Stem and leaf anatomy of six species of Verbena native to Buenos Aires province, Argentina

Abstract

There are 21 species of Verbena L. in Argentina, six of which are found in the Buenos Aires province: Verbena gracilescens (Cham.) Verter; V. bonariensis L.; V. intermedia Gillies and Hook; V. litoralis Kunth; V. montevidensis Spreng. and V. rigida Spreng. These species are used in popular medicine for their hepatoprotective, digestive, anti-inflammatory, antidiarrheal and healing properties. The stems and leaves of these species were fixed and transverse sections were cut for observation with a light microscope in order to differentiate their anatomical features. Likewise a scanning electron microscope and an environmental scanning electron microscope were used to study the leaf surface of these species. The type and density of stomata and glandular and nonglandular trichomes were analysed in all species, as well as the internal structure of the leaf and stem. The Verbena species of Buenos Aires province have been characterized using these features to help in their identification.

Keywords:

• Verbena
• Verbenaceae
• anatomy
• trichomes
• medicinal plants
• Argentina

Introduction

In recent years, the return to using natural products in therapeutics has been facilitated by factors such as the serious side effects of synthetic drugs, greater chemical and pharmaceutical knowledge of plant-based drugs and their derivatives and the development of analytical methods that guarantee better quality control (Cañigueral et al. 2003). This resurgence raises the need for correct identification and appropriate selection of medicinal plants.

Even though medicinal plants of the native flora of Argentina have become very important for use in popular medicine for various treatments, as well as in the formulation of herbal medicines, there is only a partial understanding of their characterization, either as mono herbs or commercially available mixtures (Barboza et al. 2001). Only a small group of species has been included in the Farmacopea Nacional Argentina VI ed. (1978) (Leonardi et al. 2002).

In Argentina there are 21 species of Verbena, six of which are native in the province of Buenos Aires: Verbena gracilescens (Cham.) Verter; V. bonariensis L.; V. intermedia Gillies and Hook; V. litoralis Kunth; V. montevidensis Spreng. and V. rigida Spreng. (Troncoso 1974; Múlgura 1999; O’Leary 2007). Numerous authors report the medicinal properties of V. litoralis, V. bonariensis, V. intermedia and V. gracilescens. However, these properties have not been cited for V. montevidensis and V. rigida. The four species mentioned are used for their hepatoprotective, digestive, emmenagogue, healing, anti-inflammatory, antineuralgic, antidiarrheic, anti-gangrene and antimicrobial properties, among other uses (Arenas & Moreno Azorero 1977; Ratera & Ratera 1980; Martínez Crovetto 1981; Alonso 1998; Cáceres 1999; Alonso & Desmarchelier 2005; Barboza et al. 2006).

Most anatomical studies of the Verbenaceae are of genera such as Lippia (Nunes et al. 2000; Barboza et al. 2001; Bonzani et al. 2003), Aloysia (Matesevach Becerra et al. 2000; Barboza et al. 2001; Bonzani et al. 2003), Acantholippia (Carmona & Ancibor 1995; Barboza et al. 2001; Bonzani et al. 2003), Glandularia (Botta 1993) and Junellia (Botta 1989). Metcalfe and Chalk (1979) performed comprehensive anatomical revisions of the Verbenaceae. Inamdar (1969) described the epidermis and the ontogenesis of stomata of different genera of the Verbenaceae. However, few anatomical studies have been carried out on Verbena. Bhoj (1983) determined the pollen characteristics of the genus. Souza et al. (2005) studied the stem and leaf anatomy of V. litoralis to find diagnostic features for its detection in pharmaceutical products. Munsif et al. (2007) analysed the epidermal surfaces of various Verbena species from the Middle East. Mathew and Shah (1983) studied taxonomic and structural aspects of the trichomes present on nine species of Verbena and also their development. In Argentina O’Leary (2007) and O’Leary et al. (2007) presented a taxonomic revision of the genus on the basis of morphological characters. However, no information was included for using their anatomical features to differentiate the Verbena species, especially the natives of Buenos Aires province. The study of anatomical characters is a useful tool for the identification and systematic revision of taxa (Metcalfe & Chalk 1979). Moreover, it allows proper identification of herbal medicines used in popular medicine (Barboza et al. 2001).

The objective of this study is to describe the anatomical features of leaves and stems of Verbena native species of Buenos Aires province, as a contribution to future taxonomical studies of the genus and for its characterization as a crude drug.

Materials and methods

Plant material

Whole specimens of each species were prepared and deposited in the herbarium of the Instituto de Botánica Darwinion (SI), Buenos Aires, Argentina (Table 1).

Table 1  List of specimens of each species collected in Buenos Aires province, Argentina.

Verbena bonariensis L.	ARGENTINA, Buenos Aires Province	San Andrés de Giles County, Loc. Solís, Real Way and National Road 8	7-XII-2007	Morcelle 333 (SI)
		Luján County, Provincial Road 6 and West Highway	31-I-2008	Morcelle 334 (SI)
V. litoralis Kunth.	ARGENTINA, Buenos Aires Province	Luján County, National Road 7 km 71 Rural Education Center N° 1	24-III-2008	Morcelle 338 (SI)
		Luján County, Nationals Roads 5 and 7. Nacional University of Luján Campus	20-II-/2008	Morcelle 335 (SI)
V. intermedia Gillies & Hook.	ARGENTINA, Buenos Aires Province	Baradero County, Provincial Road 41, Access Road Baradero. Field of Mr. Luis Calvá	6-XII-2007	Morcelle 332 (SI)
		San Andrés de Giles County, loc. Solís, Field of Mr José Luis Badano,	17-X-2007	Morcelle 330 (SI)
V. montevidensis Spreng.	ARGENTINA, Buenos Aires Province	Zárate County, Access Road Las Palmas, Field of Mr. Fabián Simoncini	27-II-2008	Morcelle 336 (SI)
		Luján County, Luján City, Sargento Cabral Street 1254		Morcelle 339 (SI)
V. rigida Spreng.	ARGENTINA, Buenos Aires Province	Martín García Island. Río de la Plata		Morcelle 340 (SI)
V. gracilescens (Cham.) Verter.	ARGENTINA, Buenos Aires Province	Baradero County, Provincial Road 41, Access Road of Baradero, Field of Mr. Luis Calvá	6-XII-2007	Morcelle 331 (SI)
		Zárate County, Access Road of Las Palmas, Field of Mr. Fabián Simoncini	27-II-2008	Morcelle 337 (SI)

Light microscopy

Transverse section

Pieces of fresh stem were cut at the height of the fourth internode from the apex from each species under study. This material was cut in 20 µm transverse sections with a MICROM HM 400 R sliding microtome (Microm, Germany), stained with safranin-fastgreen and mounted in synthetic balsam. Also stems and leaves were fixed in FAA (ethanol 96%:distilled water:formaldehyde:acetic acid, 10:7:2:1), embedded in paraffin, cut in 15–20 µm transverse sections with a MICROM HM 325 rotary microtome (Microm, Germany), stained with saffranin-fastgreen and mounted in synthetic balsam. Likewise, small pieces of the leaves of these specimens were fixed in 4% glutaraldehyde, post-fixed in 1.5% osmium tetroxide in 0.1 M phosphate buffer, embedded in epoxy resin and cut with an ultramicrotome in transverse sections of 1–2 µm. Subsequently, these sections were stained with 1% toluidine blue and mounted in synthetic balsam (PMYR, Instrumental Pasteur, Argentina).

Clearing

Expanded leaves from the fourth node from the apex were cleared using Dizeo de Strittmater’s method (D’Ambrogio 1986). This technique involves: removal of chlorophyll with boiling ethanol for 30 s; the dissolution of protoplasm using boiling 5% NaOH and ethanol (1:1) for 1 min and washing three times with distilled water; removal of brown coloration by bleaching with 50% sodium hypochlorite and washing three times with water; clearing with 2.5 g cm⁻³ chloral hydrate 2–3 min; and staining with diluted safranine and then mounting with glycerin-gelatin.

Microchemical tests

Fresh transversal sections of leaves were used to determine cellular contents. Thus, cresyl blue was used for mucilages and 2M HCl for calcium carbonate (cystoliths) (Martínez et al. 2008).

Observations, drawings and photographs were made using an UNICO light microscope with a computerized system for the capture of images (Intellicam; Matrox Electronic Systems, Canada) and an OPTIMA 6.5 image processor of the Luján National University (Buenos Aires, Argentina).

Scanning electron microscopy

Pieces of leaves (0.5 cm×0.5 cm) of the different species under study were fixed in FAA, dehydrated in an ascending series of alcohol and were dried to the critical point. Subsequently they were coated with a gold-palladium alloy so that they could be observed and photographed with a Phillips XL-30 SEM microscope (Microscopy Service, Bernardino Rivadavia Natural Sciences Museum, Buenos Aires, Argentina).

Environmental scanning electron microscopy

Fresh leaves of the species under study were observed and photographed using an ESEM Electroscan Phillips 2010 microscope (Microscopy Service, CITEFA, Buenos Aires, Argentina).

Statistical analysis

Stomatal and glandular trichomes density was measured in 30 randomly chosen optical microscope fields on 10 slides from each species. The results for each parameter were analysed with ANOVA and compared with Tukey's multiple comparison test, using SPSS v.12.0 software (SPSS Inc., Chicago, Illinois, USA).

Results

Leaf

Surface view

The leaves of the Verbena species under study show isodiametric epidermal cells with wavy borders on both leaf surfaces. The stomata are anisocytic and anomocytic in all species, rarely diacytic in V. montevidensis. The stomatal density calculated shows significant differences between the adaxial and abaxial surfaces (Figs. 1A–1E; Table 2). The highest values were recorded for V. litoralis, 318.7/mm² on the abaxial surface and 139.7/mm² on the adaxial surface (Table 2).

Figure 1  Leaf surface of Verbena native species of Buenos Aires province. A, Adaxial epidermis of V. bonariensis (SEM). B, Abaxial epidermis of V. bonariensis (SEM). C, Anomocytic stomatal apparatus, adaxial epidermis of V. bonariensis (SEM). D, Adaxial epidermis of V. bonariensis (SEM). E, Adaxial epidermis of V. montevidensis (LM). F, Vascularization, abaxial epidermis of V. rigida (LM). Abbreviations: g, glandular trichomes; LM, Light Microscopy; ng, nonglandular trichomes; s, stomata; SEM, scanning electron microscopy. Bars: A, B, F, 200 µm. C, D, E, 50 µm.

Table 2  Density of stomata and glandular trichomes in the leaves of Verbena native species of Buenos Aires province, Argentina.

Leaf epidermis	Species	Stomatal density^1	Glandular trichomes density^2
Adaxial surface	V. litoralis	139.7 a	35.6 a
	V. bonariensis	9.4 d	12.9 ab
	V. intermedia	59.4 c	9.7 b
	V. rigida	86.2 bc	6.1 b
	V. montevidensis	96.0 b	20.4 b
	V. gracilescens	53.3 c	11.7 b
Abaxial surface	V. litoralis	318.7 a	24.5 a
	V. bonariensis	234.3 bc	8.9 a
	V. intermedia	274.4 ab	25.6 a
	V. rigida	190.1 cd	27.6 a
	V. montevidensis	285.9 ab	10.2 a
	V. gracilescens	138.6 d	22.4 a
^1Number of stomata/mm^2. ^2Number of glandular trichomes/mm^2. Note: Different letters indicate significant differences (P=0.05) between species for the same leaf epidermis. Tukey's multiple comparison test.

The cuticle on both the adaxial and abaxial epidermis is slightly striated, the striations being more marked over the mid-vein (Fig. 2F).

Figure 2  Glandular and nonglandular trichomes of Verbena native species of Buenos Aires province, leaf surface view. A, General view of abaxial epidermis of V. litoralis (ESEM). B, Detail of appressed nonglandular trichome, adaxial epidermis of V. gracilescens (ESEM). C, Long-stalked glandular trichome and nonglandular patent trichome, abaxial epidermis of V. bonariensis (SEM). D, Long-stalked glandular trichome adaxial epidermis of V. rigida (SEM). E, Long-stalked glandular trichome, adaxial epidermis of V. intermedia (ESEM). F, Four-celled head of short-stalked glandular trichome, abaxial epidermis of V. rigida (SEM). G, Multicellular head of short-stalked glandular trichome, abaxial epidermis of V. montevidensis (ESEM). Abbreviations: ESEM, environmental scanning electron microscopy; g, glandular trichome; LM, light microscopy; ng, nonglandular trichome; SEM, scanning electron microscopy. Bars: A, 250 µm. B, 150 µm. C–E, 50 µm. F, G, 10 µm.

All species have nonglandular and glandular trichomes in both epidermis (Figs. 1A, 1B, 1F; Fig. 2A; Fig. 3).

Figure 3  Anatomical features that differentiate the leaves of Verbena native species of Buenos Aires province, Argentina. Abbreviations: Ab, Trichomes in the abaxial epidermis; Ad, Trichomes in the adaxial epidermis. The number inside the oval indicates number of cells in head of glandular trichomes.

The nonglandular trichomes have unicellular body and two multicellular and radially arranged basal cells. The body of these trichomes is elongated, with a pointed tip and smooth or verrucose walls. They are distributed over both epidermal surfaces of the leaves and may be appressed or slightly appressed. Moreover, there are many unicellular patent trichomes (Figs. 1A, 1B, 1D, 1F; Figs. 2A–2E).

The glandular trichomes have a multicellular head and a stalk of varying length and they are distributed on both epidermal surfaces (Fig. 2A, Figs. 2C–2G).

All the leaves of these species have cladodromous venation, with incomplete vascularization on the leaf blade and margin, and pentagonal to polygonal areoles, generally with ramified veins (Fig. 1F).

Transverse section

The mesophyll is dorsiventral in all species studied, with three layers of palisade chlorenchyma. The epidermis has stomata on both surfaces (amphistomatic leaf) (Figs. 4A–4D). The cuticle is thin and slightly striated.

Figure 4  Leaf transverse section of Verbena native species of Buenos Aires province (LM). A, General view of V. litoralis. B, Detail mid-vein of V. montevidensis. C, Detail of abaxial epidermis and spongy chlorenchyma of V. bonariensis. D, Detail of adaxial epidermis and palisade chlorenchyma of V. bonariensis. E, Patent nonglandular trichome in V. rigida. F, G, Nonglandular trichome with cystoliths in the basal cells of V. bonariensis. H, Short-stalked glandular trichome of V. litoralis. I, Sunken short-stalked glandular trichome of V. litoralis. J, Long-stalked glandular trichome and nonglandular trichomes of V. intermedia. K, Long-stalked glandular trichomes of V. bonariensis. L, Adaxial epidermis of V. bonariensis. Abbreviations: lg, long-stalked glandular trichome; LM, light microscopy; ps, parenchymatous sheath; s, stomata; sg, short-stalked glandular trichome; black arrow, cystoliths. Bars: A, B, F, J, L, 100 µm. C–E, K, 50 µm. G, H, I, 25 µm.

The nonglandular trichomes have a multicellular base with cystoliths detected by microchemical test. Mucilages were not detected in these cells. The wall of the body cell is lignified (Figs. 4F–4G). The glandular trichomes are formed of a basal cell, a stalk cell of varying length and a bi- to multicellular head. If the stalk cell of glandular trichomes is short it may be sunken (Figs. 4H–4L). Each species has a particular type, density and distribution of trichomes (Fig. 3; Figs. 4A, 4B, 4E, 4F, 4H–4L).

Vascular bundles surrounded by a parenchymatous sheath were seen in all species studied (Fig. 4B).

Stem

Transverse section

The stems of all species have tetragonal to quadrangular transverse sections (Fig. 5; Fig. 6A). Nonglandular and glandular trichomes were observed in the unistratified epidermis as described for the leaf. The type of glandular trichome present in the stem is the same as that described for the leaf of the same species.

Figure 5  Stem transverse section of Verbena native species of Buenos Aires province. A, V. gracilescens. B, V. montevidensis. C, V. bonariensis. D, V. litoralis. E, V. intermedia. F, V. rigida. Bars: 500 µm.

Figure 6  Stem transverse section of Verbena native species of Buenos Aires province. A, General aspect of V. montevidensis (LM). B, Detail of stem angle in V. gracilescens (LM). C, Detail of the stem side in V. bonariensis (LM). D, Epidermis and hypodermis in V. litoralis (LM). E, Endodermis with Casparian bands in V. gracilescens (LM). F, Vascular bundle and cap of fibres in V. intermedia (LM). Abbreviations: c, collenchyma; e, endodermis; f, fibres; h, hypodermis; LM, light microscopy; s, stomata; black arrow, Casparian bands. Bars: A, 250 µm. B, C, 100 µm. D–F, 25 µm.

There is a layer of hypodermic cells underneath the epidermis (Figs. 6C–6D). Caps of collenchyma and sclerenchyma are found in the stem angles (Fig. 5; Figs. 6A–6B) and the chlorenchyma of cortex is interrupted in these areas (Figs. 6A–6C). All species studied have a continuous endodermis surrounding the parenchymatous cortex and vascular system (Figs. 6A–6C, 6E). Casparian bands have been observed in the endodermis in V. gracilescens and V. montevidensis (Fig. 6E), but no thickening of this type was seen in the rest of the species studied (Fig. 6C).

The vascular bundles are collateral and have caps of phloem fibres (Figs. 6A, 6B, 6F). All species studied show incipient secondary growth at the height of the fourth node from the apex (Fig. 5; Figs. 6A, 6C).

Discussion

The stems of the species studied show the characteristics of the Verbenaceae, such as the square transverse section and the presence of sclerenchyma and collenchyma caps at the angles, coinciding with that observed by Metcalfe and Chalk (1950), O’Leary et al. (2007) and O’Leary (2007).

According to Schnack and Covas (1944) and Botta (1989), the stem of Verbena has discontinuous cortical parenchyma. On the other hand, O’Leary et al. (2007) and O’Leary (2007) described two types of cauline anatomy for the genus: Type A: with continuous cortical parenchyma (V. rigida and V. gracilescens); Type B: with discontinuous cortical parenchyma (V. bonariensis, V. litoralis, V. montevidensis and V. intermedia). However, in our study all species including V. rigida and V. gracilescens show stems with discontinuous parenchyma (Type B), whereas both types of parenchyma distribution are observed in V. montevidensis and V. bonariensis (Types A and B).

Caps of angular collenchyma are seen in the angles of all stems. However, Souza et al. (2005) reported the presence of lamellar collenchyma in the stem angles of V. litoralis. The presence of a hypodermis in the stem of Verbena was also reported by Metcalfe and Chalk (1950) and Souza et al. (2005). On the other hand, some authors indicate that there is continuous collenchyma around the perimeter of the Verbena stem (Botta 1989; O’Leary 2007; O’Leary et al. 2007) rather than a hypodermis, but this has not been seen in the species considered in this study.

The endodermis is seen to be continuous around the perimeter of the stem in all species and Casparian bands are present in the endodermal cells of V. gracilescens and V. montevidensis. These features are cited for the Verbenaceae and Verbena by Metcalfe and Chalk (1950, 1979), O’Leary et al. (2007) and O’Leary (2007). However, Souza et al. (2005) considered that this layer of cells was a starch parenchymatic sheath.

The sclerenchyma, represented by caps of fibres in the stem angles and phloem fibres, of Verbena species studied is similar to that described for other genera of the family (Barboza et al. 2001; Souza et al. 2005; O’Leary et al. 2007).

The leaves of Verbena species considered in this study present the general characteristics of the Verbenaceae family and specifically those of the Verbena genus (Metcalfe & Chalk 1950, 1979; Inamdar 1969; Souza et al. 2005; O’Leary et al. 2007): nonglandular and glandular trichomes of different types, stomata on both epidermal surfaces and heterogeneous mesophyll.

The presence of anisocytic and anomocytic stomata has also been reported for other genera of the Verbenaceae (Metcalfe & Chalk 1979), in Acantholippia, Aloysia and Lippia (Barboza et al. 2001) and in Glandularia (Botta 1993). However, Souza et al. (2005) reported paracytic stomata in V. litoralis and Inamdar (1969) found diacytic ones in V. venosa (Syn. V. rigida Spreng.). The high stomatal density seen in V. litoralis shows a ratio of 3:1 stomata (abaxial surface:adaxial surface), as cited by Souza et al. (2005) for Verbena species.

The species in this study show nonglandular unicellular trichomes with multicellular bases with cystoliths as in other representatives of the family (Inamdar 1969; Metcalfe & Chalk 1979; Botta 1993; Barboza et al. 2001). This type of trichome has been previously cited for the Verbena genus (Inamdar 1969; Mathew & Shah 1983; Cañigueral et al. 1998; Souza et al. 2005; O’Leary et al. 2007).

The different types of glandular trichomes observed were previously cited for representatives of the family (Metcalfe & Chalk 1950, 1979; Inamdar 1969; Mathew & Shah 1983; Botta 1993; Barboza et al. 2001). Cañigueral et al. (1998) report the presence of long-stalked glandular trichomes and others with a short stalk and tetracellular head in V. officinalis. On the other hand the presence of long-stalked glandular trichomes is refuted by Souza et al. (2005).

Although the general anatomical features of all Verbena species considered in this study coincide with those reported for the Verbenaceae, some anatomical parameters and characteristics have been found that are valid for differentiating between the species of Buenos Aires province. These distinctive anatomical characters can be applied to taxonomic revisions of the genus and also aid the identification of Verbena native species of Buenos Aires province as crude drugs.