Morphological diversity of foliar trichomes in Asteraceae from Sand-fields of the Pampa biome, Rio Grande do Sul State, Brazil

ABSTRACT Asteraceae is the largest family of angiosperms and occurs predominantly in grassland areas. This study aimed to identify and characterize the foliar trichomes of 34 Asteraceae species from Sand-fields of the Pampa biome, by means of epidermal analyzes (front and transverse view) under light and scanning electron microscopy. Eleven types of trichomes were identified and characterized: three glandular (recurved on the epidermis, erect-capitate and uniseriate to multiseriate vesicular-capitate) and eight non-glandular (simple conical, flagellate-filiform, aseptate-flagellate, whip-like, oblique-flagellate, branched with one arm, branched with two T-shaped arms, branched with three or more arms). The most representative glandular type was the uniseriate to multiseriate vesicular-capitate (58%) and the non-glandular type was the simple conical (35%). A large number of trichomes is an adaptive strategy to the adverse conditions of the Pampa biome and its morphological diversity can be useful in the family systematics.


Introduction
Asteraceae is the largest of the angiosperm families, with approximately 23,000 species distributed in over 1,600 genera, 13 subfamilies and 44 tribes (Funk et al. 2009, Panero et al. 2014. Its representatives are predominantly herbaceous, with simple and usually lobed leaves, inflorescence of the capitulum type and fruit of the cypsela type, which, along with the pappus, characterize its unit of dispersion (Roque & Bautista 2008, Funk et al. 2009). In Brazil there are 2,100 species, 290 genera and 28 tribes of the family (Flora do Brasil 2020), with Vernonieae, Eupatorieae, Heliantheae, Astereae, and Gnaphalieae being the richest tribes according to floristic surveys of the family in some localities (Nakajima & Semir 2001, Almeida et al. 2005, Ritter & Baptista 2005, Heiden et al. 2007, Beretta et al. 2008, Fernandes & Ritter 2009, Borges et al. 2010.
Asteraceae is one of the most successful plant families, because of the great diversity of secretory structures, the specialized methods of pollination and dispersion and phenotypic plasticity that contribute to the high number of species and their distribution in almost all habitats and vegetation formations (Smiljanic 2005, Beretta et al. 2008. However, a greater occurrence of the family is observed in open and arid areas (grasslands and savannas) and of high altitude and, less commonly, in forest environments (Barroso et al. 1991, Anderberg et al. 2007. In Brazil, Asteraceae is distributed predominantly in the grassland formations of the Pampa biome (Ritter & Baptista 2005, Cancelli et al. 2007, which in the country is restricted to the State of Rio Grande do Sul and presents mixed vegetation of grassland formations and forests, with a predominance of grasslands (Overbeck et al. 2007). In addition, the Pampa biome presents different phytophysiognomic units (Seasonal Forest, Central Fields, Barba-debode Fields, Deep-soil Fields, Savannoid Vegetation, Coastal Fields, Sand-fields, Parkland Vegetation, Shallow Soils and other formations) , among which the Sand-fields (southwest of the State) are distinguished by the high species richness of Asteraceae (77 spp.) in relation to the other families, being Astereae (17), Eupatorieae (12), Gnaphalieae (10) and Vernonieae (nine) the most representative tribes .
The high representativeness of Asteraceae in the Sand-fields is possibly a result of the morphoanatomical adaptations of these plants, which allow their survival under extreme and limiting local environmental conditions such as sandy and low-fertility soils, water deficit, strong winds, high temperatures and high luminosity (Azevedo & Kaminski 1995, Klamt & Schneider 1995, Marchiori 1995, Freitas et al. 2009, Overbeck et al. 2015. These adaptations, usually xeromorphic, involve the presence of narrow leaves to reduce the surface area exposed to sunlight, presence of xylopodia and/or thick subterranean organs, presence of secondary metabolites to protect against predation, and a large number of trichomes on the vegetative organs (Lindman & Ferri 1974, Boldrini 2009).
Trichomes have a fundamental role in protection against high temperatures, excessive light, pathogens and herbivores (Werker 2000, Wagner et al. 2014). In addition, they are one of the most useful characters used in taxonomic studies of many angiosperms due to their almost universal presence among taxa, easy observation and morphological diversity (Faust & Jones 1973, Payne 1978, Adedeji & Jewoola 2008, Krak & Mráz 2008.
Trichomes can occur on vegetative and reproductive organs, varying in type and density over all parts of the plant and across different taxonomic levels (Werker 2000, Adedeji & Jewoola 2008. Due to the high diversity of forms, sizes, functions, modes, and types of secretion, the classification of trichomes is wide, and the distinction between glandular (secretory) and non-glandular trichomes (non-secretory) is one of the most widely used categorizations (Werker 2000, Evert 2007, Krak & Mráz 2008, Wagner et al. 2014.
Considering the ecological importance of trichomes and representativeness of Asteraceae in the Sand-fields of Rio Grande do Sul (important area for conservation of native vegetation), this study aimed to identify and characterize the foliar trichomes observed in the species of the family occurring in this place, contributing to the knowledge of these structures and supporting taxonomic work in the Asteraceae family.

Material and methods
The 34 Asteraceae species studied (table 1) were collected in the southwestern region of Rio Grande do Sul -Brazil, in the municipalities of Alegrete, Manoel Viana and São Francisco de Assis (between latitudes 29°00'S and 31°00'S and longitudes 54°30'W and 58°45'W), in areas of natural grasslands of sandy soils susceptible to the sandy patch process, with or without influence of livestock activity .
In this region the climate is humid subtropical (Moreno 1961) with an average annual rainfall of 1400 mm (Nimer 1979); however, rains are poorly distributed, with periods of reduced rainfall interspersed with heavy rains that can concentrate rain in just one day (Freitas et al. 2009). Due to high evaporation, periods of water deficit can occur (Berlato et al. 2006). The mean annual temperature is 14.3 °C in winter and 26.3 °C in summer (Nimer 1979), although absolute minima and maxima are -4 °C and 40 °C, respectively (Cordeiro & Soares 1977).
The relief, originating from the Botucatu sandstone formation, is gently undulating punctuated with flat areas. The soil is Ortic Quartzarenic Neosol (Streck et al. 2008), with arenous and silty-arenous texture, high acidity, phosphorus and potassium deficiency, an excess of aluminum and difficulty in water retention (Azevedo & Kaminski 1995, Klamt & Schneider 1995, Verdum 2004). These characteristics reduce its fertility, making it highly susceptible to erosion and water scarcity in periods of drought (Azevedo & Kaminski 1995, Klamt & Schneider 1995. The collections were carried out in the months of May  For the anatomical analyses in light microscopy (LM), three fertile individuals of each species were collected, and fully expanded leaf blades were fixed in FAA 50 (37% formaldehyde, glacial acetic acid and 50% alcohol in a ratio of 1:1:18) (Johansen 1940) and preserved in 70% alcohol. For leaf blade observation in frontal view (FV), fragments of the leaf medial region were dissociated with hydrogen peroxide and acetic acid at a concentration of 1:1 (Franklin 1945, modified) and then stained with basic fuchsin in 50% alcohol. The trichomes were also analyzed in transverse view of the leaf blade, and cross-sections (CS) were performed by hand, with the aid of a razor blade, in the median region of the leaf blades of the species under study. Cross-sections were clarified with 50% sodium hypochlorite, stained with alcian blue and basic fuchsin (Kraus et al. 1998) and then mounted on semi-permanent, glycerinated gelatin (Kaiser 1880).
All FV and CS images were captured using a DP041 digital camera coupled to an Olympus Bx70 photomicroscope, using the DP Controller program, at the Photomicroscopy Laboratory of the Center for Biological Sciences and Health of the Universidade Estadual do Oeste do Paraná -Unioeste, Cascavel, PR.

Results and Discussion
Based on the proposal of Ramayya (1962) and complementary literature, 11 types of trichomes were identified and characterized, of which three were glandular and eight non-glandular. Most of the analyzed species present both glandular and nonglandular trichomes on the same leaf blade, except for Senecio leptolobus DC. and Senecio selloi (Spreng.) DC., which presented only glandular trichomes and Vernonanthura chamaedrys (Less.) H.Rob. that had only non-glandular trichomes (table 2). As for distribution, the trichomes (glandular and nonglandular) occur generally on both surfaces of the leaf blade and, when restricted to one side, this is predominantly the abaxial (table 2).
The variety of trichome types observed in the species of this study corroborates with other studies already done in the family that highlight the diversity of trichomes in Asteraceae (Solereder 1908, Ramayya 1962, Castro et al. 1997, Amrehn et al. 2013. Faced with this morphological diversity, studies on trichomes in the family have resulted in a variety of terminologies that often overlap in meanings and make it difficult to compare the types of trichomes found in the various family representatives (Wagner et al. 2014). Another factor that hinders these comparisons is the use of the same terminology for different types of trichomes (Wagner et al. 2014). In this way, the identification and characterization of trichomes in the family is not an easy task. The types of trichomes identified in this study are characterized and discussed below.
Glandular trichomes -In most species they occur on both surfaces of the leaf blade (table 2, figures 1-2), generally in depressions in the epidermis (figures 1g-h, 2b-l), corroborating with other studies that reveal glandular trichomes of Asteraceae in this position (Solereder 1908, Metcalfe & Chalk 1950, Trindade 2013.
Three types of glandular trichomes were identified and characterized in the species studied here: recurved on the epidermis, erect-capitate and uniseriate to multiseriate vesicular-capitate.
Recurved on the epidermis (G1) -multicellular, uniseriate, filamentous, recurved on the epidermis; foot and stalk with 3-4 cells; head unicellular, varying from globoid to spatulated (figure 1a-c). They have been observed on both leaf surfaces of the species of tribe Eupatorieae (figure 1a-c), except for Mikania thapsoides DC., in Aspilia montevidensis (Spreng.) Kuntze and Dimerostemma grisebachii (Baker) M.D.Moraes (Heliantheae tribe) (table 2). Studies with other representatives of the Eupatorieae (Amorin et al. 2014, Trindade et al. 2014, Fernandes et al. 2016 and Heliantheae tribes (Empinotti 2005, Oliveira et al. 2013) also indicate the occurrence of this type of trichome, which can be interpreted as a probable affinity between these tribes (Castro et al. 1997, Fernandes et al. 2016. Erect-capitate (G2) -multicellular, uniseriate or biseriate, filamentous, erect and capitate (figure 1d-l). The number of foot and stalk cells, as well as the morphology (semigloboid, globoid, oblong-ovate or ovate) and the number of head cells (unicellular, bicellular or multicellular) may vary. The foot and stalk are uniseriate with 2-4 cells in Calea uniflora  These trichomes occur on both sides of the leaf blade in most species and only on one side in some species (table 2). In Senecio leptolobus these trichomes are restricted to the region of midrib depression and in Calea uniflora they occur together with uniseriate to multiseriate vesicular-capitate glandular trichomes -G3 (figure 1h).
Erect-capitate glandular trichomes were quite representative in this study, occurring in 50% of the analyzed species. Trichomes with an oblong-ovate head similar to those observed in Lessingianthus macrocephalus were also described in Lessingianthus buddleiifolius (Mart. ex DC.) H. Rob. (Santos 2013), although these are indicated as structures unpublished by the author. Thus, information about these trichomes is increased for the genus Lessingianthus. Similar trichomes to the semigloboid head were reported by Castro et al. (1997), Martins (2003) and Duarte et al. (2011) in other species of Asteraceae. Globoid head trichomes were also reported in other representatives of the Senecioneae tribe (Martins 2003, Rojas-Leal et al. 2017) and trichome heads with this shape have already been described in Calea uniflora by Rob., Gyptis crassipes and Eupatorium subhastatum, respectively. d-e. Erect-capitate glandular trichome (G2) with oblong-ovate head in Lessingianthus macrocephalus and Vernonanthura nudiflora, respectively. f-g. Erect-capitate glandular trichome (G2) with semigloboid head in Aspilia montevidensis and Lucilia acutifolia, respectively. h-j. Erect-capitate glandular trichome (G2) with globoid head in Calea uniflora, Chaptalia integerrima and Gyptis crassipes, respectively. k-l. Erect-capitate glandular trichome (G2) with globoid head in Senecio selloi. Budel et al. (2006). In this way, knowledge about the occurrence and morphological variation of this representative type of trichome in the Asteraceae species is increased.
In addition to their wide distribution and morphological diversity in the family, glandular trichomes are responsible for the secretion of different compounds of secondary metabolism (mucilage, resinous substances, tannins, essential oils, among others), which perform important biological and pharmacological activities conferring on the family great economic importance in the production of bioactive compounds (Metcalfe & Chalk 1950, Trindade 2013, Fernandes 2014. Non-glandular trichomes -Most species present only one type of non-glandular trichome, except Senecio oxyphyllus DC. and Orthopappus angustifolius (Sw.) Gleason, both with two types (table 2). In most species, they occur on both sides of the leaf blade (table 2) and are generally densely distributed on the leaf surface.
Eight types of non-glandular trichomes were identified and characterized: simple conical; flagellatefiliform; aseptate-flagellate; whip-like; obliqueflagellate; branched with one arm; branched with two T-shaped arms; branched with three or more arms (figures 3-4). Due to the morphological similarity of the head (long, flagellate and acute apex) of the non-glandular trichomes of the flagellate-filiform, aseptate-flagellate, whip-like and oblique-flagellate type, in the literature (Ramayya 1962, Narayana 1979, Mussury et al. 2007, Sancho et al. 2010, Redonda-Martínez et al. 2012, the same terminology can be used in the identification and characterization of different trichomes thus hindering comparison with the trichomes described in this study. Simple conical (N1) -uniseriate; simple or compound foot and entire body (undifferentiated) consisting of 2-5 (figure 3a-e) or up to eight cells as in Stenachaenium riedelli (figure 3f); basal cells broader than long and the apical cell with a conical or pointed shape (figure 3a-e). The lateral walls are usually thick and may have adornments as in Aspilia montevidensis and Dimerostemma grisebachii (figure 3c). These  trichomes occur on both sides of the leaf blade of all species of the tribe Eupatorieae, Heliantheae, Millerieae, Neurolaeneae and in Stenachaenium riedelli (Inuleae) and Senecio oxyphyllus (Senecioneae) (table 2).
This type was the most representative among the non-glandular trichomes, occurring in 12 of the 34 species studied (35% of species). Trichomes of this type are considered common in the family (Smiljanic 2005) and described for members of distinct tribes, such as Tithonia diversifolia (Hemsl.) A.Gray (Heliantheae tribe) (Empinotti 2005), Mikania lanuginosa DC. (Amorin et al. 2014) and Trichogonia cinerea (Fernandes et al. 2016) in the tribe Eupatorieae, in some species of the subtribe Leiboldiinae (tribe Vernonieae) (Redonda-Martínez et al. 2016) and in species of Podocoma (tribe Astereae) (Sancho et al. 2010). In these studies, the authors reveal the occurrence of variations in the number of cells of the simple conical trichomes, corroborating with this study which found the largest number of cells in Stenachaenium riedelli in relation to the other species with simple conical trichomes. In addition, the description of these trichomes in Acanthospermum australe (Loefl.) Kuntze and Calea uniflora corroborates with the identification and characterization of the simple conical trichomes observed by Araújo et al. (2013) and Martins et al. (2006) in Acanthospermum australe and Budel et al. (2006) in Calea uniflora.
Flagellate-filiform (N2) -uniseriate, filiform; simple or compound foot and body differentiated into stalk and head; stalk with 2-4 cells generally isodiametric or with 1-2 basal cells broader than long (figure 3g-h); unicellular, flagellate and long head, tapering to a pointed apex (figure 3i-j). These trichomes occur on both surfaces of Lessingianthus macrocephalus and all species of the Gnaphalieae tribe (except in Lucilia acutifolia (Poir.) Cass.) and only on the abaxial side of Pterocaulon alopecuroides (table 2). As for the abaxial surface of most species, the trichomes cover the entire leaf surface of this side.
Trichomes like the flagellate-filiform observed in this study were also found in other species of the Gnaphalieae (Smiljanic 2005, Mussury et al. 2007 and Vernonieae (Narayana 1979, Santos 2013, Redonda-Martínez et al. 2016 tribes, although in these studies the authors used the term "whip" for characterization of the apical cell, whereas in our study this term describes another type of non-glandular trichome. Aseptate-flagellate (N3) -uniseriate, flagellate; simple or compound foot and body differentiated into stalk and head; stalk consisting of 2-3 cells, one or two basal cells being broader than long and the other(s) being longer than broad; head unicellular, clearly delimited from the stalk, long and tapering upward with a pointed apex (figure 3k-l). They occur on both sides of the leaf blade only in Noticastrum acuminatum (table 2). Similar trichomes were observed in the leaf margin of Calendula officinalis L. (Calenduleae) (Ramayya 1962) and on the stem and leaves of Gnaphalium polycaulon Pers. (Gnaphalieae) (Jana 2014).
Whip-like (N4) -uniseriate, flagellate; simple or compound foot and body differentiated into stalk and head; stalk with two cells forming a enlarged base upwards (figure 4a); the head is composed of a generally long, whip-shaped flagellate cell whose base is broad and tapering upward to a pointed apex ( figure   4a-b). These trichomes occur on both leaf surfaces of Lucilia acutifolia and Vernonanthura chamaedrys (table 2). In the first, they cover the whole leaf surface of both sides and in the second only on the abaxial surface, whereas on the adaxial surface the trichomes are sparsely distributed.
This type of trichome was described for the genus Lucilia by Metcalfe & Chalk (1950) and was also characterized as a peculiar trichome in Lucilia nitens Less. (Duarte et al. 2011), although in our study this species did not present this trichome, but rather the flagellate-filiform trichome (N2).
Oblique-flagellate (N5) -uniseriate, flagellate; simple or compound foot and body differentiated into stalk and head; stalk consisting of 2-5 cells with at least one basal cell broader than long; unicellular or multicellular head with a cell(s) longer than broad, flagellate, continuous with the stalk or clearly delimited from it by an oblique septum (figure 4c-d). They occur on both sides of the leaf blade of Pterocaulon angustifolium, Pterocaulon polystachyum DC., Vernonanthura nudiflora and only on the abaxial surface of Senecio oxyphyllus (table 2). In Pterocaulon angustifolium they cover the entire surface of the abaxial surface and are more sparsely distributed on the adaxial surface.
Trichomes like the oblique-flagellate type were observed by Cabrera & Ragonese (1978) in American species of the genus Pterocaulon (including Pterocaulon angustifolium and Pterocaulon alopecuroides, however, in our study this last species did not present this type of thichome), but these authors use the term "non-septate flagelliform" to identify this trichome. Freire et al. (2002) also indicated the occurrence of these trichomes in some species of the genus Gochnatia (Gochnatieae).
Branched witch one arm (N6) -uniseriate; simple or compound foot and body differentiated into stalk and head; stalk consisting of 1-3 cells, one or two of the basal cells being broader than long; unicellular head with a flat arm channeled into the base, forming an angle with the stalk, pointed apex (figure 4e-g) and thick lateral walls. They occur on both surfaces of the leaf blade of some representatives of the Vernonieae tribe (Chrysolaena cognata, Chrysolaena flexuosa (Sims) H.Rob., Lessingianthus brevifolius and Orthopappus angustifolius) (table 2). Similar trichomes were described for Elepanthopus mollis Kunth (Empinotti 2005, Empinotti & Duarte 2008 and for some species of the genus Vernonia (Narayana 1979).
Branched witch two T-shaped arms (N7) -uniseriate; simple or compound foot and body differentiated into stalk and head; stalk with 1-5 or more cells with at least one of the basal cells broader than long and the other cells generally cylindrical; unicellular head with two arms generally flat, channeled in the base and which may vary in shape (figure 4h-i). In the case of Orthopappus angustifolius (abaxial surface) and Centratherum camporum (Hassl.) Malme (both sides) (figure 4h-i) the two arms are short, having unequal sizes and forming a T-shaped structure together with the stalk (table 2). In Chaptalia integerrima (both surfaces) the arms are long and spread laterally as in the T-shaped, but tend to curl (table 2). The apex of the arms is pointed or rounded. These trichomes cover the entire leaf surface of the abaxial surface of Chaptalia integerrima.
Trichomes of this type are frequent in the species of the Vernonieae tribe and can be used in the taxonomy of the representatives of this tribe (Narayana 1979, Redonda-Martínez et al. 2012, Wagner et al. 2014. Similar trichomes were also observed in some species of the genus Gochnatia (Gochnatieae) (Freire et al. 2002).
Branched witch three or more arms (N8) -uniseriate; simple or compound foot and body differentiated into stalk and head; stalk composed of 1-2 cells longer than broad; branched unicellular head with 3-5 long and thin arms that spread apart at different angles (figure 4j). They occur only in Hieracium commersonii, on both sides of the leaf blade (table 2). Similar trichomes have been described in some species of the genus Gochnatia (Gochnatieae) (Freire et al. 2002) and the subtribe Lychnophorinae (Vernonieae tribe), where they are considered frequent (Wagner et al. 2014).
Due to their direct contact with the environment, glandular and non-glandular trichomes can influence many physiological and ecological aspects of plants, protecting them against stressful biotic and abiotic factors (Johnson 1975, Werker 2000, Krak & Mráz 2008, Trindade et al. 2014. Glandular trichomes are responsible for the secretion of different substances of secondary metabolism, which can form a continuous layer on the surface of the plant and thus act in reflecting light and in decreasing the temperature of the leaf (Wagner 1991, Perveen et al. 2016).
In addition, the secreted substances can act in the chemical defense against pathogens and herbivores (Wagner 1991, Trindade et al. 2014.
Non-glandular trichomes play an important role in protecting the leaves against pathogens, herbivores, high temperatures, excessive light and excessive water loss (Johnson 1975, Werker 2000, Wagner et al. 2004, Trindade et al. 2014). The functions developed by them depend on their morphology, orientation, and density (Werker 2000, Wagner et al. 2004. Longer, non-glandular trichomes which cover the entire leaf surface, as observed in most of the species studied, may act as a mechanical barrier against pest attack (Levin 1973), decrease water loss through reflection (Ehleringer 1984, Wagner et al. 2014) and influence the wettability of the leaves making them repellent to water and thus preventing the growth of pathogenic microorganisms, the deposition of pollutants and favoring photosynthetic yield (Wang et al. 2015, Rojas-Leal et al. 2017.
Considering that the species of this study occur in Sand-fields, which are characterized by high temperatures, high light intensity, water scarcity, sandy soils and strong winds, the high frequency of glandular and non-glandular trichomes in these species can constitute an adaptive strategy to these adverse environmental conditions (Costa et al. 2011). From a taxonomic point of view, the morphological diversity and the distribution of trichomes in the Asteraceae family can contribute to systematics studies at different taxonomic levels of the family, since they have been shown not to be influenced by environmental and climatic factors, thus constituting reliable taxonomic characters (Narayana 1979, Wagner et al. 2014. In our study, the morphological diversity of trichomes seems to be useful in the taxonomy of the family at the tribal level. Considering the tribes with the largest number of species sampled, we verified that the recurved on the epidermis glandular trichomes (G1) are more frequent in the Eupatorieae tribe (83% of the species studied) whereas erect-capitate glandular trichomes (G2) are more frequent in the Gnaphalieae (83%) and Senecioneae (all species) tribes. Uniseriate to multiseriate vesicular-capitate glandular trichomes (G3) are more frequent in the Vernonieae (87%), Eupatorieae (83%) and Inuleae (all species) tribes. Among the non-glandular trichomes, the simple conical type (N1) is more frequent in the Eupatorieae tribe, while the flagellate-filiform (N2) and the branched witch one arm type (N6) are the most frequent in the Gnaphalieae tribe (83%) and among members of the Vernonieae tribe (50%), respectively. Trichomes of the aseptate-flagellate (N3) and branched witch three or more arms (N8) type are restricted to the Astereae and Cichorieae tribes, respectively, although in each of these tribes only one species has been sampled.
These taxonomic inferences are closely related to the number of species sampled in each of the tribes studied, and the tribes with the highest frequency of some type of trichome are also the ones with the highest species richness and the highest sampling rate in our study: Vernonieae (23%), Eupatorieae and Gnaphalieae (18%). Thus, the value of trichomes as a taxonomic character at the tribal level in this study can be greatly increased by sampling more species in each tribe, especially those in which only one or two species were sampled (Astereae, Cichorieae, Heliantheae, Millerieae, Mutisieae, Neurolaeneae) and also with the joint analysis of other characters relevant to taxonomy. In addition, studies that develop a more detailed terminological analysis and, if possible, the standardization of the nomenclature used in the identification and characterization of trichomes in Asteraceae would facilitate their use in taxonomic studies of the family.