Secretory structures in Tagetes minuta (Asteraceae, Helenieae) - Sociedad Argentina de Botánica

ISSN0373-580X

Bol.Soc.Argent. Bot. 37 (3-4): 181

-

191. 2002

SECRETORY

STRUCTURESIN

TAGETES

MINUTA

(ASTERACEAE,

HELENIEAE)

P. M.SIMON13,L. KATINAS12,and A. M.ARAMBARRI2

Summary: Tagetes minuta is an important worldwide resource due to itsagrochemical and

pharmacological properties.Despiteof the great number of chemical studieson this species, the secretory structures arescarcelystudied.Ananatomicalsurveyofall organsof T.minutawas carried out.Three types of secretory structurescoexist in T. minuta:(1)cavitiesinfoliarblade and involucral

phyllaries;(2)ductsinroot, stem,petioles, midvein,capitula peduncle, corollas,andstyles; and(3) glandular trichomeson stem, leaves,phyllariesofthe involucre, and corollas. Ducts havean uniseriate orbiseriate epithelium, generally surroundedby aparenchyma sheath. Secretory cavities have a

multilayeredepithelium,and lack a parenchyma sheath. There is a lack of continuity between ducts and cavities.

Key words: Asteraceae, Helenieae, Tagetes minuta, secretory structures.

Resumen: Estructurassecretorasen Tagetesminuta (Asteraceae,Helenieae). Tagetes minutacons¬

tituyeun importante recurso a nivel mundial debido a suspropiedadesagroquímicasyfarmacológicas.

Sibiense han realizado numerosostrabajos químicossobreesta especie,las estructuras secretoras han sido poco estudiadas. Un examen anatómico de todos losórganosde T.minutademuestra'que

existen trestipos deestructurassecretoras:(1) Cavidades en segmentos foliares y filados del involu¬ cro; (2) conductos en raíz, taílo,pecíolo,vena media, pedúnculo del'capítulo, corolasyestilos; y (3) tricomas glandularesen tallo,hojas,pedúnculos, filados ycorolas. Losconductos tienen epitelio uniseriado o biseriado y están generalmente rodeados de vainaparenquimática. Las cavidadestienen epitelio

multiseriadoy no poseen, vaina parenquimática. No existe continuidad entreconductosy cavidades. Palabras clave: Asteraceae, Helenieae, Tagetesminuta, estructuras secretoras.

externaland internalstructures(Cutler,1969; Esau, 1976) is confusing since individual structures Secretory structures involve a wide variety of sometimes transcendparticular categories (Cutler, elements suchascavities, ducts, individual cells, and 1969). Forexample,someauthors consider glandsas

trichomes. Asecretorycavity (glandor sac)isa more internalstructures(e.g.. Cutler, 1969)whereas others

or less isodiametric internal space or lumen, consider themasexternal (e.g., Esau,1976). surrounded byanepithelium ofsecretorycells(Fahn,

1979).When thespaces areelongated they constitute secretorycanals(orducts) (Esau, 1976). In otherca¬

ses, the epidermal cells give rise to glandular trichomes of various degrees of complexity, which release the secretion between the cell wall and the cuticle.Lerstenand Curtis (1989)concludedthat there isnoqualitative difference between ducts and

cavities,and that thesetermsmerely identify thetwo extremesofarangeof reservoirs. Inaddition, the traditional classification of these structures into

INTRODUCTION

Lerstenand Curtis areamongmodem authors who have contributedmost to theknowledge of secretorystructuresin Asteraceae,e.g.,in Solidago and Conyza (Lersten & Curtis, 1987, 1989),in

Eupatorium (Curtis & Lersten, 1986;Lersten & Curtis, 1986), and in Ambrosia (Lersten & Curtis, 1988), belonging tothe tribes Astereae, Eupatorieae and Heliantheae, respectively. On the other hand,

secretorystructuresingeneraofthetribe Heleniae havenotbeen extensively studied.

The subtribe Pectidinae (Robinson,1981) isone of themosteasilyrecognized oftheHelenieae,mainly byits conspicuous, large,secretorycavities in leaves andinvolucralphyllaries. ThegenusTagetesL., with , 55 species (Soulé,1996),constitutes, together with Pectis,oneofthelargestgeneraof Pectidinae.It includes the popular TagetesminutaL.,commonly knownas“chinchilla” and “Mexican marigold”,

DivisiónPlantas Vasculares,Museo de La Plata, Paseo del Bosque s/n,1900La Plata. E-mail: [email protected]; [email protected]

Morfología Vegetal,Facultad deCiencias Agrariasy Fo¬ restales, Casillade Correo 3>, 1900 La Plata.E-mail:

[email protected]

Corresponding

author

Bol.

distributed in thetemperategrasslandsandmontane herbarium (see Appendix 1) and livingspecimens.

regionsofsouthernSouth America,including Peru,

Bolivia,Paraguay, Chile andArgentina.Itsaffinity for reconstitutedinboilingwater.Whole foliarsegments

disturbedsites hasallowedT.minutatocolonizemany andphyllarieswerecleared accordingtoStrittmatter areasaround the world suchasEurope,Asia,Africa, (1973). The midregion of plantorgans wereisolated Madagascar,India,Australia andHawaii(Soulé, 1993). and freehandcut transversely.Transverse serial Tagetesminuta is aneconomically important sectionsinwhole organs suchas florets and fruits worldwideresourceinmanycountries duetoits oilwith were also made. Some sectionswere stained with agrochemical andpharmacological properties.The Safranin while therestwere leftunstained. Material insecticidal activity offloral, foliar,androotextractsof T. wasalso fixed in formalin-acetic'acid-alcoholand minutaagainst cropspestssuchasweevils (Sriharanet processed by the usual techniques of paraffin

al.,1994; Weaveretal.,1994),mosquitoes(Perichetal., infiltration.Transverseserial sectionswere cut 10-1994, 1995;Maradufuetal.,1978),nematodes(Kumaret 15pmthickand stainedwithCresylviolet.

al.,1998; Zumarán etal., 1994), fungi(Bii etal., 2000),

Samples from herbarium specimens were

Light microscope observations and drawings bacteria (Hethelyietal.,1986),and viruses (Hudson, were carriedouton aLeitz SMLux microscope

1990)arewidelyrecognized.Variousmedicinalproperties equippedwitha camera lucida. Selected light anduseas acondiment and in the essential oil industry microscope imageswere transferred electronically (as“Tagetesoil”)arefully demonstrated andexploited fromthemicroscopetothecomputerusing the Photo (Neher,1968;Martijenae/«/., 1998;Craveirocía/., 1988). Express version 1.0 software, and then sized,

'_Due

totheeconomic valueofT.minutamany processedandorganizedintoplates withCorelDraw chemical studies have beenperformed (e.g., Kaplan, 9, Corel Photo-Paint10and MicrosoftPowerPoint 1960;Lilipov,1997;Israilev&Seeligmann, 1983, 1985; computerprograms. Leaf and phyllary tissueswere Maradufu etal., 1978; Font Quer, 1981; Russinetal., prepared for scánning electron microscopy by 1988). The species is richinmany secondary sputter-coating with gold/paladium, and examined compounds, including monoterpenes, sesquiterpenes, andphotographedwithaJeolJSM-T100 Scanning flavonoids, thiophenes, and aromatics. Differences in ElectronMicroscope.

the oil activity between organs of T.minuta,suchas

between leaves and florets,havebeendocumented(Bii “ducts” for the elongated internal secretory structures,and “secretory cavities” for themore or Wefollow Fahn (1979) in theuseofterms,i.e.,

etal.,2000).

Secretory cavities, ducts andsecretorycells have less isodiametrical secretory structures. Both been reportedinthegenusTagetes (VanTieghem, 1872; structures aredefinedasincluding the epithelium Thouvenin, 1884;Leblois, 1887;Col, 1903;Metcalfe & and lumen.Allsecretorystructuresaredescribed in Chalk, 1950), focusingonTageteserecta(Thouvenin, transection,exceptwhenindicated.

1884)and T.patula (Van Tieghem, 1872). Untilnow, secretory structureshave been scarcely analyzed in T.

minuta.Adevelopmentalstudy in T.minutaby Del

RESULTS

Fueyo (1986) identified glands (=secretorycavities) in

leaves andphyllaries,andsecretoryducts in petiole,

andmidvein,interpreting themasschizogenous. The _{the vascular cylinder (Fig. 1A, 2A). Cortex ducts}: presence of thesestructuresinroots,capitulapeduncle, _{Fourtosix,ranging from 10pmto}25 pmdiam., situated florets, and fruits,onthe other hand, hasneverbeen _{amongthe endodermis and the parenchyma cells of}

investigated.

In thispaperweexamine theocurrence,structure _{periderm inrootswith secondary growth.Uniseriate}

and distribution ofsecretory structuresinroot,stem, _{epithelium, with 5-6}epithelialcells. Parenchyma

leaves,capitulapeduncle,involucre,florets, and fruits _{sheath is absent. Vascular cylinder ducts: Ten}to12, 15 pm to 40 pmdiam., situated inthephloem.

Uniseriate epithelium, with 5-7 epithelial cells. Parenchymasheath isabsent.

Root.Ducts aredistributed in thecortexand in

thecortexinrootswith primary growth,orin the

of T.minuta.

MATERIAL

AND METHODS

Roots,stems,leaves,capitulapeduncle,phyllaries Stem.Ducts: Twelveto 20,ca.30 pmdiam., oftheinvolucre,floretsand fruitswereobtained from alternatingwith thephloem andassociatedwith

P.M.Simoneta!., Secretorystructuresin Tagetes minuta(Asteraceae)

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Fig. 1. Lightmicrographsofcrosssectioninorgansof Tagetesminutashowing thesecretory structures(arrows). A:root

showingtwosecretoryducts,onein theperiderm andoneinthe phloem; B: enlarged' view ofstemshowingthecortexwhere

thesecretoryductsare found; C:secretoryduct instem cortex,associated with sclerenchymatissue;D:biseriate capitate

glandulartrichome instem.E: transitionareabetween the foliar blade and the petioleshowingducts (arrow)andonecavity; F: enlarged view of the foliarsecretorycavity. A, E, F: Simon 1078 (LP); B-D:Simon 20 (LP). Scale bars: 200 pm on A,B,

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corresponds to the capitate hairs or “biseriate capitateglandular hair” ofRamayya (1962), andare verywidelydistributedinAsteraceae.

schlerenchymatissue,inthe corticalparenchyma(Fig. IB).Uniseriate epithelium(Fig. 1C), with 6-10epithelial cells, surrounded by a parenchyma sheath. Trichomes (Fig. ID, 2B)L Differentiatedinto stalk and

head,70-200 pmlong.Stalkbiseriate (occasionally uniseriate); uniforminbreadth; oblongorbarrel¬

shaped;contentstranslucent; 4-7-celledineachrow; with cells suboppOsiteoropposite;

cell

walls thinor

thick;outerwallsstraightorslightlyconvex,smooth; a1-2-cellularbaseimbedded among the epidermal

cells;cells of the basal tiers usually broader than longor isodiametrical. Head sharply or slightly demarcated fromthe stalk;roundedinshape;shorter than the stalk; contents dense, generally more notorious inthe terminal tier; 2-4-tiered, cells

isodiametrical,ofvariousshapes;cellwalls thinor

thick;outerwalls smooth. This typeoftrichome

Leaf

petiole. Ducts: Threetofive, rangingfrom

35 pmto50pmdiam.,situated abaxially inthepetiole,

between the vascularbundles,in theparenchyma

(Fig. IE, 3A).Uniseriatetobiseriateepithelium,with 5-12epithelial cells,surrounded byaparenchyma

sheath(Fig. 3B). Occasionally, cavitiesare present

intransitionalareasbetweenpetioleand blade(Fig. IF).Trichomes: Similartothestemtrichomes.

Leaf

midvein.Ducts:Threetofive,ranging from 20 pmto50pmdiam. (Fig. 3C). Ductsareseen

at various stages of development (Fig. 3D). Developedducts withuniseriatetobiseriateepithelium,

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Fig.2.Secretory structuresinTagetesminuta(arrows). A:secretoryducts intheroot cortex(above) andphloem(below)

showingthe uniseriateepithelium;B: biseriatecapitate glandular hairsin differentviews;C:secretorycavityinaninvolucral

phyllary showingthe multiseriate epithelium;D,secretoryduct in the style incontact withperiphericvascular and central . stigmatoidtissue. A: Simon 1078 (LP);B: Simon20(LP); C:Ringuelet185(LP); D:Mangieri11 (LP). Scalebars: 100 pm.

P. M.Simoneta!., Secretory structuresin Tagetesminuta (Asteraceae)

Leaf

blade. Cavities. Surface view: Leaves in with3-5epithelialcells,surroundedbyaparenchyma

sheath(Fig. 3E).Trichomes: Three-5-celled, oblong, T. minuta are pinnatisect, constituted by 8-20 segments; the secretorycavities areconspicuous, similartothestemtrichomes.

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Fig. 3.Lightmicrographs of cross section in organs of Tagetesminutashowing thesecretory structures(arrows). A: enlarged view of the petiole showingtwosecretory ductsatboth sides of one small vascularbundle;B:detail of one petiolesecretory

duct showing the biseriateepitheliumand the surroundingparenchymasheath;C: enlargedview of the midveinshowingthe

areabetween the vascular bundles where thesecretoryductsatdifferentstagesof developmentarefound; D:secretoryduct of the midveinindevelopmentstage;E:maturesecretoryductofthe midvein showing biseriateepitheliumandthe surrounding parenchyma sheath. F: enlarged view of thecapitulapeduncle showing theareabetweenthe vascular bundles where the

Bol.Soc. Argent.Bot.37 (3-4)

large,pellucid,bulgingabove the surface of the thephloem,inthe parenchyma(Fig. 3F).Uniseriate

segments,rounded-elliptictolinearinshape,ten to epithelium,with 6-8epithelialcells,surrounded bya 15 per segment. They occurintworegions,the parenchyma sheath. Trichomes:Eight-10-celled,

slightlylargercavities (ca. 1mmlong.)arefound oblong,similartothestemtrichomes. alongthe blade margin, and the smallerones(0.1-0.3

mmlong.) areclustered ingroups about midway

between themarginand the midvein. Despite their InvolucreinT.minutais constitutedbyasingleseries differencesinposition andsize,thesecretorycavities of3-4connatephyllaries.Likeintheleaves,thesecretory are identical instructure. Transection: There is cavitiesareconspicuous,large,pellucid,

bulging

above commonlyone secretorycavityineach halfofthe thesurface ofthe phyllaries (Fig. 4A), lineartooblong segment,atthe level ofboth,palisade and spongy inshape, 0.2-2mmlong.,twotofiveperphyllary.They

mesophyll, or just at the levelof the spongy aredistributed in thecenterofthe phyllary,nearthe mesophyll,nearthe margin,paralleltothemidvein, margin,and also inthejunctionareabetweentwo

They range from 170 pmto 350 pm long. The phyllaries.Transection: One to fiveinthe whole

epitheliumis multilayered (insomepreparationsthe involucretransection, rangingfrom 50pm to230 pm border of the cavities appears disintegrated) (cfr.Fig. long.,attheleveloftheparenchymatissue (mesophyll), IF). Parenchymasheathnotseen. Trichomes:Two- oroccupyingthe whole phyllarytransection,fromthe 3-celled, sparse,similartothestemtrichomes.

Phyllaries

_of

theinvolucre.Cavities. Surface view:

externaltothe internalepidermis.Epitheliumsimilarto those of the cavities in leaf blade. Parenchyma sheath Capitulapeduncle.Ducts:Tento15,ca.30 pm is absent (Fig. 2C,4B, C). Trichomes:Four-10-celled,

diam. alternating with the vascularbundles,next to oblong,similartothestemtrichomes.

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Fig.4. A-B: SEM micrographsofsecretorycavities inTagetesminuta.A:secretory cavityinthe externalsurface ofa phyllary ofthe involucre; B: crosstransection«ofa secretorycavityinaphyllary ofthe involucre. C-D: lightmicrographs_of

crosssection inorgansofTagetesminutashowingthesecretory structures(arrows). C:phyllary oftheinvolucre showingone

secretorycavity bulgingabove the externalsurface;D:corollawithasecretoryduct adaxialtothe vascularbundle, A,B:

P.M. Simon et at., Secretory structuresin Tagetes minuta(Asteraceae)

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Fig. 5. Diagrammatic overview of Tagetesmiijutawith the organs in transection, showing the ocurrence andpositionof the

secretory structures.Frombottomtotop: root, stem,petiole, midvein, blade,capitulapeduncle, capitulum.c=corolla; p=

Floralorgans.Secretory structures arepresent (1872),the corticalducts in therootsof Asteraceae in corolla and style, but they areabsent from the appearasmerely

intercellular

spaces formed in close connection with the endodermis. Tetley (1925) Corolla. Ducts:Fourtosix percorolla,ranging classified the ducts inroots of Asteraceae as from20 pmto30 pmdiam.,adaxially situated and endodermal (situatedinthecortex, closeto the

alignedtothe vascular bundles,adjacenttothe xylem. endodermis),and non-endodermal (usually found in Uniseriate epithelium, with 4-6 epithelial cells, thesecondary phloem).Thisauthor suggested that surrounded by aparenchyma sheath (Fig. 4D). infacttheendodermal ductsarebordered by four

Trichomes:Distributedontheexternal epidermis,5- cellswhich,intransversesections, look exactlythe

9-celled,linear-oblong,similartothestemtrichomes. same astherestofthe endodermal andcorticalcells. Style. Ducts: One,rangingfrom 15pm to20 pm Theseductsmayremainborderedbythese four cells

diam.,situatedinternallytothe vascular tissue andin orinsomecasesbecome surrounded bythin-walled

contactwithacentralmassofcells withcollenchymatous epithelial-likecells.Inaddition,the formationof walls that would correspondtothestigmatoidtissue epithelial-likecells surrounding the non-endodermal (Esau, 1976).Uniseriateepithelium,with4-6epithelial ducts is ofcommon ocurrence(Tetley, 1925). Our examinations in T.minutaagree with Tetley’s (1925)

observations,showingendodermal (in the cortical Ductsandcavities connection. Serialtransverse parenchyma,orintheperiderm) andnon-endodermal sections and clearedsegmentsand phyllaries show (in the phloem) rootducts, bothtypesbordered by that there isnocontinuitybetweensecretoryducts epithelial cells (Fig. 2A).

and

cavities'

which results from the.absence ofany

typeofinterconnections between them. androecium and ovary (and thus fruit).

cells. Parenchyma sheath is absent (Fig. 2D).

Rootductsarethe onlystructuresin T.minuta that areboth,extravascular and imbeddedinthe vascular tissue (Fig. 1A). In therestofthe organs secretory structures areextravascular wich,in the caseofthe leaves, coincides with other genera of the tribe Helenieae such asPectis and Flaveria

DISCUSSION

Threetypesofsecretory structurescoexistinT (Petenatti &Del Vito, 20Ó0).Ductsincorollas of T. minutaaredistributed adaxialtothe vascular bundles minuta(Fig. 5): (1)secretorycavities infoliarblade

and involucralphyllaries;(2)secretoryducts inroot, (Pig- 4D), andarecontinuous from the base of the stem,petioles,midvein,capitulapeduncle, corollas, corolla tubetotheapexofthe limb. Discontinuity of and styles; and (3) biseriatevesicular glandular thesecretory ducts in florets of other genera of

trichomeson stem,leaves,peduncles, phyllariesof Asteraceae, such as Grindelia, is an unusual feature(Giraux & Susplugas, 1935). Ductsinstyles

theinvolucre,and corollas.

Atthispoint,wecanestablishmorepreciselya

(Pig-

2D)areonlyseenin theareaofthe staminal filamentsandnot atthe level of anthers or the distinction betweensecretoryducts and cavities in

T.minuta.Besides their different distribution in the apical appendages.

This studyprovided the firstcomprehensive

plant,asremarkedabove,thesecretorycavitiesare „

wide (90-300 pm diam.),bulgingabovethe organ’s anatomicaldescription of thesecretory structures surfacè (Fig. 4A). In transection they have a of Tagetesminuta,mainlythose aspectsrelated multilayeredepithelium(Fig. 2C), lackaparenchyma to the secretory ducts. It was clarified or.

sheath,andarefather separated from

the

vascular established the presence andpositionofsecretory ducts in differentplant organs,the anatomical

tissue byparenchymaor sclerenchyma. Ducts,on

the otherhand,areless wide (10-50 pm diam.), and aspects of ducts were elucidated, and clear neverbulgeabove the organs surface. Intransection,

theyhaveauniseriateorbiseriateepithelium (Figs.

1C, 3B, E),aresurrounded byaparenchymasheath, demonstrated differences in theanatomy,size,and orthe sheat is absent, andarerelativelyclosetothe distribution of secretory structures of an economicallyimportant and still promisingspecies

Someadditionalremarkscanbemadewithrespect ’°f Asteraceae that can helpto anchor the great

tothe duct characteristics inrootsand the location amountofchemicalinformationabout Tagetesmi¬

nutato sites andstructures.

differences between ducts and cavities were established. In summary, this study has

vascular tissue.

P.M.Simonet at., Secretory structuresinTagetes minuta (Asteraceae) ISRAILEV, R. L. A. & P. SEELIGMANN. 1983. Distribu¬ ciónde los flavonoides foliaresen tresespeciesde Tagetes (Compositae)ysu significadoquimiosistemático.Lilloa 36: 5-14.

ISRAILEV, R. L. A. & P. SEELIGMANN. 1985. Los flavonoidesfloralesentresespecies de Tagetes. Lilloa 36: 187-190.

KAPLAN, L. 1960. Historical and ethnobotanicalaspects

ofdomesticationin Tagetes. Econ. Bot.14:200-202. KUMAR, A., F. V. DUNKEL & -S.SR1HÀRAN. 1998.

Effectiveness ofrootexudatesofthe Mexican marigold, Tagetesminuta, forthemanagement ofthe sugarbeet

cystnematode,Heterodera schachtii. Abstract_ofthe Annual Meeting_ofEntomologicalSociety_ofAmerica, Las Vegas,Nevada.

LEBLOIS, A. 1887. Recherches sur I’origine et le développement des canaux sècrèteurs etdespoches

sècrètrices. Ann. Sci. Nat., Bot. 7e série,6: 247-330. LERSTEN, N. R. & J. D. CURTIS. 1986. Tubular cavitiesin

white snakeroot Eupatoriumrugosum(Asteraceae). Amen J.Bot.73: 1016-1021.

LERSTEN, N. R. & J. D. CURTIS. 1987. Internalsecretoryspaces

inAsteraceae.A review and originalobservationsonConyza

canadensis (tribeAstereae).LaCellule74:179-196. LERSTEN, N. R. &J. D. CURTIS. 1988. Secretory reservoirs

(ducts) oftwokindsin giantragweed (Ambrosia_trífida; Asteraceae). Amer. J. Bot. 75:1313-1323.

LERSTEN, N. R. &J.D. CURTIS. 1989.Foliar oil reservoir

anatomy and distribution in Solidago canadensis

(Asteraceae,tribe Astereae). Nordic J. Bot. 9: 281-287. MARADUFU,A.,R. LUBEGA &F. DORN. 1978. Isolation of (5 E)-ocimone: A mosquito larvicide from Tagetes

minuta.Lloydia 41: 181-182.

MARTIJENA, J. D., D. A. GARCIA, R. H.MARÍN,M. A. PERILLO& J. P. ZYGADLO. 1998. Anxiogenic-like

and antidepressant-like effects of the essential oil from Tagetesminuta.Fitoterapia 69: 161-164.

METCALFE, C.R.& L. CHALK. 1950. Anatomy_ofthe Dicotyledons. 2. Oxford Clarendon Press.

NEHER,R. T. 1968. The ethnobotany of Tagetes. Econ.

Bot. 22: 317-325.

PERICH, M„ K. E.TREDWAY,C. WELLS &W. BERTSCH. 1994.ToxicityofextractsfromthegenusTagetesagainst adult and larvalstatesofAedesaegyptiandAnopheles stephensi(Diptera, Culicidae). Med. Entomol. 3L 833. PERICH,M., C. WELLS, W. BERTSCH&K.E. TREDWAY.

1995. Isolation of the insecticidal components of Tagetes minuta(Compositae)againstmosquito larvae andadults. Journal_ofthe American mosquito Control Association 11: 307-3 10.

PETENATTI, E. M. & L. A. DEL VITTO. 2000.Estructu¬ ra Kranz en las especies argentinas de Flaveria. (Asteraceae-Helenieae). Kurtziana 28: 251-257. RAMAYYA, N. 1962. Studies in the trichomes of some

Compositae.I.Generalstructure.Bull. Bot. Surv. India 4: 177-188.

AKNOWLEDGEMENTS

WearegratefultoJorge V. Crisci, SusanaE. Freire, Richard Keating, andtwoanonymousreviewers for usefulcommentsuponthemanuscript. Special thanks aregiventoJohn Curtis for his valuablecomments

and suggestions. Víctor H. Calvetti is also acknowledged for the drawing of figure 5, and

curators ofherbaria LP and LPAG for loan of specimens. This workwassupported(L.K.)by Con¬ sejo Nacional de Investigaciones CientíficasyTéc¬ nicas (CONICET), Argentina, and the National Geographic Society (Grant5776-96).

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Recibido el 22 Julio de 2002, aceptado el 12 deSeptiembre

P. M. Simon etal.,Secretorystructures in Tagetes minuta(Asteraceae)

Appendix 1. Collection data (country,province, locality,date ofcollection,collector andnumber,and herbarium abbreviation) ofthe anatomicallyexamined specimens analyzedof Tagetesminuta.

ARGENTINA.Prov.BuenosAires.LaPlata,1995,Ringuelet 185 (LPAG),V-1995,Arambarri156(LPAG), 22-IV-1931,Finasteras.n.(LP);PuntaLara, 6-IV-1966,Rossis.n.(LPAG); Berisso,balnearioBagliardi, 1996,

Bayón 318 (LPAG). Prov. Chaco. Dpto. Nueve deJulio,Las Breñas, 16-XI-1998, Simon 20 (LP).Prov. Misiones.Caá-Guazú,IX-1940,Mangieri11(LP).

BOLIVIA.Dpto. Tarija,Cainguá, 15 kmNde VillaMontes, 4-VI-1971,Krapovickasetal.s.n.(LP);Dpto. Santa Cruz,vicinityofabandoned old JardínBotánico,alongRio Pirai and roadsidesonW side of Santa Cruz, 17° 47' S,63° 13'W,8-VII-1987,Nee35074(LP).

PARAGUAY.Dpto. Cordillera,Altos Colonia Bernal Cué, 18-VI-1973, Schininis.n.(LP).