981). Z. coco does tcalfe & Chalk, 1950) and also parameters not present this behaviour and it is different of the hydric conditions. from Z. armatum because shows a bigger development of the palisade parenchyma. Acknowledgements This could indicate that it is better for high intensities of light of the region, due to a We are grateful to the curators of the herbaria higher development of palisade parenchyma listed under Materials and Methods for per- is related with habitats that are exposed to a mission to study and/or loans of specimens. drought and intense sun radiation (Bosa- We want also to thank Gerard Thijsse, Chief

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Collections Manager, Nationaal Herbarium, Important Plants from Galliyat Areas, Nederland, and Raul Pozner, Instituto de NWFP, Pakistan. Ethnobotanical Le- Botánica Darwinion, Argentina for helping aflets: 2004(1): Article 6. Available us to locate the typus of Zanthoxylum arma- at: http://opensiuc.lib.siu.edu/ebl/ tum. This research was supported by grants vol2004/iss1/6. from SECYT- Universidad Nacional de Río Cuarto, Argentina. Barkatullah, M. I., y N. Muhammad, 2011. “Evaluation of Zanthoxylum armatum LITERATURE CITED DC for in-vitro and in-vivo pharmaco- logical screening”. African Journal of Abbasi, A.M.; M.A. Khan, M. Ahmad, M. Pharmacy and Pharmacology, 5(14): Zafar, S. Jahan, y S. Sultana, 2010. 1718-1723. “Ethnopharmacological application of medicinal plants to cure skin diseases Barkatullah, M.I.; G. Jelani, y I. Ahmad, and in folk cosmetics among the tribal 2014. “Leaf, Stem Bark And Fruit communities of North-West Frontier Anatomy of Zanthoxylum armatum Province”. Pak. J. Ethnopharmacol., Dc. (Rutaceae)”. Pak. J. Bot., 46(4): 128(2): 322-335. 1343-1349.

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