Braz. J. Bot (2014) 37(4):505–517 DOI 10.1007/s40415-014-0101-2

Anatomy of vegetative organs in Aldama tenuifolia and A. kunthiana (: )

Edilmara Michelly Souza da Silva • Adriana Hissae Hayashi • Beatriz Appezzato-da-Glo´ria

Received: 14 February 2014 / Accepted: 9 September 2014 / Published online: 26 September 2014 Ó Botanical Society of Sao Paulo 2014

Abstract The South American species of the Vi- tuberisation process (roots). Regarding the environmental guiera (Asteraceae) have been transferred to Aldama based adaptation, both species share the presence of a xylopodi- on molecular studies. However, the circumscription of um with a high bud shoot-forming potential, fructan Aldama tenuifolia and A. kunthiana has not been well accumulation in the tuberised roots, root-mycorrhizal established because the two species are morphologically associations, the occurrence of secretory structures, such as similar. Both occur in areas of the Cerrado domain, espe- glandular trichomes (stems and leaves), internal secretory cially in ‘‘campos sujos’’, ‘‘campos limpos’’ and ‘‘campos spaces (roots, xylopodia, stems and leaves) and hydathodes rupestres’’, which are characterised by intense solar irra- (leaves). diation, water scarcity during the autumn and winter, and frequent fires. The aim of the present study is to analyse the Keywords Compositae Secretory structures Tuberous anatomy of the vegetative organs of both species in order to roots Xylopodia identify features that may be useful in their circumscription and in understanding their environmental adaptations. Samples of leaves, stems, xylopodia and roots of each Introduction species were collected, fixed, and processed according to the usual methods for light and scanning electron micros- The Asteraceae family is the most numerous group within copy. The anatomical features useful to delimit the two the Angiosperms, with about 23,000 species and 17 tribes species are the contours of the epidermal cell walls and in (APG III 2009). In Brazil, 14 tribes have been reported and the occurrence of secretory ducts in the primary phloem Heliantheae is the third most predominant featuring 41 and fundamental parenchyma of the midrib (leaves), the genera (Mondin 2004). Based on molecular analyses, occurrence of secretory ducts in the primary and secondary Schilling and Panero (2011) have proposed to transfer the phloem (stems) and the degree of cambial activity in the South American species of the genus Viguiera Kunth (Heliantheae), a non-monophyletic genus, to the genus Aldama La Llave. The taxonomic study on 35 Brazilian E. M. S. da Silva species of Aldama included three new combinations for Programa de Po´s-Graduac¸a˜o em Fisiologia e Bioquı´mica de Plantas, Universidade de Sa˜o Paulo, Escola Superior de binomials and two for varieties, besides a new variety, 20 Agricultura ‘‘Luiz de Queiroz’’, Piracicaba, SP, Brazil new synonyms and 25 lectotypifications (Magenta and Pirani 2014). However, some Aldama species show a dif- A. H. Hayashi ficult delimitation due to their morphological similarities Nu´cleo de Pesquisa em Anatomia, Instituto de Botaˆnica, Caixa Postal 68041, Sa˜o Paulo, SP 04045-972, Brazil (Magenta 2006). The anatomy and chemical composition of the essential B. Appezzato-da-Glo´ria (&) oils of aerial organs are useful in delimiting the species Departamento de Cieˆncias Biolo´gicas, Universidade de Sa˜o Aldama filifolia (Sch.Bip. ex Baker) E.E.Schill. & Panero, Paulo, Escola Superior de Agricultura ‘‘Luiz de Queiroz’’, Caixa Postal 9, Piracicaba, SP 13418-900, Brazil A. linearifolia (Chodat) E.E.Schill. & Panero and A. e-mail: [email protected] trichophylla (Duse´n) Magenta (Bombo et al. 2012). 123 506 E. M. S. da Silva et al.

Vegetative anatomy is also useful to identify Aldama Niquelaˆndia, GO (S 14°23029.500,W48°25053.600;S arenaria (Baker) E.E.Schill. & Panero from A. robusta 14°25023.000,W48°26012.000) and at Parque Estadual da (Gardner) E.E.Schill. & Panero (Oliveira et al. 2013). Serra Dourada, GO (S 16°04027.900,W50°11011.300). A. Amongst the anatomical features used to distinguish Ald- tenuifolia was collected at Sa˜o Sebastia˜o do Paraı´so, MG ama species, the occurrence of secretory structures is par- (S 20°58053.000,W46°58052.000); Capito´lio, MG (S ticularly useful. Indeed, the anatomy of secretory structures 20°39007.000,W46°18043.800) and Parque Nacional da Serra and trichomes (glandular and non-glandular) is considered da Canastra, MG (S 20°15019.200,W46°25000.800). These a great taxonomic value to Asteraceae due to their specific sites are open physiognomies of the Cerrado domain and distribution in each organ (Solereder 1908; Metcalfe and showed signs of previous fires (personal communication). Chalk 1950; Carlquist 1958; Castro et al. 1997; Luque et al. Voucher specimens were deposited in the ESA Her- 1999; Ciccarelli et al. 2007). Moreover, when thickened barium (A. kunthiana - ESA 122873, ESA 122874 and ESA subterranean organs were analysed, Bombo et al. (2014) 122875; A. tenuifolia-ESA 122869, ESA 122870 and ESA and Oliveira et al. (2013) have found that root tuberisation 122871). in Aldama species differs from that previously described in other Asteraceae (Machado et al. 2004; Vilhalva and Ap- Anatomical and ultrastructural studies pezzato-da-Glo´ria 2006; Hayashi and Appezzato-da-Glo´ria 2005, 2007; Appezzato-da-Glo´ria et al. 2008a). Young and fully expanded mature leaves were analysed in Aldama tenuifolia (Gardner) E.E.Schill. & Panero the median region of the leaf blade. For aerial stems, in- (= Viguiera tenuifolia Gardner) and Aldama kunthiana ternodes of different diameters were analysed by taking the (Gardner) E.E.Schill. & Panero (= Viguiera kunthiana minimum (1st internode), mean and maximum diameters of Gardner), which belong to section Paradosa, series Tenu- each individual. For the subterranean organs, xylopodia ifolieae according to the classification of Blake (1918), are and tuberous and non-tuberous roots were analysed. morphologically similar (Magenta 2006). Both occur in Samples of leaves, aerial stems, xylopodia and tuberous open physiognomies of the Cerrado domain, especially in and non-tuberous roots were fixed in FAA 50 (formalde- ‘‘campos sujos’’, ‘‘campos limpos’’ and ‘‘campos rupes- hyde, acetic acid and 50 % ethanol) (Johansen 1940)or tres’’, which are characterised by intense solar irradiation, Karnovsky solution (Karnovsky 1965), dehydrated in a frequent fires and water scarcity during the autumn and graded ethanol series and stored in 70 % ethanol. winter (Ratter et al. 1997). These species shed their aerial The fixed samples were dehydrated in a graded ethanol organs under unfavourable conditions but remain alive due series and embedded in plastic resin (Leica HistoresinÒ), to their underground organs, which resprout under and the blocks were sectioned (5–8 lm thick) using a Leica favourable conditions. This strategy enables these species RM 2045 rotary microtome. The sections were stained with to escape to water deficit by adjusting their vegetative and 0.05 % toluidine blue O in citrate–phosphate buffer, pH 4.5 reproductive phenology to the most favourable seasons (Sakai 1973) and mounted in EntellanÒ synthetic resin (Aronne and Wilcock 1997). In addition to phenological (Merck, Darmstadt, Germany). Prior to embedding in his- time, drought resistance relies on adaptive strategies toresin thick stem and xylopodium samples were softened based on structural traits related to (1) increasing water in 10 % ethylenediamine (Carlquist 1982). uptake and storage, (2) reducing water loss during dry The epidermis dissociation technique using 10 % Jef- periods and (3) mechanically reinforcing tissues to prevent frey solution was applied prior to observing the leaf surface wilting, which can lead to irreversible cellular collapse and (Johansen 1940). Fragments were stained with safranin and damage (De Micco and Aronne 2012). astra blue (Bukatsch 1972) and mounted in glycerinated The aim of the present study is to analyse the anatomy gelatin. of the aerial and subterranean vegetative organs of both Histochemical analyses of all vegetative organs were species in order to identify structural features that may be performed on sections of fresh and fixed material that were useful in their circumscription and in understanding their embedded or not in historesin. The following reagents and environmental adaptations. stains were used: Sudan IV for lipophilic substances (Jensen 1962), Sudan black B for total lipids (Pearse 1968), ferric chloride for phenolic compounds (Johansen 1940), Materials and methods ruthenium red for pectin, polysaccharides and acidic mucilage (Johansen 1940), zinc-chloride iodide for starch Plant material grains (Strasburger 1913), phloroglucin in acidic medium for lignin (Johansen 1940) and NADI reagent for essential Aerial and subterranean organs were collected from three and resinous oils (David and Carde 1964). Inulin crystals individuals at each site. A. kunthiana was collected at were visualised under polarised light, and their presence 123 Anatomy of vegetative organs in Aldama (Asteraceae) 507 was confirmed using thymol-sulphuric acid reagent (Jo- mesophyll (Figs. 10 and 11), and their chlorophyllous hansen 1940). parenchyma extends to the lateral portions of the central Using fresh material of all vegetative organs, longitu- midrib (Figs. 15 and 16). Lipid droplets were observed in dinal and cross-sections (25–60 lm thick) were prepared the chlorophyllous parenchyma cells only in A. tenuifolia by hand using a razor blade or sliding microtome, cleared (Fig. 14). Both species exhibit collateral vascular bundles in 20 % sodium hypochlorite and washed in distilled water. (Fig. 10) and may also exhibit bundle sheath extension The sections were stained with Congo red and green iodine facing the upper and/or lower epidermis (Figs. 10 and 11); (Dop and Gautie´ 1928) or safranin and astra blue (Bukatsch however, secretory ducts occur only on the adaxial sheath 1972) and mounted in glycerine gelatine to prepare semi- extension (Figs. 10, 12 and 13). permanent slides. The leaf margin is slightly flexed in A. kunthiana only Photomicrographs were taken with a Leica DM LB (Fig. 12). In both species, the dorsiventral mesophyll and microscope equipped with a Leica DC 300F camera. lateral vascular bundles nearly reach the epidermis at the For scanning electron microscopy analyses, stem and leaf margin (Figs. 12 and 13). leaf samples were fixed in Karnovsky solution (Karnovsky In A. kunthiana and A. tenuifolia, hydathodes occur on 1965), dehydrated in a graded ethanol series and critical- the leaf margin (Fig. 17). Hydathodes have water pores, an point drying with CO2 (Horridge and Tamm 1969), incomplete parenchymatous sheath that surrounds the thin mounted on aluminium stubs and coated with gold layer walled cells of the epithem, and the terminal tracheids of (30–40 nm). The samples were examined under a LEO VP the vascular bundle (Fig. 17). 435 scanning electron microscope (SEM) at 20 kV. Glan- In both species, the midrib is prominent on both leaf dular trichomes were classified according to Castro et al. faces but more pronounced on the abaxial face (Figs. 15 (1997). and 16). The vascular system is organised on one large collateral bundle and two small collateral bundles sur- rounded by lignified cells (Figs. 15, 16, 18 and 19). In only Results A. kunthiana, there are ducts in the primary phloem that secrete lipophilic substances (Fig. 20). In the fundamental In frontal view, the epidermal cells on both leaf surfaces of parenchyma of A. kunthiana, there are four secretory ducts A. tenuifolia have straight anticlinal walls (Fig. 1, Table 1). of similar size (two facing the adaxial surface and two In A. kunthiana, the anticlinal walls are sinuous (Fig. 2). facing the abaxial surface) (Fig. 18) with a narrow lumen, Both species exhibit anomocytic stomata (Figs. 1, 2)on uniseriate epithelium (Fig. 18, inset) and lipophilic secre- both sides of the leaf, two types of glandular trichomes tions (Fig. 20). In A. tenuifolia, however, there are only (Type II, Figs. 4, 7 and Type VI, Fig. 5, 8 and 9) and one two tiny secretory ducts facing the abaxial surface type of multicellular non-glandular trichome (Figs. 3 and (Fig. 19), whose secretions react positively with pectic 6). The non-glandular trichomes (Figs. 3 and 6) occur on substances (Fig. 21). In only A. tenuifolia, the parenchyma both leaf surfaces and consist of three cells: a dilated basal cells near the vascular bundles exhibit starch grains on the cell, a cylindrical intermediate cell of variable size and a abaxial surface (Fig. 22). terminal cell with an acute apex. The walls of the basal and The tissues in the primary structure of the first internode intermediate cells are adorned by wart-like structures of the aerial stem are more differentiated in A. tenuifolia composed of pectin (Figs. 3 and 6). At the base of each (Fig. 23) than in A. kunthiana (Fig. 24). In both species, non-glandular trichomes, there are two sets of epidermal the stem epidermis is uniseriate and covered with a thin cells that exhibit pectin thickening (Fig. 1). Type II tric- cuticle. The cells accumulate phenolic compounds and homes are linear and uniseriate, with a spatulate terminal have pectin-thickened walls in only A. tenuifolia. The cell showing phenolic content (Figs. 4 and 7) and occur on stems have two trichome types similar to those described both leaf surfaces. Type VI trichomes (Figs. 5, 8 and 9) are for the leaves: non-glandular and glandular type II. In A. capitate and biseriate, consist of five to six pairs of cells kunthiana, non-glandular trichomes can have up to five (Fig. 8), secrete lipophilic substances (Fig. 9) that are cells (Fig. 25). The stems have few stomata (Fig. 23). stored in the subcuticular space located near the most distal The cortex consists in two to three layers of subepidermal pair of cells and occur only on the abaxial epidermis. collenchyma and one to two layers of parenchyma in A. On both leaf surfaces of A. tenuifolia and A. kunthiana, tenuifolia (Fig. 23) and in four to five layers of collenchyma the single-layered epidermis consists of cells with pectin- and four to six layers of parenchyma in A. kunthiana thickened external periclinal walls (Fig. 10) that are cov- (Fig. 24). In both species, the cortical cells may exhibit ered by a thin cuticle. The stomata, on both surfaces, are at phenolic compounds, and secretory ducts occur in the cor- the same level as the other epidermal cells (Figs. 10 and tical parenchyma region (Figs. 23 and 24). In A. tenuifolia, 11). Both species exhibited dorsiventral heterogeneous the cortex is bounded internally by endodermis (Fig. 26) 123 508 E. M. S. da Silva et al.

Figs. 1–9 Aldama tenuifolia (1, 3, 4, 6, 9) and Aldama kunthiana (2, and glandular type IV (5) trichomes. 6 Non-glandular trichome 5, 7, 8). 1, 2 Surface view of the upper leaf epidermis (arrows = pari- showing wart-like parietal ornamentation (arrow). 7 Type II trichome etal thickening of epidermal cells around the bases of non-glandular with phenolic content stain with ferric chloride. 8–9 Type IV trichomes) stained with toluidine blue. 3–5 Scanning electron trichomes stain with toluidine blue (8) and Sudan IV (9). micrographs of non-glandular (3, arrow), glandular type II (4, arrow) Bar = 10 lm (5, 7–9), 20 lm (4), 30 lm (1, 2, 6), 50 lm (3) with Casparian strips already visible at the first internode. In In A. kunthiana, lignification occurs only at later stages of A. kunthiana (Fig. 27), the endodermis exhibits Casparian development (Figs. 30 and 32). In only A. kunthiana,there strips only at subsequent internodes (Fig. 28). Starch grains are secretory ducts in the primary phloem (Fig. 27). In the are found in the endodermis in both young and mature stems. perimedullary region, there are secretory ducts of varying In the vascular cylinder of A. tenuifolia, the open col- diameters with a uniseriate epithelium that secrete lipophilic lateral bundles (Figs. 26 and 27) exhibit early-differentiating substances. In A. kunthiana, these ducts were visible in all pericyclic fibres opposite to the primary phloem (Fig. 26). analysed individuals (Figs. 24 and 33).

123 Anatomy of vegetative organs in Aldama (Asteraceae) 509

Table 1 Major anatomical features distinguishing Aldama tenuifolia from A. kunthiana Organ Feature A. tenuifolia A. kunthiana

Leaf Anticlinal walls of epidermal cells Straight Sinuous Secretory duct in the primary phloem of the vascular Absent Present bundle of the midrib Secretory ducts in the colourless parenchyma of the 2 abaxial 2 adaxial and 2 abaxial midrib Stem Secretory ducts in the primary and secondary phloem Absent Present Secretory ducts in the pith Rare and difficult to visualise Frequent and highly visible Root Secretory duct in the secondary phloem Present from the onset of Rare; present only in tuberised secondary structure secondary structure Pronounced cambial activity in the tuberisation process Present Absent

In the region of largest stem diameter, the epidermis of Numerous spaces secreting lipophilic substances are both species is maintained as a covering tissue, and the present in the cortex (where it persists), secondary phloem subepidermal cells are lignified (Figs. 29 and 30). The (Fig. 37) and medullary parenchyma (Fig. 38, inset). cortical ducts remain active, and the endodermis exhibits Fructans are not detected in the xylopodium. starch grains and Casparian strips (Fig. 28). The cambial The root systems of A. kunthiana and A. tenuifolia activity is similar in the fascicular and interfascicular consist of non-tuberised (Fig. 39) and tuberised adventi- portions (Figs. 29 and 30), but the interfascicular second- tious roots (Fig. 40) that originate from the xylopodium. ary phloem is positioned more externally than the fascic- In the primary structure of the non-tuberised root of both ular secondary phloem (Figs. 29 and 30). This species, the uniseriate epidermis (Fig. 41) has cells with characteristic is more evident in A. tenuifolia (Fig. 29). pectin-thickened external periclinal walls. The uniseriate Secretory ducts occur in the secondary phloem in A. kun- exodermis of the cortex exhibits tall juxtaposed cells thiana (Fig. 32) but not in A. tenuifolia (Fig. 31). (Fig. 41) with parietal suberin thickening. The cortical The expansion of the pith is not significant, and the parenchyma has four to five layers of cells, some of which ducts secreting lipophilic substances are preferentially show mycorrhizal fungus association (Fig. 41). The cortex positioned in the peripheral medullary region (Figs. 31 and contains ducts that secrete lipophilic substances and that 33). are formed by four cells: two cells from the cortical The xylopodium (Fig. 34) of both species occurs in the parenchyma and two endodermal cells (Fig. 41). The vas- upper soil layers and is responsible for producing tuberous cular cylinder is solid, with a uniseriate pericycle and few (Fig. 39) and non-tuberous adventitious roots (Fig. 40). protoxylem poles (Fig. 41). Due to its high budding potential (Fig. 34), the xylopodium In the secondary structure of the non-tuberised portion emits aerial branches during favourable periods of devel- of the roots in both species (Figs. 42, 43, 44, 45, 46, 47 and opment. This process increases the anatomical complexity 48), the epidermis and exodermis are replaced by several of the organ because self-grafting occurs at the base of the layers of cells with parietal suberin thickening (Fig. 44) branches in the proximal region (Figs. 35 and 36). The resulting from periclinal divisions of the cortical paren- buds located in the proximal region (Fig. 34) are axillary chyma cells located under the exodermis. The cortex buds at the bases of the self-grafted branches (Fig. 35). begins to show groups of sclereids between the paren- The covering tissue of the xylopodium is similar to that chyma cells, forming an almost-continuous ring around the of the tuberous roots, resembling stratified cork originating vascular cylinder (Figs. 42, 43, 47 and 48). The endoder- in the subepidermal layers. As the organ thickens, the mal secretory ducts found at the onset of secondary- cortical parenchyma cells show periclinal divisions, fol- structure development (Figs. 45 and 46) still exhibit four lowed by suberisation of the inner cell layers (Fig. 37). At epithelial cells. With root thickening, the number of epi- later stages of development, the outer layers of cortex are thelial cells of the duct increases due to divisions of the eliminated, as the secondary phloem is located immedi- cells that surround the lumen of the duct. In the vascular ately below the covering tissue (Fig. 37). cylinder, the secondary phloem of A. tenuifolia exhibits The secondary xylem is well developed, contributing to secretory ducts from the onset of secondary-structure the lignification of the organ (Fig. 38). The endarch pri- development (Fig. 47). In A. kunthiana, however, phloem mary xylem occurs at the periphery of the pith, which is ducts are rare and occur only in the tuberised secondary undeveloped and can become sclerified (Fig. 38, inset). structure. The secondary xylem features more visible

123 510 E. M. S. da Silva et al.

Figs. 10–22 Cross-sections of the leaf blade in Aldama tenuifolia (arrows = secretory ducts in the parenchyma, arrowhead = secre- (10, 13, 14, 16, 19, 21, 22) and A. kunthiana (11, 12, 15, 17, 18, 20). tory duct in the primary phloem in 18). Detail of a secretory duct 10, 11 Uniseriate epidermis (Ep), palisade parenchyma (Pp), spongy (inset in 18). 20 Ducts in the primary phloem (arrowhead) and parenchyma (Sp) and collateral bundles (arrowheads = stomata, parenchyma (arrows) with lipophilic secretions stained by Sudan IV. arrows = secretory duct). 12, 13. Leaf margin (arrows = secretory 21 Secretory ducts on the abaxial surface of the midrib stained by ducts). 14 Lipid droplets in chlorophyllous parenchyma stained with ruthenium red. 22 Starch grains (arrows) after reaction with zinc- Sudan IV. 15, 16 Midrib. 17 Hydathode, consisting of tracheary chloride iodide in parenchyma cells near the vascular bundles. elements, epithem (Ept), aquiferous pore (Ap) and incomplete sheath Bar = 20 lm (18, inset), 30 lm (14, 18), 50 lm (10–13, 17, 19–22), (arrows). 18, 19 Detail of the vascular bundles of the midrib 100 lm (15, 16) vascular rays in A. tenuifolia (Figs. 42 and 47). Undevel- In the secondary structure of the tuberised portion of the oped pith parenchyma is found in the centre of the organ roots in both species (Figs. 49, 50, 51, 52, 53 and 54), the (Figs. 42 and 43). covering also consists of cells with parietal suberin

123 Anatomy of vegetative organs in Aldama (Asteraceae) 511 thickening arranged in radial rows resulting from periclinal kunthiana and in Aldama linearifolia (Bombo et al. 2012), divisions of the cortical parenchyma cells (Fig. 55). In the A. arenaria and A. robusta (Oliveira et al. 2013), but they cortical parenchyma, the number of sclereid layers are absent in A. filifolia and A. tricophylla (Bombo et al. increases (Fig. 56). The endodermal ducts and the endo- 2012). The occurrence of linear glandular trichomes (type dermis with Casparian strips found in the non-tuberised II) varied amongst Aldama species. They occur in both portion of the root remain unchanged. The most obvious studied species and also in A. arenaria and A. robusta changes occur in the vascular cylinder. In both species, (Oliveira et al. 2013) but they are absent in A. filifolia, A. tuberisation results from cell proliferation in the pith linearifolia and A. tricophylla (Bombo et al. 2012). These parenchyma (Figs. 49, 50 and 51). However, significant linear trichomes reacted positively for terpenoids in He- cambial activity occurs during this root-thickening process lianthus annuus (Aschenbrenner et al. 2013), but they in A. tenuifolia (Figs. 52 and 53) but not in A. kunthiana accumulate phenolic compounds in both studied species. (Fig. 54). This cambial activity displaces the tracheal ele- In A. tenuifolia and A. kunthiana, the hydathodes on the ments towards the inner portions of the organ (Fig. 50) due leaf margins show structural features similar to those to the formation of xylem parenchyma arranged in radial described in other Aldama species (Bombo et al. 2012; Oli- rows (Figs. 52, 53). During tuberisation, phloem ducts veira et al. 2013) and other Asteraceae (Lersten and Curtis begin to form in A. kunthiana (Fig. 57), and cavities form 1985). These hydathodes consist of stomata-like water pores, in the expanded pith of both species (Fig. 58). These epithem surrounded by an incomplete sheath and tracheids. cavities originate from the division of a single pith paren- Although both A. tenuifolia and A. kunthiana are adapted to chyma cell (Fig. 59). Substantial fructan accumulation environments with water stress, aerial organs with hydath- occurs, especially in the vascular cylinder around the tra- odes are produced only during the rainy season, when the cheal elements (Fig. 60) and in the lumen of the internal relative humidity is high, providing ideal conditions for secretory spaces (Fig. 61). guttation. A similar phenomenon has been observed in Aldama arenaria and A. robusta (Oliveira et al. 2013). Internal secretory spaces that produce lipophilic com- Discussion pounds, as observed in the studied species, have been described in the underground organs of A. arenaria and A. In the Asteraceae family, leaf anatomy has been used to robusta (Oliveira et al. 2013) and several other Asteraceae characterise tribes and some genera (Carlquist 1959a, (Appezzato-da-Glo´ria et al. 2008b; Cury and Appezzato- 1959b; Anderson and Creech 1975; Breitwieser 1993). The da-Glo´ria 2009; Fritz and Saukel 2011). with well- contours of the epidermal cell walls in frontal view, which developed secretory ducts, such as A. tenuifolia and A. are straight in A. tenuifolia and sinuous in A. kunthiana, are kunthiana, are less vulnerable to herbivore attack than one feature that can help to distinguish Brazilian species of plants with reduced duct systems since their lipophilic the genus Aldama (Bombo et al. 2012; Oliveira et al. secretions aid to deter herbivores from feeding or to reduce 2013). The epidermal cell contour can vary with the growth (Farrell et al. 1991). intensity of light and humidity of the plant growing sites A subterranean system formed by a xylopodium bearing (Stace 1965). Calolisianthus species, growing in more tuberous adventitious roots, as observed in A. tenuifolia shaded areas, showed more sinuous epidermal cells of the and A. kunthiana, has been described in other Asteraceae leaves, such as those of Calolisianthus amplissimus, that occur in the Cerrado (Appezzato-da-Glo´ria et al. whereas plants that grow under full sunlight have straight 2008a). The budding capacity of the xylopodium is a epidermal cells in C. speciosus and C. pendulus (Delgado notable characteristic of this organ (Appezzato-da-Glo´ria et al. 2011). Even though the two Aldama species grow in and Cury 2011) which is considered a bud bank in Eupa- full sunlight areas, probably the sinuosity of the epidermal torium ligulaefolium due to vegetative regeneration occurs cell walls in A. kunthiana may help to maintain the leaf from the belowground organs (Fidelis et al. 2010). structure under mechanical stress due to the increase of the According to Rachid-Edwards (1956), bud formation in contact amongst adjacent cells (Haberlandt 1928). various subterranean plant organs in open physiognomies Secretory structures have been used as diagnostic char- of the Cerrado represents an adaptive strategy towards acters that allow for the recognition of species in Astera- periodic fires and droughts, as noted at the A. tenuifolia and ceae (Solereder 1908; Metcalfe and Chalk 1979, Castro A. kunthiana collection sites. et al. 1997). Variability in the occurrence and distribution The subterranean organs are covered by stratified cork, of secretory ducts in the leaf midrib and stem verified in the as previously described in Aldama linearifolia, A. tricho- present study has been used to distinguish other Aldama phylla and A. filifolia (Bombo et al. 2014) and in A. are- species (Bombo et al. 2012; Oliveira et al. 2013). Biseriate naria and A. robusta (Oliveira et al. 2013). One-year-old glandular trichomes occur in both A. tenuifolia and A. twigs of young Rhamnus californica plants show a 123 512 E. M. S. da Silva et al.

multilayered subepidermal phellem of suberised cells (De arranged in series to regulate water exchange at the plant– Micco and Aronne 2012), similar to the stratified cork atmosphere interface. described here. According to De Micco and Aronne (2012), Adventitious roots with tuberised portions, as found in this structure constitutes a barrier of hydraulic resistors A. tenuifolia and A. kunthiana, are a characteristic of

123 Anatomy of vegetative organs in Aldama (Asteraceae) 513 b Figs. 23–33 Cross-sections of internodes in Aldama tenuifolia (23, Fructans are also involved in supplying energy to form 26, 29, 31) and A. kunthiana (24, 25, 27, 28, 30, 32, 33). 23, 24 First aerial organs between the dormancy and flowering periods internode showing secretory ducts in the cortex (arrowheads) and pith (24, arrows). 25 Non-glandular trichome with more than three cells. and in helping individuals to tolerate stressful conditions, 26, 27 Collateral bundles. Secretory ducts in the primary phloem (27, such as water deficits during dormancy (Carvalho and arrows) and pith (27, arrowhead). 28–30 Region of largest stem Dietrich 1993). The tuberisation of adventitious roots in diameter (arrows = Casparian strips in the endodermis in 28, both species is mainly due to the proliferation of pith arrows = fascicular cambium in 29, arrows = secondary phloem in 30). 31, 33 Perimedullary secretory ducts (arrows). 32 Secretory duct parenchyma cells, as observed in other Aldama species in the secondary phloem (arrow)(Co = collenchyma; En = endo- (Bombo et al. 2014; Oliveira et al. 2013), but the pro- dermis; Ep = uniseriate epidermis, St = stomata; Fi = fibre cap; nounced vascular cambium contribution to root tuberisa- Pa = parenchyma). Bar = 20 lm (28), 30 lm (26, 27, 32), 50 lm tion in A. tenuifolia has not been reported previously. In (23, 24, 31), 100 lm (25, 29, 30, 33) other Asteraceae, tuberisation may result from the prolif- eration of the pericycle (Vilhalva and Appezzato-da-Glo´ria Aldama species in Brazil (Magenta 2006) and function in 2006; Hayashi and Appezzato-da-Glo´ria 2007) or from cell the storage of water (Fahn 1964) and fructans (carbohy- divisions in the inner cortex (Machado et al. 2004). drate reserves). The water content of these organs was The mycorrhizal associations in the primary root of both 60 % in A. robusta and accumulated up to 80 % of their species have also been observed by Bombo et al. (2014)in dry mass as fructans in A. discolor (Isejima and Figueiredo- small-diameter roots of Aldama filifolia. These associations Ribeiro 1993). Fructans are especially notable in the xylem are found in many Cerrado herbs (Detmann et al. 2008) and of the studied species, A. arenaria and A. robusta (Oliveira they can be crucial to increase water absorption in the dry et al. 2013) and Campuloclinium chlorolepis (Vilhalva season (Delgado et al. 2011). Furthermore, the positive et al. 2011). This distribution most likely occurs because correlation between mycorrhizal fungus association and fructans are osmotically active molecules and can decrease calcium, phosphorous and potassium contents in the leaves the water potential in the xylem tissue, facilitating water in Roupala montana corroborated the increase of the plant flow into the xylem (Valluru and Van den Ende 2008). nutrient uptake by mycorrhizal fungi (Detmann 2007). The

Figs. 34–38 Xylopodia of Aldama tenuifolia (34, 36–38) and A. the secondary phloem (Sph) with internal secretory spaces (*). 38 kunthiana (35). 34 Note the large number of buds (arrows). The Secondary phloem (Sph), well-developed secondary xylem (Sx) and dashed line indicates ground level. 35 Longitudinal section of two pith (Pi). Inset: Pith (Pi) showing various sclerified cells, internal aerial branches (Ab) united at the base. Note the axillary bud (arrow) secretory spaces (arrowhead) and primary xylem endarch (ellipses). at the base of one branch. 36 Proximal portion of the xylopodium, Bar = 50 lm (36), 100 lm (37), 200 lm (38), 1 mm (35), 3 mm showing various stem branches in cross-section (arrows). 37–38 (34) Cross-Sections 37 Periclinal divisions (arrows) of cells juxtaposed to

123 514 E. M. S. da Silva et al.

Figs. 39–48 Aldama tenuifolia (39, 42, 45–47) and A. kunthiana roots. 42, 43 Undeveloped pith and larger vascular rays in 42. 44 (40–41, 43–44, 48). 39, 40 Subterranean system consisting of Secondary covering tissue with suberin-thickened cell walls stained xylopodium (Xp) and adventitious roots with tuberised (*) and non- with Sudan IV (arrow). 45, 46 Endodermal secretory ducts (arrows). tuberised portions (arrows). 41 Cross-section of root in primary * Indicates Casparian strips in the endodermis in 45; lipophilic growth showing the epidermis (Ep), exodermis (Ex), cortical paren- secretions stained by Sudan IV in 46. 47, 48 Sclereids (Sc) and chyma (Cp) with fungal associations (arrowheads), secretory ducts secretory ducts in the cortex (arrows), secretory ducts in the (white arrows), endodermis (En) with Casparian strips (black arrow) secondary phloem (*) and larger xylem rays (Xr) in 47. Bar = 30 lm and pericycle (Pr). 42–48 Cross-Sections (42–45, 47, 48) and (41, 44, 45), 50 lm (48), 100 lm (46, 47), 200 lm (42, 43), 1.5 cm longitudinal Section (46) of the secondary structure of non-tuberised (39), 2.0 cm (40)

123 Anatomy of vegetative organs in Aldama (Asteraceae) 515

Figs. 49–54 Cross-sections of the secondary structure of tuberised 50. 51, 54 arrows indicate the cambium, which does not form radial roots in Aldama tenuifolia (49, 50, 52, 53) and A. kunthiana (51, 54). rows of parenchyma cells in the secondary vascular tissues (54) as 49 Onset of tuberisation. 50–54 Maximum root tuberisation observed shown in 52 and 53 (arrows). Bar = 50 lm (54), 100 lm (52), in both species. 49, 50 arrows indicate the cambium; arrowheads 200 lm (53), 250 lm (49, 51), 500 lm (50) show tracheary elements within the innermost regions of the organ in higher availability of nutrients to plants (Detmann 2007) several traits that enable these plants to adapt to the envi- can be an adaptive strategy to edaphic stress in Cerrado ronmental constraints of the Cerrado region such as the areas attributed to its acidic and aluminium-rich soil. presence of a xylopodium with a high bud shoot-forming In conclusion, our anatomical analyses suggest that the potential, fructan accumulation in the tuberised roots, root- vegetative organs of A. tenuifolia and A. kunthiana possess mycorrhizal associations, the occurrence of glandular

123 516 E. M. S. da Silva et al.

Figs. 55–61 Cross-sections of the secondary structure of tuberised phloem. 58 Secretory cavities in the pith. 59 Formation of a secretory roots in Aldama tenuifolia (55, 59, 60, 61) and A. kunthiana (56, 57, cavity via the divisions of a single pith parenchyma cell. 60, 61 Inulin 58). 55 Secondary covering tissue formed by periclinal divisions crystals observed under polarised light around the tracheal elements (arrow) of cortical parenchyma cells. 56 Cortex region showing a (60) and the lumen of the internal secretory spaces (61). larger sclereid layer (Sc) than that found in the non-tuberised root Bar = 30 lm (55, 58, 59), 50 lm (57, 60, 61), 100 lm (56) (* = cortical secretory ducts). 57 Secretory duct (*) in the secondary trichomes (stems and leaves), internal secretory spaces Appezzato-da-Glo´ria B, Cury G, Soares MKM, Rocha R, Hayashi AH (roots, xylopodia, stems and leaves) and hydathodes (2008a) Underground systems of Asteraceae species from the Brazilian Cerrado. J Torrey Bot Soc 135:103–113. doi:10.3159/ (leaves). Furthermore, some anatomical characteristics are 07-RA-043.1 useful taxonomically to distinguish the two species: the Appezzato-da-Glo´ria B, Hayashi AH, Cury G, Soares MKM, Rocha R contours of the epidermal cell walls and in the occurrence (2008b) Occurrence of secretory structures in underground of secretory ducts in the primary phloem and fundamental systems of seven Asteraceae species. Bot J Linn Soc 157:789–796. doi:10.1111/j.1095-8339.2008.00823.x parenchyma of the midrib (leaves), the occurrence of Aronne G, Wilcock CC (1997) Reproductive phenology in Mediter- secretory ducts in the primary and secondary phloem ranean macchia vegetation. Lagascalia 19:445–454 (stems) and the degree of cambial activity in the tuberi- Aschenbrenner A-K, Horakh S, Spring O (2013) Linear glandular sation process (roots). trichomes of Helianthus (Asteraceae): morphology, localization, metabolite activity and occurrence. AoB PLANTS. doi:10.1093/ aobpla/plt028 Acknowledgments We thank Conselho Nacional de Desenvolvi- Blake SF (1918) A revision of the genus Viguiera. Contr Gray Herb mento Cientı´fico e Tecnolo´gico (CNPq) for the Grants (no. 54:1–205 135820/2011-1; 302776/2010-9) and Fundac¸a˜o de Amparo a` Pesquisa Bombo AB, Oliveira TS, Oliveira ASS, Rehder VLG, Magenta MAG, de Sa˜o Paulo (FAPESP) (Thematic Project Proc. no. 2010/51454-3) Appezzato-da-Glo´ria B (2012) Anatomy and essential oils from for providing financial support. We are also grateful to Professor aerial organs in three species of Aldama (Asteraceae: Helian- Mara Angelina Galva˜o Magenta for plant identification. theae) that have a difficult delimitation. Aust J Bot 60:632–642. doi:10.1071/BT12160 Bombo AB, Oliveira TS, Oliveira ASS, Rehder VLG, Appezzato-da- Glo´ria B (2014) Anatomy and essential oil composition of the References underground systems of three species of Aldama La Llave (Asteraceae). J Torrey Bot Soc 141(2):115–12. doi:10.3159/ Anderson LC, Creech JB (1975) Comparative leaf anatomy of TORREY-D-12-00053.1 Solidago and related Asteraceae. Am J Bot 62:486–493 Breitwieser I (1993) Comparative leaf anatomy of New Zealand and Angiosperm Phylogeny Group (2009) An update of the phylogeny Tasmanian Inuleae (Compositae). Bot J Linn Soc 111:183–209. group classification for the orders and families of flowering doi:10.1111/j.1095-8339.1993.tb01898.x plants: APG III. Bot J Linn Soc 161:105–121. doi:10.1111/j. Bukatsch F (1972) Bermerkungen zur Doppelfarbung Astrablau- 1095-8339.2009.00996.x Safranin. Mikrokosmos 61:255 Appezzato-da-Glo´ria B, Cury G (2011) Morpho-anatomical features Carlquist S (1958) Anatomy and systematic position of Centauro- of underground systems in six Asteraceae species from the dendron and Yunquea (Compositae). Brittonia 10:78–93 Brazilian Cerrado. An Acad Bras Cieˆnc 83:981–991. doi:10. Carlquist S (1959a) Glandular structure of Holocarpha and their 1590/S0001-37652011005000018 ontogeny. Am J Bot 46:300–308. doi:10.2307/2439482

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