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Morphoanatomical and histochemical characterization of Larrea species from Northwestern of Argentina

Abstract

Larrea divaricata Cav., L. cuneifolia Cav. and L. nitida Cav., Zygophyllaceae, are evergreen xerophytic shrubs occurring in Northwestern Argentina used in traditional medicine. The aim of this work was to characterize the morphology, anatomy and histochemistry of the vegetative organs of three Larrea species by light and scanning electron microscopy in order to provide supporting data for their correct identification and to determine the site of synthesis and accumulation of its main active compounds. The shape, number and percentage of coalescence of leaflets, presence or absence of mucrones and rachis and the shape of the stipules represented the main botanical differences between the studied Larrea species. Anatomically three species presented amphystomatic leaves, with thick resinous slightly striated cuticle with resinous deposits, polygonal epidermal cells with straight anticlinal walls, ciclocytic, brachy-paracytic and paracytic stomatal types, non-glandular trichomes and isolateral mesophyll. The position and abundance of the sclerenchyma at the mid vein and petiole transection allows the differentiation of the three species, been more abundant in L. cuneifolia. Secondary phloem and parenchyma cells presented abundant calcium oxalate druses and solitary rhomboidal crystals. Epidermal cells and cuticle layer of leaflets and stipules of the three species presented amber resin deposits and content which stained positively for polysaccharides, phenolic compounds, flavonoids and tannins, while mesophyll palisade cells showed small refracting droplets stained positively for lipophilic substances.

Keywords:
Larrea; Morphology; Anatomy; Histochemistry; Leaf; Stem

Introduction

Zygophyllaceae family is represented approximately by 280 species almost restricted to tropical and subtropical areas. In Argentina the family is represented by seven genera (Bulnesia Gay, Kallstroemia Scop., Larrea Cav., Plectrocarpa Gill. ex Hook. & Arn., Porlieria Ruiz & Pav., Tribulus L. and Zygophyllum L.), and typically they are often dominant in the landscapea of Chaco and Monte regions (Cabrera and Willink, 1973Cabrera, A.L., Willink, A., 1973. Biogeografía de América Latina Monografía 13. Serie de Biología. Secretaría General de la Organización de los Estados Americanos. EEUU, Washington DC.; Cabrera, 1976Cabrera, A.L., 1976. Regiones fitogeográficas argentinas. In: Kugler, W.F. (Ed.), Enciclopedia argentina de agricultura y jardinería. Tomo 2. 2º edición. Acme, Buenos Aires, Argentina, p. 85.; Zuloaga and Morrone, 1999Zuloaga, F.O., Morrone, O., 1999. Catálogo de las Plantas Vasculares de la República Argentina II. Fabaceae-Zygophyllaceae (Dicotyledoneae). Monogr. Syst. Bot. Missouri Bot. Gard. Missouri.; Flora Argentina, 2018Flora Argentina, 2018. http://www.floraargentina.edu.ar/ (accessed 09.03.18).
http://www.floraargentina.edu.ar/...
). In Brazil it is represented by three genera (Kallstroemia, Larrea and Tribulus) (Engler, 1827Engler, H.G.A., 1827. Zygophylleae. In: Flora Brasiliensis, vol. XII, Part II, Fasc. Ed. von Martius C.F.P. 60, pp. 73–74. http://florabrasiliensis.cria.org.br/ (accessed 21.03.18).
http://florabrasiliensis.cria.org.br/...
).

The genus Larrea, Zygophyllaceae, comprises five species with amphitropical distribution in dry regions of South America (Argentina, Chile, Bolivia, Peru, Brazil) and North America (Mexico to Utah, United Stated of North America) (Engler, 1827Engler, H.G.A., 1827. Zygophylleae. In: Flora Brasiliensis, vol. XII, Part II, Fasc. Ed. von Martius C.F.P. 60, pp. 73–74. http://florabrasiliensis.cria.org.br/ (accessed 21.03.18).
http://florabrasiliensis.cria.org.br/...
; Hunziker et al., 1972Hunziker, J.H., Palacios, R.A., Valesi, A.G., Poggio, L., 1972. Species disjunction in Larrea: evidence from morphology, cytogenetics, phenolic compounds, and seed albumins. Ann. Missouri Bot. Gard. 59, 224-233., 1977Hunziker, J.H., Palacios, R.A., Poggio, L., Naranjo, C.A., Yang, T.W., 1977. Geographic distribution, morphology, hybridization cytogenetics and evolution. In: Mabry, T.J., Hunziker, J., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and Chemistry of Larrea in New World Deserts. , pp. 48–91.; Flora Argentina, 2018Flora Argentina, 2018. http://www.floraargentina.edu.ar/ (accessed 09.03.18).
http://www.floraargentina.edu.ar/...
). L. divaricata (common names: "jarilla", "jarilla hembra", "chamanilla", "jarilla del cerro", "yarilla"), L. cuneifolia (common names: "jarilla", "jarilla macho", "jarilla crespa", "jarilla norte-sur", "jarilla del campo") and L. nitida (common names: "jarilla", "jarilla de la montaña", "crespa", "pispa o pispita", "jarilla fina"), are represented in Northwestern Argentina forming shrubby associations called "jarillales". Among these, L. divaricata is the only Larrea species cited in the Brazilian flora (Engler, 1827Engler, H.G.A., 1827. Zygophylleae. In: Flora Brasiliensis, vol. XII, Part II, Fasc. Ed. von Martius C.F.P. 60, pp. 73–74. http://florabrasiliensis.cria.org.br/ (accessed 21.03.18).
http://florabrasiliensis.cria.org.br/...
).

Larrea species are evergreen, xerophytic, erect aromatic shrubs, 1–4 m with opposite, pubescent, sub-sessile and stipulate compound leaves which show a resinous yellowish appearance. The main botanical difference between Larrea species resides in their morphology, phenological patterns, and mating systems (Barbour et al., 1977Barbour, M.G., Cunningham, G., Oechel, W.C., Bamberg, S.A., 1977. Growth and development form and function. In: Mabry, T.J., Hunziker, J.H., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and chemistry of Larrea in New World Deserts. Dowden, Hutchinson and Ross, Stroudsburg, PA, pp. 48–91.; Simpson et al., 1977Simpson, B.B., Neff, J.L., Moldenke, A.R., 1977. Reproductive systems of Larrea. In: Mabry, T.J., Hunziker, J., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and Chemistry of Larrea in New World Deserts. Dowden, Hutchinson and Ross, Stroudsburg, PA, pp. 92–114.; Ezcurra et al., 1991Ezcurra, E., Montaña, C., Arizaga, S., 1991. Architecture, light interception, and distribution of Larrea species in the Monte desert, Argentina. Ecology 72, 23-34.). Ragonese (1960)Ragonese, A.M., 1960. Estudio anatómico de las especies Argentinas de Larrea (Zygophyllaceae). Rev. De Inv. Agric. 14, 355-370. noted some differences based on anatomical characters mainly the proportion and disposition of sclerenchyma tissues.

Larrea divaricata, L. cuneifolia and L. nitida are used in Argentinean traditional medicine as anti-inflammatory, antirheumatic, hypotensive, rubefacient, diaphoretic, febrifuge, oxytocic, emenagogo, odontalgic, antitussive and to treat fungal and bacterial infections (Alonso and Desmarchelier, 2006Alonso, J., Desmarchelier, C., 2006. Plantas medicinales autóctonas de la Argentina. Editorial Fitociencia, Buenos Aires, Argentina.; Alonso, 2007Alonso, J., 2007. Tratado de fitofármacos y nutracéuticos. Editorial Corpus, Rosario, Argentina.; Barboza et al., 2009Barboza, G., Cantero, J., Nuñez, C., Pacciaroni, A., Ariza Espinar, L., 2009. Medicinal plants: a general review and a phytochemical and ethnopharmacological screening of the native Argentine Flora. Kurtziana 34, 7-365.).

A wide range of pharmacological activities were previously described for these species indicating their potential usage as alternative or complementary medicine. Aqueous and/or alcoholic extracts from L. divaricata showed antibacterial (Stege et al., 2006Stege, P.W., Davicino, R.C., Vega, A.E., Casali, Y.A., Correa, S., Micalizzi, B., 2006. Antimicrobial activity of aqueous extracts of Larrea divaricata Cav (jarilla) against Helicobacter pylori. Phytomedicine 13, 724-727.; Zampini et al., 2007Zampini, I.C., Cudmani, N., Isla, M.I., 2007. Actividad antimicrobiana de plantas medicinales argentinas sobre bacterias antibiótico-resistentes. Acta Bioquim. Clin. Latinoam. 41, 385-393.), antitumoral (Anesini et al., 1996aAnesini, C., Genaro, A., Cremaschi, G., Zubillaga, M., Boccio, J., Sterin-Borda, L., Borda, E., 1996. In vivo and in vitro antitumoral action of Larrea divaricata Cav. Acta Physiol. Pharmacol. Ther. Latinoam. 46, 33-40., 2001Anesini, C., Ferraro, G., Lopéz, E., Borda, P., 2001. Different intracellular signals coupled to the antiproliferative action of aqueous extract from Larrea divaricata Cav. and nordihydroguaiaretic acid on a lymphoma cell line. Phytomedicine 81, 1-7.; Davicino et al., 2010Davicino, R., Manuele, M.G., Turner, S., Ferraro, G., Anesini, C., 2010. Antiproliferative activity of Larrea divaricata Cav. on lymphoma cell line: participation of hydrogen peroxide in its action. Cancer Invest. 28, 13-22., 2011Davicino, R., Manuele, M.G., Turner, S., Ferraro, G., Anesini, C., 2011. Larrea divaricata Cav. scientific evidence showing its beneficial effects and its wide potential application. Bol. Latinoam. Caribe Plant. Med. Aromaticas 10, 92-103.; Martino et al., 2016Martino, R., Barreiro Arcos, M.L., Alonso, R., Sülsen, V., Cremaschi, G., Anesini, C., 2016. Polyphenol-rich fraction from Larrea divaricata and its main flavonoid quercetin-3-methyl ether induce apoptosis in lymphoma cells through nitrosative stress. Phytother. Res. 30, 1128-1136.), antioxidant (Carabajal et al., 2017Carabajal, M.P.A., Isla, M.I., Zampini, I.C., 2017. Evaluation of antioxidant and antimutagenic activity of herbal teas from native plants used in traditional medicine in Argentina. S. African J. Bot. 110, 258-265.) and inmunomodulatory (Anesini et al., 1996bAnesini, C., Genaro, A., Cremaschi, G., Sterin Borda, L., Cazaux, C., Borda, E., 1996. Immunomodulatory action of Larrea divaricata Cav. Fitoterapia 67, 329-333.; Davicino et al., 2007Davicino, R., Mattar, A., Casali, Y., Porporatto, C., Correa, S.G., Micalizzi, B., 2007. In vivo immunomodulatory effects of aqueous extracts of Larrea divaricata Cav. Immunopharmacol. Immunotoxicol. 29, 352-366.) activity. Organic solvent extracts were active against phytopathogenic fungi (Quiroga et al., 2001Quiroga, E.M., Sampietro, A.R., Vattuone, M.A., 2001. Screening antifungal activities of selected medicinal plants. J. Ethnopharmacol. 74, 89-96.; Svetaz et al., 2010Svetaz, L., Zuljan, F., Derita, M., Petenatti, E., Tamayo, G., Cáceres, A., Cechinel Filho, V., Giménez, A., Pinzón, R., Zacchino, S.A., Gupta, M., 2010. Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin American countries. J. Ethnopharmacol. 127, 137-158.; Vogt et al., 2013Vogt, V., Cifuente, D., Tonn, C., Sabini, L., Rosas, S., 2013. Antifungal activity in vitro and in vivo of extracts and lignans isolated from Larrea divaricata Cav. against phytopathogenic fungus. Ind. Crops Prod. 42, 583-586.). Whereas L. cuneifolia showed larvicidal (Batallán et al., 2013Batallán, G., Torre, R., Flores, F., Konigheim, B., Ludueña-Almeida, F., Tonn, C., Contigiani, M., Almirón, W., 2013. Larvicidal activity of crude extracts from Larrea cuneifolia (Zygophyllaceae) and of its metabolite nordihydroguaiaretic acid against the vector Culex quinquefasciatus (Diptera: Culicidae). Rev. Soc. Bras. Med. Trop. 46, 84-87.) and antioxidant properties (Torres et al., 2003Torres, R., Urbina, F., Morales, C., Modak, B., Delle Monache, F., 2003. Antioxidant properties of lignans and ferulic acid from the resinous exudate of Larrea nitida. J. Chil. Chem. Soc. 48, 61-63.; Carabajal et al., 2017Carabajal, M.P.A., Isla, M.I., Zampini, I.C., 2017. Evaluation of antioxidant and antimutagenic activity of herbal teas from native plants used in traditional medicine in Argentina. S. African J. Bot. 110, 258-265.). A synergistic antifungal effect of L. nitida and Zuccagnia punctata Cav. was also reported (Butassi et al., 2015Butassi, E., Svetaz, L.A., Ivancovich, J.J., Feresin, G.E., Tapia, A., Zacchino, S.A., 2015. Synergistic mutual potentiation of antifungal activity of Zuccagnia punctata Cav. and Larrea nitida Cav. extracts in clinical isolates of Candida albicans and Candida glabrata. Phytomedicine 22, 666-678.).

Martino et al. (2016)Martino, R., Barreiro Arcos, M.L., Alonso, R., Sülsen, V., Cremaschi, G., Anesini, C., 2016. Polyphenol-rich fraction from Larrea divaricata and its main flavonoid quercetin-3-methyl ether induce apoptosis in lymphoma cells through nitrosative stress. Phytother. Res. 30, 1128-1136., Carabajal et al. (2017)Carabajal, M.P.A., Isla, M.I., Zampini, I.C., 2017. Evaluation of antioxidant and antimutagenic activity of herbal teas from native plants used in traditional medicine in Argentina. S. African J. Bot. 110, 258-265., Agüero et al. (2011)Agüero, M.B., Svetaz, L., Sánchez, M., Luna, L., Lima, B., López, M.L., Zacchino, S., Palermo, J., Wunderlin, D., Feresin, G.E., Tapia, A., 2011. Argentinean Andean propolis associated with the medicinal plant Larrea nitida Cav (Zygophyllaceae). HPLC–MS and GC–MS characterization and antifungal activity. Food Chem. Toxicol. 49, 1970-1978. and Blecja et al. (2007)Blecja, J.E., Anderson, M., Chow, J., Guevarra, C., Pender, C., Penaranda, C., Zavodovskaya, M., Youngren, J., Berkman, C., 2007. Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs. Bioorg. Med. Chem. Lett. 17, 4026-4029. reported the presence of nordihydroguaiaretic acid, essential oils and flavonoids as main constituents in some Larrea species. Several flavonoids including quercetin, apigenin and kaempferol derivatives were identified in organic extracts of L. cuneifolia (Valesi et al., 1972Valesi, A., Rodriguez, E., Vander Velde, G., Mabry, T., 1972. Methylated flavonoids in Larrea cuneifolia. Phytochemistry 11, 2821-2826.).

Bioactive compounds of Larrea species are presumably found in the resin that covers their leaves and stems. Ragonese (1960)Ragonese, A.M., 1960. Estudio anatómico de las especies Argentinas de Larrea (Zygophyllaceae). Rev. De Inv. Agric. 14, 355-370. observed a gradual decline in the resin content of older leaves and stems, and suggested that the resin is synthesize in the stipules from where it spills on nearby organs.

Pointing to the great potential of these species and their traditional use, the aim of this study was to characterize the morphoanatomy and histochemistry of the vegetative organs of L. divaricata, L. cuneifolia and L. nitida utilized in folk medicine of Northwestern Argentine, to identify anatomical diagnostic characters for their correct identification, and to determine the site of synthesis and accumulation of its main active compounds.

Materials and methods

Plant material

Aerial parts of Larrea cuneifolia Cav. and L. divaricata Cav., were collected in April 2015 at Amaicha del Valle, Tucumán, Argentina at 2000 m.a.s.l. Samples of L. nitida Cav. were collected in April 2015 at Vinchina, La Rioja, Argentina at 3485 m.a.s.l. Voucher specimens of each collection were deposited at the Herbarium of Fundación Miguel Lillo (LIL). Herbarium numbers of specimens are as follows: L. cuneifolia: LIL 614829; L. divaricata: LIL 614299 and L. nitida: LIL 615845.

Light microscopy

Samples of leaves and stems of five plants of each species were fixed in FAA (formalin, acetic acid, 50% ethanol, 5:5:90 v/v/v) and stored during one week after processing. Sections (10–25 µm) were obtained with a rotation microtome, subsequently treated with 50% NaClO solution, washed with distilled water and stained with astra blue-safranin and then mounted in 50% glycerol (Zarlavsky, 2014Zarlavsky, G.E., 2014. Histología vegetal: Técnicas simples y complejas. Sociedad Argentina de Botánica, Buenos Aires.). Sections were visualized with a Zeiss Axiolab optic microscope equipped with a polarized light filter and a Zeiss Axiocam ERc 5s digital camera.

Measurements were made using AxioVision software version 4.8.2 (Carl Zeiss Ltd, Herts, UK).

Histochemistry

The main classes of chemical compounds of the leaves and stipules were investigated in transverse microtome sections of fresh material. Fresh leaves and stipules were place between dental wax supports and sectioned at 20–25 µm with a rotation microtome.

Vanillin–sulphuric acid (Gaucher et al., 2013Gaucher, M., Dugé de Bernonville, T., Lohou, D., Guyot, S., Guillemette, T., Brisset, M.N., Dat, J.F., 2013. Histolocalization and physico-chemical characterization of dihydrochalcones: insight into the role of apple major flavonoids. Phytochemistry 90, 78-89.) and Neu's reagent (2-aminoethyl-diphenylborinate, Sigma) 10% in absolute methanol (Neu, 1957Neu, R., 1957. A new reagent for differentiating and determining flavones on paper chromatograms. Naturwissenschaften 43, 82.), were used to visualized flavonoids. Sections stained with Neu's reagent were analyzed under a fluorescence microscope (Nikon Optiphot) with UV light (filter UV-1A: 365 nm excitation filter, 400 nm barrier filter). Under these conditions, flavonoids were detected by a yellowish fluorescence (Mondolot-Cosson et al., 1997Mondolot-Cosson, L., Andary, C., Guang-Hui, D., Roussel, J.L., 1997. Histolocalisation de substances phénoliques intervenant lors d'interactions plante-pathogène chez le tournesol et la vigne. Acta Bot. Gallica 144, 353-362.). Photographs were taken with a digital Nikon Coolpix 4500 camera. Nadi reagent was used to detect terpenoids, essential oil and oil resins (David and Carde, 1964David, R., Carde, J.P., 1964. Coloration differentielle des inclusions lipidiques et terpeniques des pseudophylles du pin maritime au moyen du reactif nadi. Comptes rendus hebdomadaires des seuances de l' Academie des Sciences. Paris 258, 1338-1340.). Ferric chloride (10%) in methanol (Zarlavsky, 2014Zarlavsky, G.E., 2014. Histología vegetal: Técnicas simples y complejas. Sociedad Argentina de Botánica, Buenos Aires.) and Vainillin–HCl (Gardner, 1975Gardner, R.O., 1975. Vanillin–hydrochloric acid as histochemical test for tannin. Stain Technol. 50, 315-317.) were used to visualize phenolic compounds and tannins respectively. Toluidine blue O was used for the detection of polysaccharides (Heslop-Harrison and Heslop-Harrison, 1981Heslop-Harrison, Y., Heslop-Harrison, J., 1981. The digestive glands of Pinguicula: structure and cytochemistry. Ann. Bot. 47, 293-319.).

Some of the sections were treated with 50% sodium hypochlorite and washed with distilled water, prior to dyeing with Sudan IV for the detection of lipids (Zarlavsky, 2014Zarlavsky, G.E., 2014. Histología vegetal: Técnicas simples y complejas. Sociedad Argentina de Botánica, Buenos Aires.; D'Ambrogio de Argüeso, 1986D'Ambrogio de Argüeso, A., 1986. Manual de técnicas en histología vegetal. Hemisferio sur S.A, Buenos Aires.) and ruthenium red for pectins (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York, London.; Zarlavsky, 2014Zarlavsky, G.E., 2014. Histología vegetal: Técnicas simples y complejas. Sociedad Argentina de Botánica, Buenos Aires.). Iodine potassium iodide (IKI) (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York, London.) was employed for the detection of starch. Standard control procedures were carried out simultaneously.

Scanning electron microscopy

Samples of leaves were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 4–6 h at 4 °C. Following rinsing in the same buffer, the material was dehydrated in a graded acetone series and sputter coated with gold. Observations were carried out on a field emission scanning electron microscope (FESEM-ZEISS SUPRA-55 VP). Electronic microscopy observations were performed at the Centro Integral de Microscopía Electrónica (CIME), CONICET, Tucuman, Argentina.

Results

Morphology and anatomy

Larrea species are evergreen xerophytic, erect aromatic shrubs 1–4 m with opposite, composite, sub-sessile, pubescent, and stipulate leaves, with a succulent, resinous yellowish appearance.

Larrea cuneifolia presents leaves (4.5–13.2 × 2.5–16.0 mm) formed by two acute asymmetric oblong-ovate leaflets (2–4 × 1–2 mm) joined along two thirds of their internal edge culminating in a reflex apex with a filiform, vascularized mucro (0.3–0.5 mm) (Fig. 1A and D). Two stipules (squamous, subtriangular, reddish) (1.2–3.1 mm) are inserted at the base of the leaves (Fig. 1A and J).

Fig. 1
Photographs of the plant material. (A) Larrea cuneifolia. Leaf detail leaflets joined along two thirds of their internal edge. (B) L. divaricata. Divaricated leaf detail. (C) L. nitida. Odd pinnately compound leaves detail. (D–M) SEM images of leaf surface of Larrea sp. with abundant non glandular unicellular antrorse-appressed trichomes. (D) L. cuneifolia reflex apex with mucro. (E and F) L. divaricata adaxial and abaxial leaf surfaces respectively. (G) L. nitida adxial leaflet surface. (H and I) L. nitida raquis adaxial and abaxial surfaces, respectively. (J) L. cuneifolia stipules. (K) L. cuneifolia adaxial surface with slightly striated cuticle, non glandular unicellular trichomes, arrow shows brachy-paracytic stomata. Detail of trichome base with 4–6 cells with actinocytic appearance. (L) Ciclocytic stomata. (M) Paracytic stomata.

Larrea divaricata shows divaricated leaves (7.3–17.8 × 2.5–4.5 mm) formed by two oblong-acute divergent leaflets joined at the base in a third of its total length (Fig. 1B), apex reflex with a short and, vascularized mucro (0.3–0.4 mm). The stipules are obtuse and rounded similar to those previously described for L. cuneifolia (Fig. 1E and F).

Larrea nitida presents odd pinnately compound leaves (7.2–13.1 × 3.5–5.0 mm) (Fig. 1C), with 11–17 sub-opposite, sessile, oblong leaflets with rounded to convex apex (3.7–7.5 × 1.4–3.5 mm); the terminal leaflet is sometimes larger with acuminated to emarginated apex (Fig. 1G). Pubescent rachis (Fig. 1H and I) and acuminated stipules were observed (1–3 mm).

The three species presented amphistomatic leaves, with thick resinous slightly striated cuticle (Fig. 1K), polygonal epidermal cells with straight anticlinal walls, ciclocytic (Fig. 1L), brachy-paracytic (Fig. 1K) and paracytic stomatal types (Fig. 1M). Unicellular non glandular trichomes, antrorse-appressed with thick wall, striated ornamentation, acute-rounded apex (241.31 ± 56.84 µm) and surrounded by 4–6 cells at their base were observed (Fig. 1K). Trichomes appeared bi-refringent under polarized light. L. nitida appeared less pubescent than the other two species.

In transection leaflets presented thick cuticle, uniseriate epidermis with thick walled epidermal cells. Trichomes bases were inserted in depressions between the epidermal cells (Fig. 2H). Stomata appeared raised above the epidermal surface, with strongly projecting cutinized outer ledge (Fig. 2I). Mesophyll is isolateral, with 3–4 layers of tightly packed adaxial palisade cells; 1–2 layers of shorter abaxial palisade cells and a narrow central zone of spongy mesophyll interrupted by collateral vascular bundles. Parenchymatous sheath and sclerenchyma caps at phloem poles were observed in the vascular bundles of higger order (Fig. 2AE). The sclerenchymatous sheaths of the vascular bundles were extensively developed in L. cuneifolia (Fig. 2A) and L. divaricata (Fig. 2BD) and less developed or absent in L. nitida (Fig. 2E). Numerous calcium oxalate druses, refractive under polarized light, were observed in the mesophyll of all studied species (Fig. 2G).

Fig. 2
Photomicrographs of examined Larrea species leaves. (A) L. cuneifolia. (B) L. divaricata proximal leaf at region where the leaflets are fused, near the petiole. (C) L. divaricata leaf at mucro level, where leaflets become separated. Note mucro vascularization. (D) L. divaricata distal region, free leaflet at the mid vein region. (E) L. nitida leaflet. (F) L. nitida raquis. (G) Druses, thick walled trichomes and vessels show characteristic bi-refringence under polarized light. (H) Trichome insertion. (I) Raised stomata with strongly projecting cutinized outer ledge. Abbreviations: d, druse; ep, epidermis; ivb, inverted vascular bundle; lf, leaflet; mu, mucro; pp, palisade parenchyma; ps, parenchymatous sheath; pvb, primary vascular bundle; ra, raquis; s, stomata; sc, sclerenchyma cap.

An abaxial collateral vascular bundle and an adaxial smaller inverted vascular bundle, with a parenchymatic pith between them, sometimes accompanied by one or two small latero-adaxial collateral bundles was observed in L. cuneifolia and L. divaricata mid vein in the region where the leaflets are fused near the petiole, and in L. nitida raquis (Fig. 2F). The vascular bundles of the mayor order veins (primary and secondary) presented sclerenchyma cups at the phloem poles (Fig. 2A, B and F). L. cuneifolia and L. divaricata transection at mucro level, presented a small collateral vascular bundle (Fig. 2C).

Larrea divaricata and L. cuneifolia leaflets at their free endings, presented midvein with a single collateral vascular bundle with a less or more developed sclerenchymatous cap at phloem pole, surrounded by a parenchymatous sheath (Fig. 2D and E).

The stipule anatomy was similar in the three species. They presented navicular shaped stipules, sessile, with obtuse apex, pubescent on the abaxial surface with unicellular non glandular trichomes identical to those previously described for the leaflet. In transection thick cuticles and deposits of resin were observed. The uniseriate adaxial or inner epidermis consisted of elongated, columnar cells whereas the uniseriate abaxial or outer epidermis was formed by more isodiametric polyhedric cells. In both, epidermis and mesophyll idioblasts are evidenced by their amber content. Mesophyll presented 1–2 adaxial layers and a single abaxial layer of short palisade cells and homogeneous compact rounded central parenchyma with thick walls and abundant druses, interrupted by collateral vascular bundles (Fig. 3AC).

Fig. 3
(A–C) Photomicrographs of the stipules transection. (D–F) Photomicrographs of the petiole transection. (A, D) Larrea cuneifolia. (B, E) L. divaticata. (C, F) L. nitida. Abbreviations: ab ep, abaxial epidermis; ad ep, adaxial epidermis; arrows indicate druses; fi, fibers; gp, ground parenchyma; vb, vascular bundle.

The petiole outline of the three species was oval to circular, occasionally truncated adaxially (Fig. 3DF). Transection showed uniseriate epidermis, three adaxial layers and seven abaxial layers of ground parenchyma form by elliptical to round cells with abundant calcium oxalate druses, which sometimes form a ring around the vascular tissues. The vascular bundle resembles an ectophloic siphonostele, accompanied in L. cuneifolia and L. divarivata by two small latero-adaxial bundles. L. cuneifolia presented a strong adaxial cup of fibers and some abaxial clusters of lignified cells (Fig. 3D); L. divaricata showed a less developed adaxial cup of fibers and isolated abaxial clusters of fibers or sclereids in some cases not lignified (Fig. 3E), finally L. nitida presented no lignified tissue or sometimes some isolated abaxial fibers or sclereids (Fig. 3F).

Primary stem transection showed, elliptic outline, sometimes with prominent ribs in L. divaricata, thick cuticle, uniseriate epidermis form by quadrangular cells with thick walls. Non glandular trichomes were observed. Stomata raised above the epidermal surface. Cortex with 1–2 layers of chlorenchymatous cells tangentially elongated and 10–14 layers of rounded parenchymatous cells with druses and rhombic crystals. A complete or incomplete ring of fibers was observed; occasionally associated with brachysclereids clusters, internally a parenchymatous ring formed by 4–5 layers of rounded parenchymatic cells rounds the vascular cylinder. The vascular system is a continuous cylinder of phloem, follow by cambium and xylem and a cross shaped pith form by isodiametric parenchymatous cells with druses (Fig. 4AC).

Fig. 4
Photomicrographs of stem transection. (A–C) Early stage of secondry growth. (D–F) Secondary growth. (A, D) Larrea cuneifolia. (B, E) L. divaricata. (C, F) L. nitida. (G) Complete cylinder of schlerified cells rounds the phloem, follow centrifugally by 2–3 layers of large quadrangular parenchymatous cells and centripetally by 4–5 layers of smaller rounded parenchyma cells with abundant druses and rhomboidal crystals. (H) Crystals under polarized light. (I) Secondary phloem 1 seriated rays. (J) Secondary xylem 1–2 seriated rays weakly storied. (K) Lignified pith. Abbreviations: c, cambium; co, cortex; ep, epidermis; fi, fibers; ip, incipient periderm; p, pith; ph, phloem; x, xylem.

In older stems (Fig. 4DF) a complete cylinder of sclerenchymatous fibers rounds the phloem. Periderm begins to differentiate in the outer cortex, followed by 2–3 layers of large quadrangular, thin-walled parenchymatous cells and 4–5 layers of smaller rounded parenchyma cells with abundant druses and rhomboidal crystals (Fig. 4G and H). The secondary phloem presented abundant druses and solitary rhomboidal calcium oxalate crystals, 1 seriated heterocelullar rays with square marginal cells (Fig. 4I). The secondary xylem was constituted by thick walled solitary vessels, rarely in pairs; accompanied by many fiber-tracheids and 1–2 seriated rays (Fig. 4J). The pith becomes lignified (Fig. 4K).

Histochemistry

Epidermal cells and cuticle layer of leaflets and stipules of the three species presented an amber content and resin deposits, while mesophyll palisade cells showed small refracting droplets. Cellulose and pectates in the cellular walls (Fig. 5AC) was detected with ruthenium red and toluidine blue O. Toluidine blue O also stained positively the content of the epidermal cells of the stipules (Fig. 5D).

Fig. 5
Leaf and stipule histochemical characterization. (A, B) Ruthenium red revealed pectates in leaf and stipules cell walls respectively. (C, D) Toluidine blue O stain positively blue-violet cellulose and lignin in leaf and stipules, respectively. (E, F) Resin deposits, and palisade cells stained positively (black) with ferric trichloride for phenolic compounds in leaf and stipules respectively. (G, H) Vanillin–HCl stained positively black for tannins in the leaflets and negative in the stipule.

The resin deposits and the content of palisade and epidermal cells stained positively (black) with FeCl3 for phenolic compounds (Fig. 5E and F) and vainillin–HCl for tannins (Fig. 5G and H) both in leaflets as in stipules.

Flavonoids were detected with vanillin–sulfuric acid (stained bright yellow to orange) in resin deposits in the cuticles and droplets at the palisade and mesophyll cells of the leaflets and stipules (Fig. 6A and B). These results were also confirmed for the resin deposits of the leaflets stained with Neu's reagent which under UV light induced a bright yellow fluorescence (Fig. 6C). Nadi reagent for terpenoids resulted in a faint violet staining of the leaflet mesophyll droplets (Fig. 6D) and did not react in the stipules.

Fig. 6
Leaves and stipules histochemical characterization. (A, B) Resin deposits in the cuticles and droplets in the palisade and mesophyll cells of leaflets and stipules, stained bright yellow to orange with vainillin–sufuric acid, indicating the presence of flavonoids. (C) Resin deposits and cuticle stain positively for flavonoids revealing bright yellow under fluorescence with Neu's reagent. (D) Nadi reaction for terpenoids resulted in a faint violet staining of the leaflet mesophyll droplets. (E, F) Mesophyll refrigent droplets and cuticles stained positively for lipophilic substances staining red with Sudan IV in leaflet and stipules respectively.

Mesophyll droplets and cuticle stained positively (red) for lipophilic substances with Sudan IV (Fig. 6E and F). Iodine potassium iodide test carried out to detect starch gave negative results (not shown).

Discussion and conclusion

The morphological characters observed in the present work are coincident with those cited in the Flora of Brazil, Chile, Argentina and North America Flora for the genus and the particular species (Engler, 1827Engler, H.G.A., 1827. Zygophylleae. In: Flora Brasiliensis, vol. XII, Part II, Fasc. Ed. von Martius C.F.P. 60, pp. 73–74. http://florabrasiliensis.cria.org.br/ (accessed 21.03.18).
http://florabrasiliensis.cria.org.br/...
; Reiche, 1896Reiche, K., 1896. Zygophyllaceae. Flora de Chile, vol. I., Parte 2 Imprenta Cervantes, Santiago de Chile.; Flora Argentina, 2018Flora Argentina, 2018. http://www.floraargentina.edu.ar/ (accessed 09.03.18).
http://www.floraargentina.edu.ar/...
; North America Flora, 2018North America Flora, 2018. http://www.efloras.org (accessed 20.03.18).
http://www.efloras.org...
). Larrea divarivata presents similar morphological characters than North American Larrea, L. tridentata Sessk & Moc. ex DC (Yang et al., 1977Yang, T.W., Hunziker, J.H., Poggio, L., Naranjo, C.A., 1977. Hybridization between South American jarilla and North American diploid creosote bush (Larrea Zygophyllaceae). Pl Syst Evol. 126, 331-346.; Laport and Ramsey, 2015Laport, R.G., Ramsey, J., 2015. Morphometric analysis of the North American creosote bush (Larrea tridentata Zygophyllaceae) and the microspatial distribution of its chromosome races. Plant Syst. Evol. 301, 1581-1599.). There has been much controversy as to whether the North American species is a separate species from the diploid South American L. divaricata, and in some papers it has been called L. divaricata subsp. tridentata. However, L. tridentata seems to be derived by long distance dispersal from South America, cytogenetic, isozyme and molecular studies confirm that they are closely related, but separate, species (Lia et al., 2001Lia, V.V., Confalonieri, V.A., Comas, C.I., Hunziker, J.H., 2001. Molecular phylogeny of Larrea and its allies (Zygophyllaceae): reticulate evolution and the probable time of creosote bush arrival to North America. Mol. Phylogenet. Evol. 21, 309-320.; Laport and Ramsey, 2015Laport, R.G., Ramsey, J., 2015. Morphometric analysis of the North American creosote bush (Larrea tridentata Zygophyllaceae) and the microspatial distribution of its chromosome races. Plant Syst. Evol. 301, 1581-1599.). Larrea ameghinoi Speg. and L. nitida are similar in their compound leaf morphology, the flower anatomy, and the merocarp texture and size (Hunziker et al., 1977Hunziker, J.H., Palacios, R.A., Poggio, L., Naranjo, C.A., Yang, T.W., 1977. Geographic distribution, morphology, hybridization cytogenetics and evolution. In: Mabry, T.J., Hunziker, J., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and Chemistry of Larrea in New World Deserts. , pp. 48–91., Simpson et al., 1977Simpson, B.B., Neff, J.L., Moldenke, A.R., 1977. Reproductive systems of Larrea. In: Mabry, T.J., Hunziker, J., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and Chemistry of Larrea in New World Deserts. Dowden, Hutchinson and Ross, Stroudsburg, PA, pp. 92–114.) but with marked differences in their growth habit (Ezcurra et al., 1991Ezcurra, E., Montaña, C., Arizaga, S., 1991. Architecture, light interception, and distribution of Larrea species in the Monte desert, Argentina. Ecology 72, 23-34.). Barbour et al. (1974Barbour, M.G., Díaz, D.V., Breidenbach, R.W., 1974. Contributions to the biology of Larrea species. Ecology 55, 1199-1215., 1977Barbour, M.G., Cunningham, G., Oechel, W.C., Bamberg, S.A., 1977. Growth and development form and function. In: Mabry, T.J., Hunziker, J.H., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and chemistry of Larrea in New World Deserts. Dowden, Hutchinson and Ross, Stroudsburg, PA, pp. 48–91.), Barbour and Díaz (1973)Barbour, M.G., Díaz, D.V., 1973. Larrea plant communities on bajada and moisture gradients in the United States and Argentina. Vegetatio 28, 335-352., and Ezcurra et al. (1991)Ezcurra, E., Montaña, C., Arizaga, S., 1991. Architecture, light interception, and distribution of Larrea species in the Monte desert, Argentina. Ecology 72, 23-34. analyzed the physiological behavior and shrub architecture of South and North American Larrea species and they stated that the mayor differences between species could be the shrub architecture rather than shrub physiology or leaf anatomy. In the present work we find that the main botanical differences between examined Larrea species from Northwestern Argentine resides in their leaf morphology, shape of leaf, leaflets and stipules; presence or absence of mucro and rachis, and percentage of coalescence of the leaflets.

Anatomical characteristics such as non-glandular trichomes, stomata types, thick striated cuticle with resinous deposits, isolateral mesophyll type, the presence of large parenchyma sheath in the minor vascular bundles and calcium oxalate crystals are common features for the examined species and other Larrea species such as L. tridentata (Ragonese, 1960Ragonese, A.M., 1960. Estudio anatómico de las especies Argentinas de Larrea (Zygophyllaceae). Rev. De Inv. Agric. 14, 355-370.; Pyykko, 1966Pyykko, M., 1966. The leaf anatomy of East Patagonian xeromorphic plants. Ann. Bot. Fenn. 3, 453-622.; Metcalfe and Chalk, 1972Metcalfe, C., Chalk, L., 1972. Anatomy of the Dicotyledons, vol. II. Clarendon Press, Oxford.; Meyer and Meola, 1978Meyer, R.E., Meola, S.M., 1978. Morphological Characteristics of Leaves and Stems of Selected Texas Woody Plants. United States Department of Agriculture. Technical Bulletin 1564, pp. 1–204.).

In accordance with Ragonese (1960)Ragonese, A.M., 1960. Estudio anatómico de las especies Argentinas de Larrea (Zygophyllaceae). Rev. De Inv. Agric. 14, 355-370., the position and abundance of the sclerenchymatic tissue at the mid vein and petiole transection allows the differentiation of these three species that coexist. L. cuneifolia possess fibers associated to the vascular bundles, while L. divaricata and L. nitida present little or undeveloped sclerified tissues. In the same way, Sheahan and Cutler (1993)Sheahan, M.C., Cutler, D.F., 1993. Contribution of vegetative anatomy to the systematic of the Zygophyllaceae R. Br. Bot. J. Linn. Soc. 113, 227-262. indicate that the vascular bundles of the petiole of L. tridentata are surrounded by collenchymatous tissue, whereas Meyer and Meola (1978)Meyer, R.E., Meola, S.M., 1978. Morphological Characteristics of Leaves and Stems of Selected Texas Woody Plants. United States Department of Agriculture. Technical Bulletin 1564, pp. 1–204. indicate the presence of fibers that almost completely encircle the leaf midrib vascular tissues.

Stem characteristics were coincident with those described for the genera by Metcalfe and Chalk (1972)Metcalfe, C., Chalk, L., 1972. Anatomy of the Dicotyledons, vol. II. Clarendon Press, Oxford. and for L. tridentata by Meyer and Meola (1978)Meyer, R.E., Meola, S.M., 1978. Morphological Characteristics of Leaves and Stems of Selected Texas Woody Plants. United States Department of Agriculture. Technical Bulletin 1564, pp. 1–204., however, unlike these authors who describes that the phellogen originates deep in the phloem, we observed periderm originating from the outer cortex as stated for L. tridentata by Sheahan and Chase (1996)Sheahan, M.C., Chase, M.W., 1996. A phylogenetic analysis of Zygophyllaceae R Br. based on morphological, anatomical and rbcL DNA sequence data. Bot. J. Linn. Soc. 122, 279-300. and Sheahan and Cutler (1993)Sheahan, M.C., Cutler, D.F., 1993. Contribution of vegetative anatomy to the systematic of the Zygophyllaceae R. Br. Bot. J. Linn. Soc. 113, 227-262.. For L. ameghinoi there are few references on its anatomy, only referred to the presence of root nodules (Medan and Tortosa, 1983Medan, D., Tortosa, R.D., 1983. Nódulos radicales en Larrea (Zygophylaceae). Bol. Soc. Argent. Bot. 22, 221-236.).

In L. tridentata, Meyer and Meola (1978)Meyer, R.E., Meola, S.M., 1978. Morphological Characteristics of Leaves and Stems of Selected Texas Woody Plants. United States Department of Agriculture. Technical Bulletin 1564, pp. 1–204., observed a thin cuticle on both surfaces of the leaflet lamina and numerous epidermal cells on both surfaces containing dark-stain which they suggest may be resins and tannin idioblasts. Ragonese (1960)Ragonese, A.M., 1960. Estudio anatómico de las especies Argentinas de Larrea (Zygophyllaceae). Rev. De Inv. Agric. 14, 355-370. suggested that the stipules were the responsible of the secretion of the resinous substance deposited in stems and leaves, however in this work we demonstrate the presence of phenolic compounds and other chemical constituents at the epidermis and the cuticle of the stipules and at the epidermis and palisade cells of the leaflets mesophyll, all these cells may act as glandular structures where the resin is synthesize and subsequently excreted to the surface.

Our study contributes to the knowledge of Larrea species anatomy, identifying morphoanatomical characters of diagnostic value such as leaflets and stipules shape; presence or absence of mucro and rachis, percentage of coalescence of the leaflets and the position and abundance of the sclerenchymatic tissue at the mid vein and petiole transection. It also allowed determining that both stipules and leaves are the site of synthesis and accumulation of secondary metabolites of interest and will lend support to further studies on their chemical constituents and its functional role in nature.

Acknowledgments

We acknowledge the Electron Microscopy Center CIME, Centro Integral de Microscopía Electrónica for assistance in preparing SEM images.

References

  • Agüero, M.B., Svetaz, L., Sánchez, M., Luna, L., Lima, B., López, M.L., Zacchino, S., Palermo, J., Wunderlin, D., Feresin, G.E., Tapia, A., 2011. Argentinean Andean propolis associated with the medicinal plant Larrea nitida Cav (Zygophyllaceae). HPLC–MS and GC–MS characterization and antifungal activity. Food Chem. Toxicol. 49, 1970-1978.
  • Alonso, J., 2007. Tratado de fitofármacos y nutracéuticos. Editorial Corpus, Rosario, Argentina.
  • Alonso, J., Desmarchelier, C., 2006. Plantas medicinales autóctonas de la Argentina. Editorial Fitociencia, Buenos Aires, Argentina.
  • Anesini, C., Ferraro, G., Lopéz, E., Borda, P., 2001. Different intracellular signals coupled to the antiproliferative action of aqueous extract from Larrea divaricata Cav. and nordihydroguaiaretic acid on a lymphoma cell line. Phytomedicine 81, 1-7.
  • Anesini, C., Genaro, A., Cremaschi, G., Zubillaga, M., Boccio, J., Sterin-Borda, L., Borda, E., 1996. In vivo and in vitro antitumoral action of Larrea divaricata Cav. Acta Physiol. Pharmacol. Ther. Latinoam. 46, 33-40.
  • Anesini, C., Genaro, A., Cremaschi, G., Sterin Borda, L., Cazaux, C., Borda, E., 1996. Immunomodulatory action of Larrea divaricata Cav. Fitoterapia 67, 329-333.
  • Barbour, M.G., Cunningham, G., Oechel, W.C., Bamberg, S.A., 1977. Growth and development form and function. In: Mabry, T.J., Hunziker, J.H., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and chemistry of Larrea in New World Deserts. Dowden, Hutchinson and Ross, Stroudsburg, PA, pp. 48–91.
  • Barbour, M.G., Díaz, D.V., 1973. Larrea plant communities on bajada and moisture gradients in the United States and Argentina. Vegetatio 28, 335-352.
  • Barbour, M.G., Díaz, D.V., Breidenbach, R.W., 1974. Contributions to the biology of Larrea species. Ecology 55, 1199-1215.
  • Barboza, G., Cantero, J., Nuñez, C., Pacciaroni, A., Ariza Espinar, L., 2009. Medicinal plants: a general review and a phytochemical and ethnopharmacological screening of the native Argentine Flora. Kurtziana 34, 7-365.
  • Batallán, G., Torre, R., Flores, F., Konigheim, B., Ludueña-Almeida, F., Tonn, C., Contigiani, M., Almirón, W., 2013. Larvicidal activity of crude extracts from Larrea cuneifolia (Zygophyllaceae) and of its metabolite nordihydroguaiaretic acid against the vector Culex quinquefasciatus (Diptera: Culicidae). Rev. Soc. Bras. Med. Trop. 46, 84-87.
  • Blecja, J.E., Anderson, M., Chow, J., Guevarra, C., Pender, C., Penaranda, C., Zavodovskaya, M., Youngren, J., Berkman, C., 2007. Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs. Bioorg. Med. Chem. Lett. 17, 4026-4029.
  • Butassi, E., Svetaz, L.A., Ivancovich, J.J., Feresin, G.E., Tapia, A., Zacchino, S.A., 2015. Synergistic mutual potentiation of antifungal activity of Zuccagnia punctata Cav. and Larrea nitida Cav. extracts in clinical isolates of Candida albicans and Candida glabrata Phytomedicine 22, 666-678.
  • Cabrera, A.L., 1976. Regiones fitogeográficas argentinas. In: Kugler, W.F. (Ed.), Enciclopedia argentina de agricultura y jardinería. Tomo 2. 2º edición. Acme, Buenos Aires, Argentina, p. 85.
  • Cabrera, A.L., Willink, A., 1973. Biogeografía de América Latina Monografía 13. Serie de Biología. Secretaría General de la Organización de los Estados Americanos. EEUU, Washington DC.
  • Carabajal, M.P.A., Isla, M.I., Zampini, I.C., 2017. Evaluation of antioxidant and antimutagenic activity of herbal teas from native plants used in traditional medicine in Argentina. S. African J. Bot. 110, 258-265.
  • D'Ambrogio de Argüeso, A., 1986. Manual de técnicas en histología vegetal. Hemisferio sur S.A, Buenos Aires.
  • Davicino, R., Manuele, M.G., Turner, S., Ferraro, G., Anesini, C., 2010. Antiproliferative activity of Larrea divaricata Cav. on lymphoma cell line: participation of hydrogen peroxide in its action. Cancer Invest. 28, 13-22.
  • Davicino, R., Manuele, M.G., Turner, S., Ferraro, G., Anesini, C., 2011. Larrea divaricata Cav. scientific evidence showing its beneficial effects and its wide potential application. Bol. Latinoam. Caribe Plant. Med. Aromaticas 10, 92-103.
  • Davicino, R., Mattar, A., Casali, Y., Porporatto, C., Correa, S.G., Micalizzi, B., 2007. In vivo immunomodulatory effects of aqueous extracts of Larrea divaricata Cav. Immunopharmacol. Immunotoxicol. 29, 352-366.
  • David, R., Carde, J.P., 1964. Coloration differentielle des inclusions lipidiques et terpeniques des pseudophylles du pin maritime au moyen du reactif nadi. Comptes rendus hebdomadaires des seuances de l' Academie des Sciences. Paris 258, 1338-1340.
  • Engler, H.G.A., 1827. Zygophylleae. In: Flora Brasiliensis, vol. XII, Part II, Fasc. Ed. von Martius C.F.P. 60, pp. 73–74. http://florabrasiliensis.cria.org.br/ (accessed 21.03.18).
    » http://florabrasiliensis.cria.org.br/
  • Ezcurra, E., Montaña, C., Arizaga, S., 1991. Architecture, light interception, and distribution of Larrea species in the Monte desert, Argentina. Ecology 72, 23-34.
  • Flora Argentina, 2018. http://www.floraargentina.edu.ar/ (accessed 09.03.18).
    » http://www.floraargentina.edu.ar/
  • Gardner, R.O., 1975. Vanillin–hydrochloric acid as histochemical test for tannin. Stain Technol. 50, 315-317.
  • Gaucher, M., Dugé de Bernonville, T., Lohou, D., Guyot, S., Guillemette, T., Brisset, M.N., Dat, J.F., 2013. Histolocalization and physico-chemical characterization of dihydrochalcones: insight into the role of apple major flavonoids. Phytochemistry 90, 78-89.
  • Heslop-Harrison, Y., Heslop-Harrison, J., 1981. The digestive glands of Pinguicula: structure and cytochemistry. Ann. Bot. 47, 293-319.
  • Hunziker, J.H., Palacios, R.A., Poggio, L., Naranjo, C.A., Yang, T.W., 1977. Geographic distribution, morphology, hybridization cytogenetics and evolution. In: Mabry, T.J., Hunziker, J., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and Chemistry of Larrea in New World Deserts. , pp. 48–91.
  • Hunziker, J.H., Palacios, R.A., Valesi, A.G., Poggio, L., 1972. Species disjunction in Larrea: evidence from morphology, cytogenetics, phenolic compounds, and seed albumins. Ann. Missouri Bot. Gard. 59, 224-233.
  • Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York, London.
  • Laport, R.G., Ramsey, J., 2015. Morphometric analysis of the North American creosote bush (Larrea tridentata Zygophyllaceae) and the microspatial distribution of its chromosome races. Plant Syst. Evol. 301, 1581-1599.
  • Lia, V.V., Confalonieri, V.A., Comas, C.I., Hunziker, J.H., 2001. Molecular phylogeny of Larrea and its allies (Zygophyllaceae): reticulate evolution and the probable time of creosote bush arrival to North America. Mol. Phylogenet. Evol. 21, 309-320.
  • Martino, R., Barreiro Arcos, M.L., Alonso, R., Sülsen, V., Cremaschi, G., Anesini, C., 2016. Polyphenol-rich fraction from Larrea divaricata and its main flavonoid quercetin-3-methyl ether induce apoptosis in lymphoma cells through nitrosative stress. Phytother. Res. 30, 1128-1136.
  • Medan, D., Tortosa, R.D., 1983. Nódulos radicales en Larrea (Zygophylaceae). Bol. Soc. Argent. Bot. 22, 221-236.
  • Metcalfe, C., Chalk, L., 1972. Anatomy of the Dicotyledons, vol. II. Clarendon Press, Oxford.
  • Meyer, R.E., Meola, S.M., 1978. Morphological Characteristics of Leaves and Stems of Selected Texas Woody Plants. United States Department of Agriculture. Technical Bulletin 1564, pp. 1–204.
  • Mondolot-Cosson, L., Andary, C., Guang-Hui, D., Roussel, J.L., 1997. Histolocalisation de substances phénoliques intervenant lors d'interactions plante-pathogène chez le tournesol et la vigne. Acta Bot. Gallica 144, 353-362.
  • Neu, R., 1957. A new reagent for differentiating and determining flavones on paper chromatograms. Naturwissenschaften 43, 82.
  • North America Flora, 2018. http://www.efloras.org (accessed 20.03.18).
    » http://www.efloras.org
  • Pyykko, M., 1966. The leaf anatomy of East Patagonian xeromorphic plants. Ann. Bot. Fenn. 3, 453-622.
  • Quiroga, E.M., Sampietro, A.R., Vattuone, M.A., 2001. Screening antifungal activities of selected medicinal plants. J. Ethnopharmacol. 74, 89-96.
  • Ragonese, A.M., 1960. Estudio anatómico de las especies Argentinas de Larrea (Zygophyllaceae). Rev. De Inv. Agric. 14, 355-370.
  • Reiche, K., 1896. Zygophyllaceae. Flora de Chile, vol. I., Parte 2 Imprenta Cervantes, Santiago de Chile.
  • Sheahan, M.C., Chase, M.W., 1996. A phylogenetic analysis of Zygophyllaceae R Br. based on morphological, anatomical and rbcL DNA sequence data. Bot. J. Linn. Soc. 122, 279-300.
  • Sheahan, M.C., Cutler, D.F., 1993. Contribution of vegetative anatomy to the systematic of the Zygophyllaceae R. Br. Bot. J. Linn. Soc. 113, 227-262.
  • Simpson, B.B., Neff, J.L., Moldenke, A.R., 1977. Reproductive systems of Larrea In: Mabry, T.J., Hunziker, J., DiFeo Jr., D.R. (Eds.), Creosotebush: Biology and Chemistry of Larrea in New World Deserts. Dowden, Hutchinson and Ross, Stroudsburg, PA, pp. 92–114.
  • Stege, P.W., Davicino, R.C., Vega, A.E., Casali, Y.A., Correa, S., Micalizzi, B., 2006. Antimicrobial activity of aqueous extracts of Larrea divaricata Cav (jarilla) against Helicobacter pylori. Phytomedicine 13, 724-727.
  • Svetaz, L., Zuljan, F., Derita, M., Petenatti, E., Tamayo, G., Cáceres, A., Cechinel Filho, V., Giménez, A., Pinzón, R., Zacchino, S.A., Gupta, M., 2010. Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin American countries. J. Ethnopharmacol. 127, 137-158.
  • Torres, R., Urbina, F., Morales, C., Modak, B., Delle Monache, F., 2003. Antioxidant properties of lignans and ferulic acid from the resinous exudate of Larrea nitida J. Chil. Chem. Soc. 48, 61-63.
  • Valesi, A., Rodriguez, E., Vander Velde, G., Mabry, T., 1972. Methylated flavonoids in Larrea cuneifolia Phytochemistry 11, 2821-2826.
  • Vogt, V., Cifuente, D., Tonn, C., Sabini, L., Rosas, S., 2013. Antifungal activity in vitro and in vivo of extracts and lignans isolated from Larrea divaricata Cav. against phytopathogenic fungus. Ind. Crops Prod. 42, 583-586.
  • Yang, T.W., Hunziker, J.H., Poggio, L., Naranjo, C.A., 1977. Hybridization between South American jarilla and North American diploid creosote bush (Larrea Zygophyllaceae). Pl Syst Evol. 126, 331-346.
  • Zampini, I.C., Cudmani, N., Isla, M.I., 2007. Actividad antimicrobiana de plantas medicinales argentinas sobre bacterias antibiótico-resistentes. Acta Bioquim. Clin. Latinoam. 41, 385-393.
  • Zarlavsky, G.E., 2014. Histología vegetal: Técnicas simples y complejas. Sociedad Argentina de Botánica, Buenos Aires.
  • Zuloaga, F.O., Morrone, O., 1999. Catálogo de las Plantas Vasculares de la República Argentina II. Fabaceae-Zygophyllaceae (Dicotyledoneae). Monogr. Syst. Bot. Missouri Bot. Gard. Missouri.

Publication Dates

  • Publication in this collection
    Jul-Aug 2018

History

  • Received
    10 Mar 2018
  • Accepted
    30 May 2018
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