Leaf anatomy of six species of Heliotropiaceae Schrad. from the Brazilian semi-arid region

anatômica A anatomia foliar Abstract The family Heliotropiaceae has more than 450 species, mainly distributed in the tropics and subtropics. In Brazil, it is represented by the genera Euploca Nutt., Heliotropium L., Myriopus Small and Tournefortia L. The aim of this study was to describe the leaf anatomy of six species of Heliotropiaceae recorded in the semi-arid region of Brazil: E. polyphylla (Lehm.) J.I.M. Melo & Semir, E. procumbens (Mill.) Diane & Hilger, H. angiospermum Murray, H. curassavicum L., M. rubicundus (Salzm. ex DC.) Luebert and M. salzmannii (DC.) Diane & Hilger. Besides contributing to the the Caatinga (Brazilian savannah), we also sought to determine and highlight the anatomical features adaptive to the region and to identify features with potential diagnostic value. Leaf structures were analyzed by light microscopy and scanning electron microscopy. Additional tests were also carried out to detect starch and total lipids. The morphological variation and the distribution of trichomes were the most relevant features for species diagnosis. The distribution of stomata in M. salzmannii and H. angiospermum differed from that described in other papers, which demonstrates the anatomical plasticity of these species. This is the irst report describing the leaf blade of M. rubicundus .


Introduction
The family Heliotropiaceae is mainly distributed in the tropics and subtropics and comprises ive genera and approximately 450 species (DIANE et al., 2002;GOTTSCHLING et al., 2004;LUEBERT et al., 2011). In Brazil, it is represented by the four genera Euploca Nutt., Heliotropium L., Tournefortia L. and Myriopus Small (MELO et al., 2009).
Tournefortia comprises approximately 150 species distributed in the Neotropics. Its representatives are allocated into two sections: Tournefortia sect. Tournefortia I.M. Johnst., with straight embryo and slightly lobed fruits; and T. sect. Cyphocyema I.M. Johnst., with curved embryo and a four-lobed fruit (CAVALHEIRO et al., 2011). In Brazil, both sections are represented by approximately 25 species (MELO et al., 2009). The inclusion of all species of T. sect. Cyphocyema in the genus Myriopus has been suggested by phylogenetic studies combined with morphological data, showing T. sect. Tournefortia as a polyphyletic group (DIANE et al., 2002). The species chosen for this study are in the genus Myriopus.
Euploca has approximately 50 species distributed in tropical, subtropical and temperate regions with centers of diversity in Africa, Australia and tropical America, especially in South America and Mexico. Its representatives are predominantly herbaceous, subshrubs and rarely shrubby SEMIR, 2010). In Brazil, it is represented by 17 species (MELO et al., 2009).
The genus Heliotropium includes approximately 300 species, distributed in tropical and subtropical regions around the world IBÁÑEZ et al., 2011). It includes herbaceous, subshrubs, and more rarely, shrub representatives, where they are mostly characterized by scorpioid inlorescences, free stamens, and schizocarp fruit with two locules and two seeds SEMIR, 2008). In Brazil, the genus is found along the coast (Restinga and Atlantic Forest formations) and on rocky outcrops and is widespread in the Cerrado as well as Caatinga (Brazilian savannah) and Pampas (Brazilian grassland) (MELO; SEMIR, 2008). Heliotropium comprises nine species (MELO et al., 2009).
Anatomical features have been used in taxonomic and phylogenetic analysis of Heliotropiaceae, but few representatives of the Brazilian lora, particularly from the Caatinga, have been included (DIANE et al., 2003;FARIÑA et al., 2003;MUHAIDAT et al., 2011). In the Caatinga, studies on Heliotropiaceae are limited to taxonomic aspects (MELO; SALES, 2005;ANDRADE, 2007;VIEIRA et al., 2013).
This study aimed to describe the leaf anatomy of six species of Heliotropiaceae found in the Brazilian semi-arid region, Caatinga biome: Euploca polyphylla, E. procumbens, Heliotropium angiospermum, H. curassavicum, Myriopus rubicundus and M. salzmannii. Besides contributing to the anatomical knowledge of the representatives of Heliotropiaceae in the Caatinga, we sought to determine and highlight the anatomical features adaptive to the semi-arid region, looking for characters with potential diagnostic value.  (JOHANSEN, 1940) and preserved in 70% ethanol. The samples were dehydrated in an ethanol series and embedded in Historesin® Leica (GERRITS; SMID, 1983). Transverse and longitudinal sections of 5-7 µm thickness were obtained with a rotary microtome and stained with 0.05% toluidine blue in acetate buffer (pH 4.7) (O'BRIEN et al., 1964). All slides were temporarily mounted in water. The images were captured with a digital camera (Olympus DP71) coupled to a light microscope (Olympus BX51). Histochemical assays for starch (BERLYN;MIKSCHE, 1976) and total lipids (PEARSE, 1980) were performed using Lugol and Sudan black B, respectively.

Material and Methods
For micro-morphological analysis, the samples were dehydrated in an ethanol series, dried by the CO 2 critical point method, mounted and metalized with gold. The observations and images were obtained by scanning electron microscopy (SEM) using a Jeol JSM 5800 LV at 10 kV with a coupled digital camera. The morphological description of the trichomes was according to Diane et al. (2003) and Metcalfe and Chalk (1979).

Results
The following descriptions are based on the results of light microscopy and SEM.

Euploca polyphylla and E. procumbens
The leaf blades of E. polyphylla and E. procumbens have a uniseriate epidermis on both surfaces ( Figure 1A-B). In paradermic sections, both have curved anticlinal walls ( Figure 1C-D). The epidermal cells are slightly rounded and have irregular size in transversal sections ( Figure 1A-B). The leaf blades are amphistomatic ( Figure  1A-D). The stomata of E. polyphylla are anomocytic ( Figure 1C), while those of E. procumbens can be anomocytic and tetracytic ( Figure 1D). E. polyphylla has dorsiventral leaves; the palisade parenchyma is single-layered and the spongy parenchyma has 5-7 layers ( Figure 1A). E. procumbens has isobilateral leaves with a layer of palisade parenchyma facing each of the sides and 3-4 layers of spongy parenchyma between the palisade parenchyma ( Figure 1B). The parenchyma cells of both species accumulate starch in their interior, evidenced by the positive reaction with Lugol ( Figure  1E-F). The midribs are plano-convex, and the midrib region follows the pattern of the mesophyll; however, the palisade parenchyma is only on the upper surface ( Figure  1G Unicellular non-glandular, simple and tapered trichomes were found in the two species (Figure 2A-F) on both surfaces. The abaxial surface of E. polyphylla has a homogeneous distribution of trichomes ( Figure  2A), while the adaxial surface has trichomes only in the central region ( Figure 2B). The trichomes of E. polyphylla are relatively short (90-250 µm), appressed to the leaf surface and have protuberances on the surface, and the adjacent epidermal cells form a high base ( Figure 2C-D). In E. procumbens, the trichomes are long (400-500 µm), curved on the surface, but not appressed or with micropapillae on the surface ( Figure  2E-F). The adjacent epidermal cells also form a high base around the trichomes ( Figure 2F). The density of the trichomes is more pronounced in E. procumbens with homogeneous distribution on the adaxial and abaxial surfaces ( Figure 2E).  Figure 3C). H. angiospermum has dorsiventral leaves, the palisade parenchyma is single-layered and the spongy parenchyma has 3-6 layers ( Figure 3A). H. curassavicum has isobilateral leaves with the palisade parenchyma showing 3-4 layers and parenchyma cells (3-5 layers) in the central region of the blade, not characterized as spongy parenchyma ( Figure 3B). Lugol tests were negative for both species. Lithocysts can be seen ( Figure 3A-B and D). Collateral vascular bundles are present in the two species (Figures 3E-F and 4B); the midrib of H. angiospermum is convex-concave and the cortical region is composed exclusively of parenchyma cells ( Figure 3E). The lateral bundles have a sheath of parenchyma cells that stains purple with toluidine blue, ( Figure 3F); histochemical tests with Sudan black B indicated the production of lipids ( Figure 4A). The midrib of H. curassavicum is plano-convex and the midrib region follows the pattern of the mesophyll ( Figure 3B).  The leaf surface of H. curassavicum is glabrous on both surfaces, while H. angiospermum has nonglandular, simple, tapered trichomes, located only on the main vein on both sides. The trichomes are long (540-600 µm), not appressed and with small micropapillae on the surface, and the adjacent epidermal cells form a high base around the trichome ( Figure 4C). In the same species, we also observed glandular trichomes consisting of peduncle and secretory portion ( Figure 4D).  Figure 5A-B). The midrib of M. rubicundus is convex-concave ( Figure 5E), and the cortical region displays angular collenchyma ( Figure 6A). In M. salzmannii, the vascular bundle of the midrib forms a semi-arc, open at the top end ( Figure 5F), and the cortical region has annular collenchyma ( Figure 6B). Unicellular, non-glandular, simple and tapered trichomes are present in the two species on both surfaces (Figures 6 C-F and 7 A-C). The trichomes of M. rubicundus are quite variable in size (280-550 µm) and appressed ( Figure 6C-D), and they have a rough surface ( Figure 6D), in addition to possessing cystoliths at their base ( Figure 6E). In M. salzmannii, the density of the trichomes is more pronounced on the abaxial surface ( Figures 6F and 7A), and two types of trichomes are distinguished. The irst one is distributed on both surfaces ( Figures 6F and 7A), has a bulbous base, is erect, measures 100-500 µm and has a smooth surface ( Figure 7B). The second type, found in a smaller number, is quite similar to the trichomes described in E. procumbens. It is long (400-500 µm), with the surface covered by micropapillae, curved in relation to the leaf surface and distributed on the abaxial surface, primarily on the main vein ( Figure 7C).  Among the species studied, we observed four types of trichomes, all simple and tapered: type 1 -appressed, with micropapillae on the surface, without cystoliths in the cells of the trichome base; type 2 -curved, with micropapillae on the surface and the adjacent epidermal cells forming a high base; type 3 -appressed, with micropapillae on the surface and with cystoliths in the epidermal cells; type 4 -upright, bulbous and smooth surface. In Table 1, we summarize the main features found in the species studied.

Discussion
This study showed that there was variation in the leaf anatomy of the species examined. However, certain features were present in all of them and are typical of the Heliotropiaceae. The presence of crystals (lithocysts) and non-glandular trichomes are important structures for species of the family (METCALFE; CHALK, 1983).
Non-glandular trichomes are often used in the separation of Heliotropiaceae species, extensively described in the work of Diane et al. (2003). The morphological variation of trichomes is broadly discussed as a useful feature for identifying species in several families of Angiosperms (WERKER, 2000), including Heliotropiaceae (METCALFE; CHALK, 1983;DASTI et al., 2003).
Variations in length, curvature and presence/ absence of cystoliths occur in the trichomes of different species of the same genus, or between species belonging to different genera CHALK, 1983). For Leaf anatomy of six species of Heliotropiaceae Heliotropiaceae, Diane et al. (2003) described seven types of non-glandular trichomes. In this study, we found three types of non-glandular trichomes itting the descriptions given by Diane et al. (2003): unicellular, unbranched trichomes with rough surface and adjacent epidermal cells forming a high base (type 1); multicellular complex -adjacent epidermal cells containing cystoliths (type 3); and unicellular, unbranched trichomes with bulbous base (type 4). Type 2 (curved, with micropapillae on the surface and adjacent epidermal cells forming a high base) was not described in the work of Diane et al. (2003). In their work, the trichomes with adjacent epidermal cells forming a high base are always appressed, whereas the type 2 is curved in relation to the leaf surface.
Crystals also have taxonomic value in the family CHALK, 1983;DIANE et al., 2003). According to Diane et al. (2003), crystals are often found in the species of the genera Tournefortia (including Myriopus) and Heliotropium. In this study, crystals were reported in the species of Heliotropium (in the lithocysts) and also in the cells of the non-glandular trichome base of M. rubicundus. Cystoliths in the basal part of the trichomes are common in representatives of the family, and sometimes in the epidermal cells adjacent to the trichomes (METCALFE; CHALK, 1983). The lithocysts observed in the two species of the genus Heliotropium are considered by Diane et al. (2003) as reduced unicellular trichomes. However, to conirm such a hypothesis, an in-depth study is required aimed at reconstructing the evolutionary history of trichome morphology in the family.
The mesophyll showed great diversity between the species examined, ranging from isobilateral to dorsiventral in a single genus. Therefore, it should not be used as a diagnostic character between species of different genera. The same is true for characteristics such as size and shape of epidermal cells and type of stomata.
Overall, the leaf anatomy of M. salzmannii described here corresponds to the observations made by Diane et al. (2003) for the same species. However, differing from the data described in the above cited work, the leaf of M. salzmannii in the Caatinga environments was hypostomatic, while the specimen examined by Diane et al. (2003) was amphistomatic. The same occurs in H. angiospermum, described by Diane et al. (2003) as amphistomatic; in our study, leaves were found to be hypostomatic. According to Kundu and Tigerstedt (1998), hypostomatic leaves are typical of xeric environments, which allow great water savings by reducing the rate of transpiration. Another different aspect was shown in relation to the types of trichomes of M. salzmannii: Diane et al. (2003) described only type 2 trichomes, and in the present investigation, we also found non-glandular trichomes of bulbous base.
In relation to Euploca, we also found differences in relation to what is described in the literature. In this study, we found only one type of non-glandular trichome in E. procumbens, i.e., curved over the surface, not appressed and with micropapillae. Diane et al. (2003) described two types of trichomes for this species: (1) trichomes with micropapillae on the surface and (2) multicellular complex of lithocysts and slender trichome with the base at the level of the epidermis. This feature may be related to the general environment of the species (Caatinga).
The leaf anatomy of H. curassavicum was in accordance with that described by Diane et al. (2003). However, the authors stated that the species had druses in the mesophyll, structures not found in the present study. The number and size of the crystals vary in different periods of the year and also according to the availability of limestone material in the soil (METCALFE; CHALK, 1983), which explains their absence in this work.
Among the species belonging to the genera studied, we found characters with potential diagnostic value. The main feature that differentiates E. procumbens from E. polyphylla is the Kranz anatomy present in the latter, which was already observed in the work of Muhaidat et al. (2011). In addition, the type and distribution of the trichomes are also important. The main difference between H. curassavicum and H. angiospermum is the absence of trichomes in the former, while the latter displays non-glandular and glandular trichomes. The species M. salzmannii and E. procumbens differ anatomically primarily by the types of trichomes they have.
The results obtained can assist in identifying the leaf features of Heliotropiaceae species found in the Caatinga. The anatomical description of M. rubicundus is presented for the irst time. Several features were different in relation to the studies already carried out for the same species, for example, the distribution of the stomata in M. salzmannii and H. angiospermum, which may be related to environmental factors, since the leaf blade is the structure that changes the most in response to environmental changes (ELIAS et al., 2003).
Because of the great importance of the morphology of trichomes in Heliotropiaceae, we suggest a study encompassing a greater number of species of this family, also including the genus Ixorhea Fenzl, in which species are monospeciic and endemic to Argentina. The overall aim would be to determine the types of trichomes and their use in phylogenetic approaches regarding this family.