ARTICLE

Anatomy of vegetative organs with an emphasis on the secretorystructures of two species of Aldama (Asteraceae–Heliantheae)Tuane Oliveira, Aline Bertolosi Bombo, and Beatriz Appezzato-Da-Glória

Abstract: Aldama arenaria (Baker) E.E.Schill. & Panero and Aldama robusta (Gardner) E.E.Schill. & Panero are aromatic plants,morphologically very similar and usually confused on the analysis of exsiccates. The present study aimed to identify thesecretory structures present in vegetative organs and to survey for anatomical features with diagnostic value between A. arenariaand A. robusta based on an examination of these organs. Aldama arenaria and A. robusta exhibit hydathodes in the dentateornamentations of the leaf margins and apices in addition to glandular trichomes, secretory cavities and ducts, all of which varywith respect to type and location in each species. Xylopodia and adventitious roots constitute the underground system. Thexylopodium is covered by epidermis or by suberised cells resulting from periclinal divisions of parenchyma cells. The tuberisa-tion process of adventitious roots results mainly from hypertrophy and hyperplasia of medullary cells, especially in A. arenaria.Through anatomical analysis, useful features for distinction between A. arenaria and A. robusta in exsiccates and fresh materialswere identified, and these features were related mainly to the position and frequency of secretory structures.

Key words: Aldama La Llave, Compositae, cavities, ducts, glandular trichomes, hydathodes, tuberous roots.

Résumé : Lors de l'analyse des exsiccata, on confond souvent l'Aldama arenaria (Baker) E.E.Schill. & Panero avec l'Aldama robusta(Gardner) E.E.Schill. & Panero, deux plantes aromatiques se ressemblant beaucoup. Dans cette étude, les auteurs ont cherché aidentifier les structures sécrétrices présentes dans les organes végétatifs et a repérer des caractéristiques anatomiques différen-tielles entre l'A. arenaria et l'A. robusta, basées sur l'examen de ces organes. L'A. arenaria et l'A. robusta montrent des hydathodesdans les ornementations denticulées des marges et des apex foliaires, en plus de trichomes glandulaires ainsi que des cavités etconduits sécréteurs, tous variant selon le type et la localisation chez chaque espèce. Des xylopodes et des racines adventivesconstituent le système souterrain. Un épiderme ou des cellules subérifiées provenant de divisions périclines des cellulesparenchymatiques, recouvrent le xylopode. Le processus de tubérisation des racines adventives provient surtout d'une hyper-trophie et d'une hyperplasie des cellules médullaires, surtout chez l'A. arenaria. Par des analyses anatomiques, les auteurs ont puidentifier des caractéristiques utiles pour distinguer l'A. arenaria de l'A. robusta sur les exsiccata ainsi que sur les spécimens frais,ces caractéristiques portant surtout sur la position et la fréquence des structures sécrétrices. [Traduit par la Rédaction]

Mots-clés : Aldama La Llave, composées, cavités, conduits, trichomes glandulaires, hydathodes, racines tubéreuses.

IntroductionSouth American species of the genus Viguiera Kunth were trans-

ferred to the genus Aldama La Llave based on the molecular anal-ysis performed by Schilling and Panero (2011). However, there arestill problems in the circumscription for the Brazilian species andanatomical studies have been useful to differentiate among thespecies as demonstrated by Bombo et al. (2012). Aldama arenaria(Baker) E.E.Schill. & Panero (= Viguiera arenaria) and Aldama robusta(Gardner) E.E.Schill. & Panero (= Viguiera robusta) are morphologi-cally very similar and usually confused based on the analysis ofexsiccates (Magenta 2006). Both species are herbs and subshrubswith potentially gemmiferous, thickened underground stemsfrom which emerge tuberised roots. They exhibit seasonal devel-opment and occur in depleted areas of Brazilian savannah(Cerrado biome) and on roadsides (Magenta 2006). The seasonal de-velopment involves the formation of aerial branches from under-ground buds during the flowering phase. These branches senesceduring the dormancy period and only the underground organsremain. The studied species are aromatic due to the production ofvolatile compounds such as essential oils that result from second-ary metabolism. The literature lacks information on the location

where these metabolites are biosynthesised; however, Da Costaet al. (2001) and Porto et al. (2009) have demonstrated the phyto-chemical potential of these species as chemical markers and theirantimicrobial activity based on the occurrence of sesquiterpenelactones and diterpenes. According to Fahn (1979), low molecularmass terpenes are produced by trichomes and ducts. These andother types of secretory structures are widely distributed in spe-cies of Asteraceae (Solereder 1908; Ramayya 1962; Metcalfe andChalk 1979; Cury and Appezzato-Da-Glória 2009). An understand-ing of the types and distribution of secretory structures in theplant body is important for the targeting of organs containingpotentially bioactive compounds; this understanding would alsoprevent the unnecessary extraction of organs lacking phytochem-ical potential. Furthermore, knowledge regarding these secretorystructures and other anatomical and morphological features hasaided in the taxonomic delimitation of Asteraceae and allowed forthe distinction among taxa (Castro et al. 1997; Zareh 2005; Adedejiand Jewoola 2008; Fritz and Saukel 2011) and among Aldama spe-cies (Bombo et al. 2012).

Therefore, the present study aimed to describe the anatomicalfeatures of the vegetative organs of A. arenaria and A. robusta with

Received 30 October 2012. Accepted 13 February 2013.

T. Oliveira and B. Appezzato-Da-Glória. Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Universidade de São Paulo, C.P. 09, 13418-900, Piracicaba,SP, Brazil.A.B. Bombo. Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Universidade de São Paulo, C.P. 09, 13418-900, Piracicaba, SP, Brazil; and Programa depós-graduação em Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brazil.

Corresponding author: Beatriz Appezzato-Da-Glória (e-mail: bagloria@usp.br).

335

Botany 91: 335–342 (2013) dx.doi.org/10.1139/cjb-2012-0271 Published at www.nrcresearchpress.com/cjb on 19 February 2013.

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a focus on identifying secretory structures and providing diagnos-tic characters thatwill facilitate the distinction between these twospecies.

Materials and methods

MaterialAldama arenaria (=Viguiera arenaria) was collected at the Estação

Ecológica de Itirapina (22°14=S, 47°51=W), São Paulo state (SP),Brazil, and Aldama robusta (=Viguiera robusta) was collected at theReserva Biológica de Mogi Guaçu, Fazenda Campininha (22°42=S,47°37=W), SP, Brazil.

Structural analysisAnatomical analyses of the leaves that were in full sunlight,

aerial stems, thickened underground stems, and adventitiousroots were performed for both species from three differentindividuals to each species. The middle region of the leaf blade(mid-rib portion, inter-rib portion, and margin) and leaf tipwere analysed. The aerial stem was analysed at the level of the3rd internode, 20th internode and the internode closest to thesoil surface. Both the tuberised and nontuberised regions ofA. arenaria adventitious roots were analysed. In A. robusta, theroots are uniformly tuberised and roots with varying thick-nesses were analysed.

The samples were fixed in FAA (formaldehyde, acetic acid, and50% ethanol in a 1:1:18 v/v/v) (Johansen 1940) or Karnovsky solution(Karnovsky 1965), placed under vacuum to remove air containedin the tissues, dehydrated with a graded ethanol series, to 70%ethanol, where they remained stored for later processing. A por-tion of the material was embedded in Leica Historesin® plasticresin (Heraeus Kulzer, Hanau, Germany), and the blocks were cutin 5–7 �m thick sections using a Leica RM 2045 rotary microtome.The sections were stained with 0.05% toluidine blue in citrate-phosphate buffer at pH 4.0–6.0 (Sakai 1973) and mounted inEntellan® synthetic resin (Merck, Darmstadt, Germany). Thickersections (60–90 �m) of fresh and fixed samples were also cut usinga Leica SN 2000 R slidingmicrotome or cutmanually using a razorblade. The sections were cleared with 20% sodium hypochlorite,rinsed in distilled water, stained with safranin and astra blue(Bukatsch 1972), and mounted on glycerinated gelatin.

Histochemical analysesThe histochemical analyses were performed using sections ob-

tained from material embedded in historesin, as well as sectionsfrom fixed material that had not been embedded in historesin.Sudan IV was used for the identification of lipophilic substances(Jensen 1962), NADI reagent for terpenes (David and Carde 1964),zinc-chloride iodide (Strasburger 1913) for the detection of starchgrains, ferric chloride for phenolic substances (Johansen 1940),phloroglucine in acid medium for lignin (Johansen 1940), andruthenium red for pectic substances (Johansen 1940). For inulin-type fructans, samples were sectioned manually and maintainedin 70% ethanol for 4 days. Inulin crystals were visualised underpolarised light, and their presence was confirmed using thymol-sulphuric acid reagent (Johansen 1940).

The epidermis dissociation technique using 10% Jeffrey solutionwas applied prior to observing the leaf surface (Johansen 1940).Fragments were stained with safranin and astra blue (Bukatsch1972) and mounted in glycerinated gelatin.

Digital images were generated using a Leica DM LB trinocularmicroscope coupled to a Leica DC 300 F video camera and cap-tured on a microcomputer using IM50 software. Stomata andglandular trichomes were classified according to Metcalfe andChalk (1979) and Castro et al. (1997), respectively.

Surface analysis (SEM)Samples were fixed in Karnovsky solution (Karnovsky 1965) and

dehydrated in a graded ethanol series (30%, 50%, 70%, 90%, and

100%). Subsequently, the samples were subjected to critical pointdrying using CO2 as transition medium (Horridge and Tamm 1969),mounted on aluminum supports and coated with a 30–40 nm goldlayer. Observations were performed and micrographs were pro-duced using a LEO scanning electron microscope (SEM) model VP435 operated at 20 kV. The scales used are printed directly on themicrographs.

Results

Leaf anatomyIn paradermal sections, the outline of the cell wall of A. arenaria

is straight on upper epidermis (Fig. 1) and sinuous on lower epi-dermis (Fig. 2). In A. robusta, the outline of the epidermal cell wallis sinuous on both epidermal surfaces (Figs. 3 and 4). Both specieshave anomocytic stomata (Figs. 1, 2, and 4), two types of glandulartrichomes (Type IV and Type II) and one type of multicellularnonglandular trichome (Figs. 5–9).

Type IV glandular trichomes are capitate and biseriate, consist-ing of two basal cells and a head with five to six pairs of secretorycells (Fig. 5). A lipophilic secretion accumulates in the subcuticu-lar space. This type of trichomes occurs only on the abaxial face ofthe leaf. In A. robusta, these trichomes are distributed throughoutthe leaf surface, whereas in A. arenaria, they are concentratedmainly at the margins and tip of the leaf. Type II glandulartrichomes are uniseriate, filamentous, curved or straight, andcontain a variable number of cells. The distal cells are generallygloboid to spatulate in shape (Figs. 6 and 7). This type of trichomeoccurs on both faces of the leaves in both A. robusta and A. arenaria.

The multicellular nonglandular trichomes observed in A. robustaand A. arenaria have a base that is formed by a variable number ofcells, which are arranged radially andmay form a pedestal (Fig. 8).At the base, three cells form a single series, and the walls of thesecells are adorned by wart-like structures composed of pectin(Fig. 8). Themost basal cell is short and dilated, whereas the apicalcell is pointed and can be short or elongated (Figs. 8 and 9). InA. arenaria and A. robusta, trichomes occur on both faces of the leaf;however, the trichomes are more elongated on the abaxial face inA. robusta (Fig. 9).

In middle region of the leaf blade in A. arenaria and A. robusta,the epidermis is uniseriate with pectin-rich outer periclinal walls.The cells on the adaxial face are more voluminous comparedwith the abaxial face, especially in A. robusta (Figs. 10 and 12).Additionally, A. arenaria is amphistomatic (Fig. 10), whereas A.robusta is hypostomatic (Fig. 12).

Both species exhibited dorsiventral heterogeneous mesophyll(Figs. 10 and 12) with cells containing lipophilic substances.

The mid-rib phloem of both species contains secretory ducts(Figs. 11 and 13), and these ducts are more evident in A. arenaria(Fig. 11). Vascular bundles are embedded in the fundamental pa-renchyma,which contains four secretory ducts on the abaxial face(Figs. 11 and 13). On the adaxial face, A. arenaria has four ducts(Fig. 11), whereas A. robusta has only one duct (Fig. 13).

Both species exhibit collateral vascular bundles and may alsoexhibit bundle sheath extensions. In A. arenaria, bundle sheathextension cells facing the upper and lower epidermis have secre-tory ducts between them (Fig. 10). In A. robusta, the secretory ductsare located only between bundle sheath extension cells facing theupper epidermis (Fig. 12).

In A. arenaria and A. robusta, hydathodes occur on both the leafmargins and tips (Figs. 14 and 15). Hydathodes have water pores,an incomplete parenchymatous sheath that surrounds the thin-walled cells of the epithem, and the terminal tracheids of thevascular bundle (Fig. 14).

All secretory ducts present in the leaves of both species secretelipophilic substances.

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Anatomy of the aerial stemThe epidermis of young stems of A. arenaria and A. robusta is

uniseriate (Figs. 16 and 17). In A. arenaria, all three types oftrichomes previously described for the leaves occur on the stems(Fig. 18), whereas in A. robusta, type IV trichomes are not present.In both species, nonglandular trichomes can consist of asmany aseight cells on top of the pedestal (Fig. 18). In the cortical region(Figs. 16 and 17), the parenchyma contains secretory ducts (Fig. 19),and the endodermis contains Casparian strips and stores starch(Fig. 19). In the vascular cylinder, the bundles are of the collateralopen type. Secretory ducts are observed among the isodiametriccells of the medullary parenchyma (Figs. 16 and 17). In A. robusta,the ducts are concentrated in the perimedullary region (Fig. 17);however, in A. arenaria, the ducts can be observed distributedthroughout the medulla including its centre (Fig. 16). Ducts in thestem secrete lipophilic substances.

Only A. arenaria exhibits periclinal divisions in the subepider-mal layer during stem thickening (Fig. 20), and both species showpericlinal elongation of the cortical parenchyma cells (Fig. 20). Inthe endodermal cells opposite the pericyclic fiber caps, slightly

conspicuous parietal suberin thickening occurs, and starch is notpresent. In the vascular bundle, pericyclic fibers are already dif-ferentiated (Figs. 20 and 21), and the primary phloem containssecretory ducts (Fig. 21). The fascicular and interfascicular regionsof the cambium have distinct activities, as follows: the fascicularportion produces secondary phloem and xylem with all elementsof the axial system in addition to rays (Fig. 20), whereas the inter-fascicular portion produces only rays that may undergo lignifica-tion (Figs. 20 and 22). In the medulla, cells of the perimedullaryregion exhibit anticlinal elongation (Fig. 20), and the peripheralducts are localised more internally, whereas the other ducts areobserved near the primary xylem of the bundles (Fig. 22).

In the thickened stem of A. arenaria, the epidermis has lenticels(Fig. 23). These structures are not observed on the stem ofA. robusta. Stratified cells with suberised walls are observed underthe epidermis only in A. arenaria. In the vascular cylinder, the cellsof the pericycle are lignified (Figs. 24 and 25). Secretory ducts areobserved between sclerified cells in both species due to lignifica-tion of the primary phloem (Fig. 24, detail). The formation ofsecretory ducts in the secondary phloem occurs only in A. arenaria

Figs. 1–9. Photomicrographs (Figs. 1–5, 7, 8) and electron micrographs (Figs. 6, 9) of Aldama arenaria (Figs. 1, 2) and A. robusta (Figs. 3–9) leaves.Surface view of the upper (Figs. 1, 3) and lower (Figs. 2, 4) leaf epidermis. Glandular trichomes of Types IV (Fig. 5) and II (Figs. 6, 7) andnonglandular trichomes (Figs. 8, 9). The arrow in Fig. 8 indicates wart-like structures composed of pectin. The insert in Fig. 9 showsnonglandular trichomes on upper epidermis of A. robusta. Scale bars: Figs. 1–5, Figs. 7–9 = 50 �m; Fig. 6 = 100 �m.

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(Fig. 24). The cells of the interfascicular parenchyma elongate peri-clinally, thus spreading out the vascular bundles (Fig. 26). Theparenchymatous medulla is more developed due to anticlinal di-visions and cellular elongation in both species (Fig. 26).

Xylopodium anatomyAldama arenaria and A. robusta have a woody xylopodium from

which tuberous adventitious roots emerge (Fig. 27). The xylopo-dium occurs in the superficial layers of the soil and has severalaxillary buds (Fig. 28) at the base of branches grown during favour-able periods of development. Gradually, these branches merge ina self-grafting process (Fig. 29) that is responsible for the complexstructure of the xylopodium. Coating of the xylopodium is per-formed in some sectors by the epidermis (Figs. 30 and 31) and inothers by cells with suberised walls formed from periclinal divi-sions of subepidermal cells (Fig. 32). Secretory cavities (Fig. 33) andsclereids (Figs. 30 and 31) can be observed among the corticalparenchyma cells. The endodermis (Fig. 33) contains Casparianstrips. In the vascular cylinder, the pericycle has several cell layerswith groups of fibers in some sectors (Fig. 30). The secondaryphloem contains clusters of sclereids in A. arenaria (Fig. 34) andsecretory ducts in both species (Figs. 34 and 35) that are moreconspicuous in A. arenaria. Contiguous ducts may merge, formingstructures with a wide lumen (Fig. 34). Secondary xylem occupiesa greater portion of the xylopodium (Fig. 29), and primary xylemexhibits centrifugal maturation (Fig. 36, detail), which confirms

the stem structure of the xylopodium. Similar to the aerial stemstructure, the secretory ducts are restricted to the perimedullaryregion in A. robusta (Fig. 36), whereas in A. arenaria, the ducts aredistributed throughout the medulla (Fig. 37). Inulin-type fructansare observed inside the vascular parenchyma cells, in the lumenof secretory ducts (Fig. 38) and inside the tracheary elements.

Root anatomyThe root system of A. arenaria and A. robusta is formed by adven-

titious roots that originate from the xylopodium. In A. arenaria,the adventitious roots have tuberised and nontuberised portions(Fig. 39), whereas A. robusta exhibits a discrete and uniform tuberi-sation (Fig. 40).

Regarding their primary structure, both species exhibit a uni-seriate epidermis. The cortex contains exodermis, isodiametricparenchyma cells (Figs. 41 and 42), and an endodermis with Cas-parian strips (Figs. 43 and 44). Additionally, ducts arising from thepericlinal divisions of endodermal cells secrete lipophilic sub-stances (Figs. 43 and 44). Cells that delimitate the lumen of ductsmay undergo periclinal and anticlinal divisions, and it was notpossible to determine the number of cells that constitute theepithelium of these structures (Fig. 43). In A. robusta, the ducts arepositioned only in areas opposite the primary phloem (Fig. 42),whereas the secretory ducts in A. arenaria surround the entirevascular cylinder. The vascular cylinder has a pericycle with onecell layer (Fig. 42) and a parenchymatic medulla (Fig. 41).

Figs. 10–15. Photomicrographs (Figs. 10–14) and electron micrographs (Fig. 15) of Aldama arenaria (Figs. 10, 11, 14) and A. robusta (Figs. 12, 13, 15)leaves. Cross sections of the leaf blade (Figs. 10, 12) and mid-rib (Figs. 11, 13) in the middle portion of the lamina. Hydathode from a leaf bladein longitudinal section (Fig. 14) and in surface view (Fig. 15) of the leaf blade. The arrows in Figs. 10–13 indicate secretory ducts. Ep, epithelium;St, stomata; Wp, water pore. Scale bars: Figs. 10–14 = 50 �m; Fig. 15 = 100 �m.

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In the secondary structure of the tuberous roots of both species,a layer of cells with suberised walls originating from periclinaldivisions of the outermost cell layers of the cortex gradually re-places the epidermis (Fig. 45).

Both species exhibit sclereids in the cortex (Fig. 47) and cavitieslocated externally to the endodermal ducts, especially in the areabelow the lenticels (Fig. 45). These cavities originate from thecortical parenchyma during the root tuberisation process. Thelumen of previously formed ducts widens, and new ducts areformed from endodermal divisions (Fig. 48). The cambium of peri-cyclic origin produces only rays (Fig. 47), whereas the cambium ofprocambial origin exhibits a different activity due to its location.When the cambium coincides with the primary vascular tissues(Fig. 47), it produces all of the elements of the axial system inaddition to rays (Fig. 46). Otherwise, the cambium produces onlyradial parenchyma, creating a false impression of vascular bun-dles in the root (Fig. 47). The secondary phloem has ducts that aremore conspicuous in A. arenaria (Fig. 46) and may merge to formstructures with a wide lumen. Secretory cavities are found in themedulla (Fig. 46) of both species; however, these structures aremore common in A. arenaria and are longitudinally shorter(Fig. 49) compared with those in A. robusta (Fig. 50).

In both A. arenaria and A. robusta, root tuberisation is duemainlyto hypertrophy and divisions of medullary cells with only a smallcontribution from the cambium (Figs. 46 and 47). In A. arenaria,

cellular divisions are more intense, which results in structureswith more pronounced tuberisations (Fig. 39). In both species,inulin crystals are observed in the cortex and in the vascularcylinder. Inulin-type fructans are observed inside the vascular pa-renchyma cells, in the lumen of secretory ducts and inside thetracheary elements (Fig. 51).

DiscussionIn this study, hydathodes were observed on the margins and

tips of leaves of both A. arenaria and A. robusta, as observed in otherspecies of Asteraceae by Lersten and Curtis (1985), Castro et al.(1997), and Milan et al. (2006). Although the underground systemof A. arenaria and A. robusta occurs in the superficial layers of thesoil in Cerrado environments, which have lower water availabil-ity, the presence of aerial organs, where the hydathodes are lo-cated, coincides with the rainy season and high relative humidity,which are ideal conditions for guttation. During the dry season,the aerial organs senesce and plants enter the dormancy phase.

The types and position of secretory structures in the plant body,in addition to the content of the material secreted, are features ofdiagnostic value that allow for the recognition of species (Solereder1908; Metcalfe and Chalk 1979). Castro et al. (1997) proposed anidentification key for genera of the family Asteraceae, includingViguiera Kunth, based on the type and position of foliar secretory

Figs. 16–26. Photomicrographs (Figs. 16, 17, 19–26) and scanning electron micrographs (Fig. 18) of the aerial stem of Aldama arenaria (Figs. 16, 20, 22,23, 24) and A. robusta (Figs. 17, 19, 21, 25, 26). The arrows in Figs. 16, 17, and 26 indicate secretory ducts in the medulla. The arrow in Fig. 19 indicates acortical secretory duct. The arrow in Fig. 21 indicates a secretory duct of the primary phloem. The arrow in Fig. 22 indicates a secretory duct formedin the parenchyma of the vascular bundle. The insert in Fig. 24 shows the presence of ducts among the pericycle fibers in primary phloem (arrow).Co, cortex; PF, pericyclic fibers; En, endodermis; Pe, pericycle; *, vascular ray. Scale bars: Figs. 16, 17, 19–26 = 50 �m; Fig. 18 = 100 �m.

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structures. The occurrence of glandular trichomes can varyamong the Aldama species. In both studied species and in Aldamalinearifolia (Bombo et al. 2012) the foliar glandular trichomes arepresent, but they are not visible in Aldama filifolia and Aldamatrichophylla (Bombo et al. 2012).

The occurrence of ducts and cavities that secrete lipophilic sub-stances is common in vegetative organs of Asteraceae species andwas reported by Solereder (1908), Fahn (1979), Lersten and Curtis(1986, 1988, 1989), Lotocka and Geszprych (2004), Milan et al.(2006), Del-Vechio-Vieira et al. (2008), Appezzato-Da-Glória et al.(2008), Cury and Appezzato-Da-Glória (2009), and Fritz andSaukel (2011). In A. arenaria and A. robusta the secretory ducts aredistributed in all vegetative organs, whereas the cavities occuronly in underground organs. Furthermore, the presence of foliarsecretory ducts in bundle sheath extensions allowed us to differ-entiate between A. arenaria and A. robusta. The distribution andsize of canals were also distinct among the Aldama species studiedby Bombo et al. (2012).

The perimedullary position of secretory structures in A. robustacorroborates the statement proposed by Solereder (1908) that se-cretory ducts in the stem medulla are restricted to the peripheryof the structure; however, in A. arenaria, ducts are distributedthroughout themedulla. The occurrence of secretory structures isin the secondary phloemand throughout themedulla of the aerialstem, which is unique to A. arenaria and can be used to distinguishA. arenaria from A. robusta.

The presence of internal secretory spaces near the endodermis,which was observed in the xylopodium and roots of A. arenariaand A. robusta, has been observed in the underground organs ofseveral Asteraceae (Appezzato-da-Glória et al. 2008; Cury andAppezzato-Da-Glória 2009; Fritz and Saukel 2011). Ducts and cavi-ties originate from divisions in the endodermal layer (Lotocka andGeszprych 2004; Fritz and Saukel 2011), and the origin of secretorycavities observed in the secondary structure of A. arenaria andA. robusta roots is similar to the origin of cortical cavities in the rootsof Pterocaulon alopecuroides (Cury and Appezzato-Da-Glória 2009).

Figs. 27–38. Photomicrographs of the xylopodium of Aldama arenaria (Figs. 27, 28, 32–34, 37, 38) and A. robusta (Figs. 29–31, 35, 36). Externalmorphology (Fig. 27), cross sections (Figs. 28–37), and a transverse section under polarised light microscopy (Fig. 38). The double arrow inFig. 30 indicates the pericycle. The arrowheads in Fig. 33 and 35 indicate secretory cavities. The arrows in Figs. 34–37 indicate secretory ducts. Thearrow in Fig. 36 (insert) indicates centrifugal maturation of the primary xylem. AR, adventitious root; B, bud; L, lacuna; SX, secondary xylem; SA,stem axis; Epi, epidermis; En, endodermis; Pe, pericycle; Co, cortex; SC, stratified suberous layer; SF, secondary phloem; Scl, sclereids; VC,vascular cambium. Scale bars: Figs. 27, 29 = 1 cm; Fig. 28 = 0.25 cm; Figs. 30–33 and Figs. 35–38 = 50 �m; Figs. 34, 37 = 100 �m.

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Both the xylopodium and tuberous roots of A. arenaria andA. robusta accumulate inulin-type fructans. According to Solereder(1908), these compounds are characteristic of Asteraceae speciesand were also reported for the genus Viguiera Kunth by severalauthors (Isejima et al. 1991; Tertuliano and Figueiredo-Ribeiro1993; Itaya et al. 1999; Van den Ende et al. 2005). Previous studieshave demonstrated that fructans are involved in supplying energyto form aerial organs between the dormancy and floweringperiods and in the resistance of individuals to stress conditionssuch as water deficit during the dormancy phase (Carvalho andDietrich 1993). Because fructans are osmotically active molecules,they can promote a drop in the water potential in xylem tissue,thus inducing the flow of water out of the soil and into the xylem(Valluru and Van den Ende 2008). This phenomenon would ex-plain the higher concentration of fructans observed in the xylemregion (including the interior of the vessel elements) in A. arenariaand A. robusta in the present study and in Campuloclinium chlorolepisby Vilhalva et al. (2011).

Aldama arenaria and A. robusta are also anatomically similar.Both species exhibit secretory structures such as hydathodes,glandular trichomes, and internal secretory spaces. Although thedegree of tuberization of the root varies between these species,the process of tuberization and the root structure are similar.Only the occurrence of stomata on the leaves and the distributionof secretory spaces among different organs varied in these speciesand, therefore, can be useful in distinguishing them.

AcknowledgementsWe thank The National Council for Scientific and Technological

Development (CNPq) for grants (302776/2010-9) and the São PauloCouncil for Research (FAPESP) (Thematic Project Proc. No. 2010/51454-3 and Proc. No. 2010/02005-1) for providing financial supportand grants to the first author. We are also grateful to ProfessorMara Angelina Galvão Magenta for plant identification and thecoordinators of the Estação Ecológica de Itirapina, SP, Brazil, andof the Reserva Biológica deMogi Guaçu, Mogi Guaçu, SP, Brazil for

Figs. 39–51. Photomicrographs of the adventitious roots of Aldama arenaria (Figs. 39, 44, 46, 49, 51) and A. robusta (Figs. 40–43, 45, 47, 48, 50).External morphology (Figs. 39, 40), cross sections (Figs. 41–43, 45–48, 51) and longitudinal sections (Figs. 44, 49–50). The arrows in Figs. 42, 46, and 48indicate secretory ducts and in Figs. 43 and 44 periclinal divisions of the endodermis. The arrowheads in Figs. 45, 46, 47, 49, and 50 indicatesecretory cavities, which are tubular in the medulla of A. robusta (Fig. 50). The circles in Figs. 46 and 47 correspond to protoxylem poles. Thearrowheads in the insert of the Fig. 46 indicate divisions in the medulla. Epi, epidermis; Ex, exodermis; En, endoderm; Le, lenticel; P, peridermis;*, vascular ray. Scale bars: Figs. 39, 40 = 10 cm; Figs. 41–45, 47, 50 = 50 �m; Fig. 46 = 500 �m (insert = 50 �m); Figs. 48, 49, 51 = 100 �m.

Oliveira et al. 341

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granting permission and facilities to collect plantmaterial for thisstudy.

ReferencesAdedeji, O., and Jewoola, O.A. 2008. Importance of leaf epidermal characters in

the Asteraceae family. Notulae Botanicae Horti Agrobotanici, 36(2): 7–16.Appezzato-Da-Glória, B., Hayashi, A.H., Cury, G., Soares, M.K.M., and Rocha, R.

2008. Occurrence of secretory structures in underground systems of sevenAsteraceae species. Bot. J. Linn. Soc. 157: 789–796. doi:10.1111/j.1095-8339.2008.00823.x.

Bombo, A.B., DeOliveira, T.S., DeOliveira, A.D.S.S., Rehder, V.L.G.,Magenta, M.A.G., and Appezzato-D, and a-Glória, B. 2012. Anatomy andessential oils from aerial organs in three species of Aldama (Asteraceae–Heliantheae) that have a difficult delimitation. Aust. J. Bot. 60: 632–642.doi:10.1071/BT12160.

Bukatsch, F. 1972. Bermerkungen zur Doppelfarbung Astrablau-Safranin. Mik-rokosmos, 61: 255.

Carvalho, M.A.M., and Dietrich, S.M.C. 1993. Variation in fructan content in theunderground organs of Vernonia herbacea (Vell.) Rusby at different phonolog-ical phases. New Phytol. 123: 735–740. doi:10.1111/j.1469-8137.1993.tb03784.x.

Castro, M.M., Leitão-Filho, H.F., and Monteiro, W.R. 1997. Utilização de estrutu-ras secretoras na identificação dos gêneros de Asteraceae de uma vegetaçãode Cerrado. Rev. Bras. Bot. 20(2): 163–174.

Cury, G., and Appezzato-Da-Glória, B. 2009. Internal secretory spaces in thick-ened underground systems of Asteraceae species. Aust. J. Bot. 57: 229–239.doi:10.1071/BT08139.

Da Costa, F.B., Schorr, K., Arakawa, N.S., Schilling, E.E., and Spring, O. 2001.Infraspecific variation in the chemistry of glandular trichomes of two Brazil-ian Viguiera species (Heliantheae; Asteraceae). J. Braz. Chem. Soc. 12(3): 403–407. doi:10.1590/S0103-50532001000300012.

David, R., and Carde, J.P. 1964. Coloration différentielle des inclusions lipidiqueet terpéniques des pseudophylles du pin maritime aumoyen du réactif Nadi.C. R. Acad. Sci. 258: 1338–1340.

Del-Vechio-Vieira, G., Barbosa, M.V.D., Lopes, B.C., Sousa, O.V., S,antiago-Fernandes, L.D.R., Esteves, R.L., and Kaplan, M.A.C. 2008. Caracter-ização morfoanatômica de Ageratum fastigiatum (Asteraceae). Rev. Bras.Farmacogn. 18: 769–776. doi:10.1590/S0102-695X2008000500023.

Fahn, A. 1979. Secretory tissues in plants. Academic Press, London.Fritz, E., and Saukel, J. 2011. Secretory structures of subterranean organs of some

species of the Cardueae, and their diagnostic value. Acta Biol. Cracov. Ser.Bot. 53(1): 62–72.

Horridge, G.A., and Tamm, S.L. 1969. Critical point drying for scanning electronmicroscopic studyof ciliarymotion. Science,163: 817–818. doi:10.1126/science.163.3869.817. PMID:17807989.

Isejima, E.M., Figueiredo-Ribeiro, R.C.L., and Zaidan, L.B.P. 1991. Fructan compo-sition in adventitious tuberous roots of Viguiera discolor Baker (Asteraceae) asinfluenced by daylength. New Phytol. 119: 149–154. doi:10.1111/j.1469-8137.1991.tb01018.x.

Itaya, N.M., Figueiredo-Ribeiro, R.C.L., and Buckeridge, M.S. 1999. Synthesisof fructans by fructosyltransferase from the tuberous roots of Viguieradiscolor (Asteraceae). Braz. J. Med. Biol. Res. 32: 435–442. doi:10.1590/S0100-879X1999000400010. PMID:10347807.

Jensen, W.A. 1962. Botanical histochemistry: principles and practice. W.H. Free-man, San Francisco.

Johansen, D.A. 1940. Plant microtechnique. McGraw-Hill, New York.

Karnovsky,M.J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolalityfor use in eletron microscopy. J. Cell. Biol. 27: 137–138.

Lersten, N.R., and Curtis, J.D. 1985. Distribution and anatomy of hydathodes inAsteraceae. Bot. Gaz. 146(1): 106–114. doi:10.1086/337504.

Lersten, N.R., and Curtis, J.D. 1986.Tubular cavities in white snakeroot, Eupato-rium rugosum (Asteraceae). Am. J. Bot. 73: 1016–1021. doi:10.2307/2444120.

Lersten, N.R., and Curtis, J.D. 1988. Secretory reservoirs (ducts) of two kinds ingiant ragweed (Ambrosia trifida; Asteraceae). Am. J. Bot. 75: 1313–1323. doi:10.2307/2444454.

Lersten, N.R., and Curtis, J.D. 1989. Foliar oil reservoir anatomy and distributionin Solidago canadensis (Asteraceae, tribe Astereae). Nord. J. Bot. 9(3): 281–287.doi:10.1111/j.1756-1051.1989.tb01003.x.

Lotocka, B., and Geszprych, A. 2004. Anatomy of the vegetative organs andsecretory structures of Rhaponticum carthamoides (Asteraceae). Bot. J. Linn. Soc.144: 207–233. doi:10.1111/j.1095-8339.2003.00251.x.

Magenta, M.A.G. 2006. Viguiera Kunth (Asteraceae, Heliantheae) na América doSul e sistemática das espécies do Brasil. Tese de Doutorado, Instituto deBiociências, Universidade de São Paulo, São Paulo, SP.

Metcalfe, C.R., and Chalk, L. 1979. Anatomy of dicotyledons: systematic anatomyof leaf and stem with a brief history of the subject. Claredon Press. V. 2.

Milan, P., Hayashi, A.H., and Appezzato-Da-Glória, B. 2006. Comparative leafmorphology and anatomy of three Asteraceae species. Braz. Arch. Biol.Technol. 49(1): 135–144. doi:10.1590/S1516-89132006000100016.

Porto, T.S., Furtado, N.A.J.C., Heleno, V.C.G., Martins, C.H.G., DaCosta, F.B.,Severiano, M.E., Silva, A.N., Veneziani, R.C.S., and Ambrósio, S.R. 2009. Anti-microbial ent-pimarane diterpenes from Viguiera arenaria against Gram-positive bacteria. Fitoterapia, 80: 432–436. doi:10.1016/j.fitote.2009.06.003.PMID:19524643.

Ramayya, N. 1962. Studies on the trichomes of some Compositae I. Generalstructure. Bull. Bot. Surv. India, 4: 189–192.

Sakai, W.S. 1973. Simplemethod for differential stainning of paraffin embeddedplant material using toluidine blue. Stain Technol. 48: 247–249. PMID:4126691.

Schilling, E.E., and Panero, J.L. 2011. A revised classification os subtribe Helian-thinae (Asteraceae: Heliantheae) II. Derived lineages. Bot. J. Linn. Soc. 167:311–331.

Solereder, H. 1908. Systematic anatomy of the dicotyledons: a handbook forlaboratories of pure and applied botany. Clarendon Press. V. 1.

Strasburger, E. 1913. Handbook of practical botany. George Allen, London.Tertuliano, M.F., and Figueiredo-Ribeiro, R.C.L. 1993. Distribution of fructose

polymers in herbaceous species of Asteraceae from the Cerrado. New Phytol.23: 741–749.

Valluru, R., and Van den Ende, W. 2008. Plant fructans in stress environments:emerging concepts and future prospects. J. Exp. Bot. 59(11): 2905–2916. doi:10.1093/jxb/ern164. PMID:18603617.

Van den Ende, W., Laere, A.V., Roy, K., Vergauwen, R., Boogaerts, D.,Figueiredo-Ribeiro, R.C.L., and Carvalho, M.A.M. 2005. Molecular cloning andcharacterization of a high DP fructan: fructan 1-fructosyl transferase fromViguiera discolor (Asteraceae) and its heterologous expression in Pichia pastoris.Physiol. Plant. 125: 419–429. doi:10.1111/j.1399-3054.2005.00579.x.

Vilhalva, D.A.A., Cortelazzo, A.L. , Carvalho, M.A.M., and F, andigueiredo-Ribeiro, R.C.L. 2011. Histochemistry and ultrastructure of Campulo-clinium chlorolepis (Asteraceae) tuberous roots accumulating fructan: evi-dences of functions other than reserve carbohydrate. Aust. J. Bot. 59: 46–52.doi:10.1071/BT10126.

Zareh, M.M. 2005. Systematic and anatomical studies of Inuleae and Plucheeaein Egypt. Feddes Repert. 116: 43–53. doi:10.1002/fedr.200311058.

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