Introduction

Dasylirion cedrosanum Trel., Commonly known as "Sotol" it is a semi-cylindrical perennial, dioecious, polycarpic and belongs to the Asparagaceae family (Trópicos.org). It is a species with greater presence in the northeast region of the country, mainly in the states of Chihuahua, Durango, Zacatecas and Coahuila. In Coahuila rosetophilous grows in scrubland, which represented 6% of all vegetation state (^{Rzedowski and Rzedowski, 1990}). It has great commercial importance, standing out in the beverage industry in the northeastern region of the country. Currently the aforementioned states have been given the designation of origin (^{Rodríguez-Gómez, 2007}). This requires an assurance of product quality to consumers but also points to a rational use.

In traditional classification systems, gender Dasylirion has been placed in different families, including Agavaceae, Liliaceae and Dracaenaceae (^{Dahlgren et al, 1985}). This has depended on taxonomic nature of the study that has been used, being morphological similarity to several species of these families. It was recently placed in the family Asparagaceae and subfamily Nolinoideae (^{USDA, ARS, National Genetic Resources Program GRIN, 2013}). The Dasylirion genus consists of 16 species, all with distinctive features (^{Melgoza and Sierra 2003}). One is the shape of the leaves. On this, ^{Bogler (1994)} mentions that the leaves of the plants of this genus have some variations such as the length and breadth, the orientation of the thorns in the margin, the presence or absence of epicuticular wax and the shape of its surface foliar. The presence of different types of crystals in the monocot mesophyll have proved taxonomic importance to distinguish between some families (^{Prychid and Rudall, 2000}).

The aim of this work is to study the morphology and anatomy of the leaves of the species D. cedrosanum at different stages of development in order to identify possible changes in the morpho-anatomical development of the species.

Materials and methods

Anatomy of the leaf surface. The experiment was conducted at the University Autonomous Agrarian Antonio Narro. The plant material used consisted of Sotol plants (6, 30, 60 and 84 months old), specimens were collected from botanical nursery at the same university. The variables studied were; abaxial and adaxial stomatal density, stomatal index abaxial and adaxial. To study the leaf anatomy two erect leaves of the middle portion of caudex four plants of each age (6, 30, 60 and 84 months), in order to differentiate the degree of development of tissues were collected. Each sheet an impression of the abaxial and adaxial surfaces for this purpose a film of transparent nail polish on the leaf surface with a brush was applied after the film was dried, it was removed with a piece of tape was obtained transparent adhesive, which is mounted on a slide.

In each print were observed at random five fields per leaf under a microscope VistaVision mark with the objective 40 X, each observation the number of stomata and epidemial cells were counted. With the data obtained stomatal density and stomatal index was estimated as follows: DE= number of stomata/0.1589 mm² (area of the visual field)= stomata per mm². The following formula is used to determine the stomatal index (IE):

IE= DES/DEP + DES x 100

Where: IE= stomatal index, DES= stomatal density (stomata per mm²), DEP= density of ordinary epidemial cells (cells per mm²) in the same unit area.

The morphological description was made according to the terminology ^{Carpenter (2005)}.

Histology of epidemial, fundamental and vascular tissues. For analysis of tissues were extracted cross and paradermal sections of the middle part of the leaves, in this section the following variables were studied: Cuticle thickness (μm), epidermal cell size (μm²), parenchymal layers of palisade, size of palisade parenchyma (μm²), layers of spongy parenchyma, size of spongy parenchyma (μm²), size sclereids (μm²), axis right-left mesophyll (μm), axis abaxial-adaxial mesophyll (μm), number of vascular bundles, xylem vessel elements (μm²), elements of the phloem tube (μm²).

The handling of tissues was performed following the methodology proposed by ^{Hernández (1990)}, first fixed in FAA (formaldehyde (36-40%) 5 ml, Ethyl alcohol (70%) 90 cc and glacial acetic acid 5 ml). which is achieved with stop cell tissue metabolism. After seven days, dehydrated at hourly intervals alcohol 60%, 70%, 85% and 96% and in mixtures absolute-xylol alcohol in proportions 3:1, 1:1 and 1:3 and they embedded in paraffin.

They cut 20 μm thick were obtained using a microtome rotation and adhered to a carrier with adhesive Haupt. The tissues were stained with safranin and fast green according to the abovementioned methodology same author. The microscopic observation were taken for five random fields in different objectives (5X, 40X and 100X). The microscope mark VistaVision was used with integrated digital camera Pixera Wiender Pro. The measurements of the images of tissues were performed with 4.6 Axion Vision software. The description of the anatomical characteristics was made according to the terminology ^{Evert (2006)}.

Results and discussion

Anatomy of the leaf surface. According to stomatal types of ^{Prabhakar (2004)}, the stomata are amphistomatic of paracytic type. In monocots the stoma paracytic type only has been observed in the species of Poaceae and Cyperaceae family (^{Zarinkamar, 2006}). ^{Stebbins and Khush (1961)} studied the stomatal complex of 192 species of monocots, finding a correlation between the number of stomata subsidiary cells of other plant characteristics such as growth habit of mature plants and geographical distribution.

The stomatal density of adaxial and abaxial surfaces ranging from 46.8-65.0 stomata mm^-2 and density between tabloids 163 and 339 cells mm^-2. Stomatal index shows values between 14.0-23.3 (Table 1). In the evaluation of the four ages revealed that the stomatal density is higher in plants Sotol of 7 years both in the adaxial surface with 63.22 stomata mm^-2 with 60.22 abaxial stomata mm^-2 and a lesser amount plants 2.5 years in the adaxial surface with 41.44 stomata mm^-2 and abaxial with 39.2 stomata mm^-2. For surfaces, stomatal density is slightly higher in the adaxial surface with an average of55.015 stomata mm^-2 per 51.705 stomata mm^-2 in the abaxial surface. In any case significant differences were observed between the two surfaces.

In assessing the stomatal index turns out to be low, aspect observed in other monocot species growing in arid areas, as is the case of the species Urginea indica has a 14-25 stomatal index, which is similar to D. cedrosanum (Liliaceae) (^{Kameshwari, 2011}). The abaxial stomatal dominance is a common feature in several species of monocots and dicotyledonous with ambistomatal stomata (^{Croxdale, 2000}); however, this difference D. cedrosanumnot observed. The numerical difference of stomata on both surfaces, rather it has been linked to environmental factors such as CO₂ concentration, light intensity or the availability of water. (^{Radoglou and Jarvis, 1990}; ^{Kouwenberg et al, 2003}).

Table 1 Mean values of the leaf surface, epidermal tissues, fundamental and vascular Sotol sheets Dasylirion cedrosanum Trel. 

†Medias. *desviación estándar. Ad= adaxial; Ab= abaxial.

Furthermore, there is evidence suggesting that depending on the composition of the epidermal wax, stomatal index may increase in one of the two surfaces (^{Holroyd et al., 2002}).

Moreover, the stomata occur differently in different environments (^{Qiang et al., 2003}).A recurring modification of plants is in the distribution and stomatal frequency, which have been used as taxonomic characters below the family level in angiosperms, as well as its phylogeny (^{Mukherjee et al, 2000}). Within the Asparagaceae family, subfamily nolinoideae has been little studied in their anatomy, and even less in the genus Dasylirion (^{Bogler, 1994}). As a result of some phylogenetic studies published and based on morphological and molecular evidence, Dasylirion has been placed as a subfamily nolinoideae within Asparagaceae (^{APG III, 2009}).

The subsidiary cells are arranged in columnar form of plates, they are arranged at the ends and sides of the axis of the stomatal opening, giving a frame. Stomata are arranged in parallel rows to the longitudinal axis of the sheet in three to six sets stomata in the middle of the sheet. The cells are elongated tabloids polygonal oriented in the same way that the stomata (Figure 1A). The epidermis is glaucous. This color is produced by the presence of epicuticular wax, which is oriented in parallel, arrangement called "Convallaria type" (^{Barthlot et al., 1998}). It presents outstanding buds, clustered and aligned with the stomata which places at a lower level.

Figure 1 Leaf anatomy of Dasylirion cedrosanum. A. adaxial surface at 10 plants X in plants with 6 months. B. Cross section at 40 X of the abaxial epidermis in plant with 6 months. C. Mesophyll at 5 X of plant with 6 months. D. Mesophyll at 5 X ofplant with 84 months: E. Face vascular first order plant mesophyll of planst with 84 months. F. Face vascular third order plant mesophyll of planst with 84 months. C= wax; Cr= crystals; Pp= papillae; Ce= epidermal cells; co= guard cells; Ce= subsidiary cells; C = substomatal camera; Ct= Cuticle; P= parenchyma; Pe= palisade parenchyma; Ps= spongy parenchyma; Hv= vascular bundle; Mx= metaxylem; Mf= metaphloem; Pf= obliterated protophloem; Px= obliterated protoxilema. 

And adaxial epidermal cells abaxial, cross-sectional views, are unistrate isodiametric with primary walls and smooth both in the epidermis abaxial and adaxial. They have on average a cuticle thicker in adaxial side (Table 1). Only epidermal cells of plants adaxial surface 84 months old, are significantly larger, while the abaxial surface shown otherwise. The stomata guard cells is sunken below the level of epidermal cells (Figure 1B).

The mesophyll is isolateral (Figure 1 C, D). The cells palisade parenchyma are of prismatic shape, and observed multilayered are developed to both surfaces. The spongy parenchyma cells form shows isodiametric. The number of layers of spongy parenchyma increases as the plant is older; adaxial side showed greater number of strata (Table 1). In plants 30, 60 and 84 months mesophyll fibrous tissue has a size between 71-103 μm, similar in all ages. These fibers are associated with vascular bands, taking the form of "Y" or inverted in abaxial and adaxial rows respectively (Figure 1 E, F), which extend towards the epidermis as beams. Furthermore, in the mesophyll it is possible to observe the presence of crystals or prismatic type styloid cuboidal shape, which are solitary in the parenchyma cells and close to the vascular bundles sclerenchymatous (Figure 1F).

Several features that have the leaves of plants, the leaf area are perhaps the most significant from the point of view systematic and phylogeny. The structure of epicuticular waxes characterizes large groups of monocots. In these has been observed in abundance two types of waxes, which have added rodlike longitudinally oriented and those in the form of parallel plates, the latter found in the family Nolinaceae (^{Chase et al, 1995}).

The mesophyll in D. cedrosanum has powers that allow the plant to adapt in arid environments. For example, the ipsilateral leaf arrangement allows the species to be able to large photosynthetic rates per unit biomass in high brightness environments even in semiarid environments (^{Knight and Robert, 1994}). The presence of fibers also aid in the transport of water through palisade tissues (^{Rotondi et al, 2003}). Another important feature is the presence of prismatic crystals cuboidal form which has been observed in other species belonging to different orders of monocots as Asparagales, Liliales and Pandanales. It is not known with certainty the role they present in tissues, often mentioned that their function is to serve as a repository of metabolic waste which could be toxic to cells or tissues (^{Prychid and Rudall, 1999}); however, it has been recognized that this type of glass can be taken as a taxonomic character (^{Prychid and Rudall, 2000}).

The vascular bundles, are organized in bands, arcuate positioned in three rows parallel to the curvature mesophyll recognized as adaxial row, central and abaxial. In particular the arrangement of each band in these plants can be categorized similar to the manner succulents. This is due to the different arrangement of xylem and phloem in each depending on their size (Cutler et al, 2007). For example, the larger representing the first order (Hv1), with the difference that the set of vessels metaxylem is separated in the middle by protoxylem obliterated. The phloem set in these bands are available to the abaxial pole (side beam) separated by a beam sclerenchymatous cells, which together with the xylem phloem enclose the whole (Figure 1E).

It is also the second order (Hv2), wherein there is obliterated and phloem protoxylem whole is divided by sclerenchyma. And the third order (Hv3), in which besides the absence protoxylem, shows that the phloem set is not divided by sclerenchymatous tissue (Figure 1F). The amount of vascular bundles is enhanced by the adaxial side and increases as the plant grows (Table 1). The size is similar in all ages. The vessel elements have simple perforation, with thickened walls.

Moreover, the arrangement of vascular tissue observed in this species has been interpreted as an adaptation to rapid water intake for short periods of water availability (^{Carlquist and Schneider, 2006}).

Conclusion

Dasylirion cedrosanum is a species that has several foliar anatomy similar to other species of the same genus characteristics. The variables stomatal density, stomatal index and density of epidermal cells do not exhibit differences between ages. The palisade parenchyma increases with age. In this study highlights some morphological characteristics of leaves in which the arrangement of mesophyll tissues could be characteristic of the species.