Rev. bras. farmacogn. vol.27 número1

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RevistaBrasileiradeFarmacognosia27(2017)9–19

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Comparative

morphoanatomical

analysis

of

Mikania

species

Valter

Paes

de

Almeida

a

_,

_Adriana

_Araci

_Hirt

b

_,

_Paola

_Aparecida

_Raeski

a

_,

_Beatriz

_Eloise

_Mika

a

_,

Bárbara

Justus

a

_,

_Vera

_Lucia

_Pereira

_dos

_Santos

c

_,

_Célia

_Regina

_Cavichiolo

_Franco

d

_,

Josiane

Padilha

de

Paula

e

_,

_Paulo

_Vitor

_Farago

e

_,

_Jane

_Manfron

_Budel

e,∗

a_Curso_de_Ciências_{Farmacêuticas,}_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil b_Curso_de_Farmácia,_Faculdades_Integradas_do_Vale_do_Iguac¸_u,_União_da_Vitória,_PR,_Brazil

c_Departamento_de_Engenharia_Saúde_e_Meio_Ambiente,_Centro_{Universitário}_{Internacional}_Uninter,_Curitiba,_PR,_Brazil d_Departamento_de_Biologia_Celular,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

e_{Pos-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26March2016 Accepted9May2016 Availableonline13June2016

Keywords:

Adulteration Asteraceae Differentiation Herbaldrug Pharmacobotany Qualitycontrol

a

b

s

t

r

a

c

t

MikaniabelongstotheAsteraceaefamilyandincludesawiderangeofpromisingpharmacological activ-ities.SeveralspeciesofMikania,whichispopularlyknowninBrazilas“guaco”,occurinSouthernBrazil andtheirexternalmorphologyissimilar.Theaimofthisstudywastoinvestigatethemorpho-anatomical characteristicsoftheleafandstemofMikaniacampanulata,Mikaniacordifolia,Mikaniaglomerata,Mikania hastato-cordata,MikaniamicropteraandMikaniasessilifoliaasameansofprovidingadditionalsupport fordifferentiatingthesetaxa.Theleavesandstemswereinvestigatedbyemployingscanningelectron microscopyandlightmicroscopytechniques.ThemorphologicalfeaturesofMikaniaspp.leavesmake itpossibletodifferentiatebetweenthespecies;nevertheless,whentheplantswerefragmentedor pul-verizedtheanatomicalfeaturesoftheleavesandstemssuppliedadditionalhelpfuldatainthisregard. Themainanatomicalcharacteristicswerepresenceofhypodermisandlensshapedepidermalcells,set oftrichomes;midrib,petioleandstemshapeandvascularpattern;sclerenchymatousringinthecortex, sclerenchymatouscellsandsecretoryductsinthepith.

Introduction

Mikania Willd. belongs to the Asteraceae family and the Eupatorieae tribe. It comprises 198 species in Brazil, ranging from the North to the South of the country, and is mainly foundin thestates of São Paulo,Rio de Janeiro, Minas Gerais, and Rio Grande do Sul (King and Robinson, 1978; Ritter and Miotto,2005;ThePlantList,2015).Mikaniaspp.hasprovideda widerangeofpromisingphamacologicalactivitiesanditcanbe describedashavingexpectorant,anti-inflammatory, bronchodila-tor, anti-hemorrhagic, antiasthmatic, analgesic, anti-mutagenic, trypanocidal, antimicrobial, antifungal, antiulcerogenic, muscle relaxant, and antirheumatic properties (Muelas-Serrano et al., 2000;Pauletal.,2000;Gasparettoetal.,2010;Pérez-Amadoretal., 2010;Mourãoetal.,2014;Ríosetal.,2014).

The inappropriate use of popular names can result in seri-ousmistakesinrelationtotheidentificationofherbaldrugs.The sameplant oftenhasseveralcommon namesand furthermore,

∗ Correspondingauthor.

E-mail:[email protected](J.M.Budel).

differentspeciesmaybeknownbythesamefolkname(American Herbal Pharmacopeia, 2011).Morpho-anatomical data areused to mitigate this problem. This technique uses morphological and anatomical features to characterize and differentiate simi-lar species (Luz et al., 2015; Wosch et al., 2015; Porto et al., 2016), especially when the botanicals are marketed in a frag-mented or powdered form (American Herbal Pharmacopeia, 2011).

Mikania campanulataGardner,Mikania cordifolia(L.f.) Willd., MikaniaglomerataSpreng.,Mikaniahastato-cordataMalme, Mika-niamicropteraDC.,andMikaniasessilifoliaDC.,whicharepopularly knownas“guaco”,occurinSouthernBrazilandtheirexternal mor-phology is similar.Hence, someconfusionand/or mistakescan resultinpopularusage.

Previous data have revealed that M. cordifolia has anti-inflammatory(Pelusoetal.,1995),insecticidal,trypanocidal(Arias etal.,1995;Muelas-Serranoetal.,2000),genotoxicand antipro-liferative (Diasetal., 2014)activities.M.glomerata isthe most common among the genus and it has many properties such as anti-inflammatory, antitussiveand bronchodilatadory agents (Gasparettoetal., 2010).M.sessilifoliais usedin folkmedicine totreat coldsand flu.Thereis nopharmacologicalor chemical

http://dx.doi.org/10.1016/j.bjp.2016.05.002

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characterizationavailableforM.microptera,M.hastato-cordataand M.campanulata.

In viewof the similarexternal morphologyof M. campanu-lata,M.cordifolia,M.glomerata,M.hastato-cordata,M.microptera and M.sessilifolia,theintention of this paperwas tostudythe morpho-anatomical data of the leaves and stems of these six speciesasameansofprovidingadditionalsupportfor differen-tiatingthesetaxaandidentifyingfeaturesofthemwhichcanbe usedas potentialcontaminantsor substitutes forothers of the genus.

Materialsandmethods

Plantmaterials

TheaerialpartsofatleastfivespecimensofMikaniacampanulata Gardner,M.cordifolia(L.f.)Willd.,M.micropteraDC.andM. hastato-cordataMalme,Asteraceae,werecollectedfromSãoMaximiano Farm,whichislocatedintheregionofSerradoSudoeste,inthe cityofGuaíba,stateofRioGrandedoSul,SouthBrazil(coordinates 30◦₁₀′_S_and₅₁◦₂₀′_W,_and₂₇_m_altitude)._M._campanulata_,_M.

cordi-foliaandM.hastato-cordatawerecollectedinSeptember2012and M.micropterainDecember2003.M.glomerataSpreng.andM. ses-silifoliawerecollectedinSeptember2014fromtheCamposGerais Region,inthecityofPontaGrossa,stateofParaná,SouthBrazil (25◦₅′ _S_and₅₀◦₆′ _W,_and₉₀₀_m_altitude)._The_botanical

materi-alswereidentifiedandtherepresentativesampleswereregistered attheRioGrandedoSulFederalUniversityundernumbersICN 175.095(M.campanulata),ICN116.543(M.cordifolia),ICN187.121 (M.glomerata),ICN129.447(M.microptera),andICN159.629(M. hastato-cordata).M.sessilifoliawasidentifiedandthevoucherwas registeredattheHerbariumoftheStateUniversityofPontaGrossa undernumberHUPG10.208.

Anatomicalanalysis

TheleavesandstemsofM.campanulata,M.cordifolia,M. glom-erata, M. hastato-cordata, M. microptera and M. sessilifoliawere cutabout10cmfromtheapex. Theywerethenputin contain-erscontainingFAA70solution(Johansen,1940),andstoredin70% ethanol(BerlynandMiksche,1976).Theplantsweresegmented byhand.Transverseandlongitudinalsectionswerestainedusing astrablueand/orbasicfuchsine(Roeser,1972).Theleaveswere clarifiedinfrontalviewoftheepidermis,usingKrausandArduin (1997)techniques.The photomicrographswere capturedby an OlympusCX31lightmicroscopeequippedwithaC7070control unit.

Histochemicalanalysis

Thecellcontentandcellwallimpregnationwereexposedby adoptingthefollowingstandard solutionsin themicrochemical tests: hydrochloricphloroglucintoreveal traces oflignin (Sass, 1951);SudanIIIfortestinglipophiliccompounds(Foster,1949); ferricchloridetotestfor phenolicsubstances (Johansen,1940); andiodine-iodidetotestforstarch(BerlynandMiksche,1976). Thesemi-permanentslideswerethenanalyzedintheLaboratory ofPharmacognosyattheStateUniversityofPontaGrossatoallow adetaileddescriptionoftheleafandstemtissues.

Scanningelectronmicroscopyandenergy-dispersiveX-ray spectroscopy(EDS)

Thescanningelectronmicroscopy(SEM)oftheleafandstem surfacewasperformedinhighvacuumandwithhighaccelerating voltage(15kV).Thisprocedurerequiredthesamplestobe pre-viouslydehydratedusingincreasingamountsofethanolandthe criticalpointofCO2.Afterthis,theyweresubmittedto

metalliza-tionwithgold(Souza,1998).

TheEDSchemical microanalysiswasrandomlyperformedin crystalsandcellswithoutcrystals(control),withanX-raydetector coupledtoSEMandunderthesameSEMoperatingconditionsthat wereusedtotaketheelectronmicrographs.Thisprocedurewas carriedoutatthemulti-userlaboratoryoftheStateUniversityof PontaGrossaand theElectronMicroscopyCenterattheFederal UniversityofParaná.

Resultsanddiscussion

ThemorphologyofM.campanulata,M.cordifolia,M.glomerata, M.hastato-cordata,M.micropteraandM.sessilifoliawassimilarand themaindifferencescanbeseeninBox1andFig.1.Theleaveswere simple(Fig.1)andthephyllotaxyhadanoppositearrangementfor allthespecies.

M.campanulata(Fig.1A)hadleavesthatwere5–10cmlongand 4–8cmwide.Theleavesweremembranousandslightlypurplishon theabaxialside.Theyweredeltatetohastateinform,with acumi-nateapex,hastatebase,paucidentatemargins,andthreevisible veins.Theyhadastraightpetiolethatmeasured5–6cminlength. M.cordifolia(Fig.1B)had leavesthat were4–8cmlong and 4–8cmwide.Thestudiedleavesweredeltoidtohastateinform, withacuminateapex,hastatetocordatebase,paucidentate mar-ginandfivevisibleveins.Thepetiolewasstraightand1.5–2cm inlength.TheleavesofM.glomerata(Fig.1C)were10–15cmlong and5–9cmwide.Theywerelanceolate–hastateinshapeandhad

Box1:MorphologicalcharacteristicsofMikaniaspp.

Leafblade M.campanulata M.cordifolia M.glomerata M.hastato-cordata M.microptera M.sessilifolia

Color Greenonthe

adaxialsideand purplishonthe abaxialside

Greenonboth sides

Size:

Length(cm) 5–10 4–8 10–15 4–6 6.5–13 3.5–8

Width(cm) 4–8 4–8 5–9 4–4.5 5.5–13 3–6

Shape Deltatetohastate Deltatetohastate Lanceolate–hastate Triangularto deltate

Triangular Widelyovate

Apex Acuminate Acuminate Acuminate Acute Acute Acutetoobtuse

Margin Paucidentate Paucidentate Entire Entire Paucidentate Crenatetoserrate

Base Hastate Hastatetocordate Cordatetohastate Cordatetohastate Hastate Slightlycordate

Venation 3 5 3–5 3 5 3

Formofpetiole. Straight Straight Curved Curved Straight Veryshort

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V.P.deAlmeidaetal./RevistaBrasileiradeFarmacognosia27(2017)9–19 11

Fig.1.Mikaniaspp.–Morphologicalappearanceofleaves–A.MikaniacampanulataGardner,B.M.cordifolia(L.f.)Willd,C.M.glomerataSpreng.,D.M.hastato-cordataMalme, E.M.micropteraDC.,andF.M.sessilifoliaDC.Scalebar=2cm.

anacuminateapex,cordatetohastatebase,entiremargins,and 3–5visibleveins.Thepetiolehadacurvingshapeandmeasured 2–2.5cminlength.

M.hastato-cordata(Fig.1D)hadleavesthatwere4–6cmlong and4–4.5cmwide.Theleavesweretriangulartodeltateinform withacuteapex,cordatetohastatebase,entiremargins,andthree visibleveins.Theyhadastraightpetiolethatmeasured2–2.5cm in length.Theleavesof M.microptera (Fig.1E)were6.5–13cm longand5.5–13cmwide.Theyweretriangularinshapewithacute apex,hastatebase,paucidentatemarginsandfivevisibleveins.The petiolewas2–2.5cminlength.

M.sessilifolia(Fig.1F)hadleavesthatwere3.5–8cmlongand 3–6cmwide.Theleaveswerewidelyovateinform,withacuteto obtuseapex,slightlycordateatthebase,crenatetoserratemargins andthreevisibleveins.Theyhadaveryshortpetiolethatmeasured 0.1–0.3cminlength.

In botanical products, substitution, impurities and contami-nantsare typesof adulteration.Adulteration canconsist ofthe partial or complete replacement of one species for a different species,excessivequantityofimpurities,deterioratedorpoor qual-itymaterial,aswellascontaminantsoranyconditionthatwould otherwise reduce thequality of the product (American Herbal Pharmacopeia,2011).

Therehavebeenmanywell-publicizedaccountsofthe unin-tentionalandintentionalcontaminationofbotanicalsbyincorrect vegetablespecies.Theproblemsofaccidentalcontaminationand economically-motivated tampering are questions that must be addressedwhen dealingwithherbaldrugsthat have been got-tenfromtradersorwhentheoriginofthebotanicalsisinexact (Reynertson and Mahmood,2015).Morpho-anatomical features havebeenusedtovouchforthequalityandauthenticityofherbal drugs(Woschetal.,2015;Araújoetal.,2015;Guerreiroetal.,2015). The botanical analysis of medicinal species is the first test conductedbytheherbalindustry and itis designed toidentify

thespeciesofplant.Itsmorphologicalfeatures areimportantin terms of authenticity. However, anatomical profiles have been used toclassify and differentiate species, especially when they arein fragmentedor powderyform.The identificationof Mika-niagenusiscomplexbecauseofitsmorphologicalsimilaritiesand thishasresultedintheindiscriminateuseofdifferentplantsfor thesametherapeuticpurposes(Amorinetal.,2014;Araújoetal., 2015).

SeveralMikaniahaveasimilarmorphology,andtheseinclude M.glomerataSpreng.andM.laevigataSch.Bip.exBaker(Budeletal., 2009),M. lanuginosaDC.and M.hirsutissimaDC.(Amorinetal., 2014),M.confertissimaSch.Bip.exBaker,M.hatschbachiiG.M. Bar-rosoandM.glomerata,andMikaniahirsutissimaandM.microlepsis Baker(Oliveiraetal.,1994).

Theanatomical characteristicsof theleavesshown inBox 2

andthestemanatomicalfeaturesshowninBox3canbeusedto differentiatetheMikaniaspecies.

Anatomically,fromafrontalviewofthefoliarblade,the anti-clinalepidermalcellwallswerethinandsinuous(Fig.2B,G,H,I) andslightlywavy(Fig.2A,C)ontheabaxialsideandslightlywavy ontheadaxialside(Fig.2D–F,J–L)forallthespecies.Inaddition, theepidermiswascoveredbyasmoothcuticle,butwasslightly striatednearthestomata(Fig.3K,N,O).

DependingontheoccurrenceofthestomataM.campanulata, M.cordifolia,M.glomerataandM.hastato-cordatahad hypostom-aticleavesandM.micropteraandM.sessilifoliahadamphistomatic leaves and anomocytic and anisocytic types of stomata were encountered(Fig.2A–C,G–I,K,L).

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Box2:AnatomicalfeaturesshowingdifferentiationbetweentheleavesofMikaniaspp.

M.campanulata M.cordifolia M.glomerata M.hastato-cordata M.microptera M.sessilifolia

Hypoder-mis Absent Absent Present Absent Absent Absent

Lens-shapedepidermalcells (adaxial)

Present Absent Absent Absent Absent Absent

Presenceofstomata Hypostomatic Hypostomatic Hypostomatic Hypostoma-tic Amphistoma-tic Amphistoma-tic

Capitateglandulartrichome Present Present(rare) Present Present Present Present

Uniseriateglandulartrichome Absent Present Present Present Present Present

Uniseriateglandulartrichome withbroad-basedcell

Present Absent Absent Absent Absent Absent

Conicalglandulartrichome Present Present Absent Present Present Present

Midribshapeincross-section Biconvexshape withobtuse projectiononthe adaxialside (prominentand roundedconvexity ontheabaxialside)

Biconvexshape, slightlyconvex ontheadaxial side(roundedon theabaxialside)

Slightlyflat,convex shape

Biconvexshape withobtuse projectiononthe adaxialside (prominentand angularconvexity ontheabaxialside)

Slightlyflat, convexshape

Midribvascularpatterns 3–5freecollateral vascularbundlesin anopenarc

3–5freecollateral vascularbundles inanopenarc

3–5freecollateral vascularbundlesin anopenarc

Onevascular bundleinthe center Petioleshapeincross-section Flat-convexshape

withtworibsonthe adaxialside

Concave-convex shapein tranversesection (roundedonthe abaxialside)

Concave-convex shapein transection (roundedonthe abaxialside)

Flat-convex shapewithtwo ribsonthe adaxialsideand threeribsonthe abaxialside Petiolevascularpattern 5–11freecollateral

vascular bundles/U-shaped

About10free collateral vascularbundles inanopenarc

5–11free collateral vascular bundles/U-shaped

5–11freecollateral vascular bundles/U-shaped

About10free collateral vascularbundles inanopenarc

Box3:AnatomicalfeaturesshowingdifferentiationbetweenthestemsofMikaniaspp.

M.campanulata M.cordifolia M.glomerata M.hastato-cordata M.microptera M.sessilifolia

Cross-sectionalstemshape Circular Hexagonal Circular Circularwith2

valeculesat oppositesides

Hexagonalwith 6acutewings

Hexagonalwith 6rounded wings

Sclerenchymatousring Absent Present Absent Absent Absent Absent

Secretoryductsinthepith Absent Present Present Absent Absent Absent

Sclerenchymatouscellsinthepith Absent Absent Absent Present Absent Absent

Prismaticcrystals Absent Present Absent Absent Absent Absent

2015).However,anisocytic(Budeletal.,2009)andactinocytictypes (NevesandSá,1991)havebeendescribedforMikaniaspecies.

In thepresent study,two types of glandulartrichomes and onetype ofnon-glandulartrichomewerefoundintheleavesof allthestudiedspecies. Withregardtotheglandulartrichomes, thefirsttype wascapitateuniseriate or biseriate andsecretion occurredwithinthesubcuticularspace(Fig.3A,G–I,K,M,Oand

Fig.4A, C, F, G). They were frequentlypositioned in a depres-sion caused by invaginations of the adjacent epidermal cells (Figs.3K,M,Oand4A,C,F,G).Thistrichomewasencounteredin allthespecies;however,itismoreabundantinM.micropteraand M.sessilifolia,andisrareinM.cordifolia.

Theotherglandulartrichomeencounteredinthestudiedspecies wasmulticellular,uniseriateandfilamentous,andformedabout sixcells.Theywereeitherstraightorcurvedandhadaterminal cellthat variedfromaspherical(Fig.3D,H–J,M)toaspatulate shape(Fig.3C).Theyoftenappearedintheepidermisdepression (Figs.3J,Mand4E).InM.campanulata,thiskindoftrichomehad broad-basedcell(Fig.3H).

Thenon-glandulartrichomewasconicalandwaslongorshort andstraight orcurved(Figs.3B,F, I,L and4B,F).It hada stri-atedcuticlethatcouldbeclearlyobservedinM.sessilifolia(Fig.3L). Inthepresentstudy,onlyM.glomeratadidnothavethistypeof trichome.

Glandularandnon-glandulartrichomesareoftenfoundin Mika-nia(Milanetal.,2006;Gasparettoetal.,2010;Appezzato-da-Glória etal.,2012;Amorinetal.,2014;Araújoetal.,2015).Inthepresent studywasobservedthatthesetoftrichomescanhelpto differen-tiatedespecies.

Inthepresentstudytheleafbladeincross-sectionhada unis-eriateepidermisinM.campanulata(Fig.5A),M.cordifolia(Fig.5B), M.hastato-cordata(Fig.5D),M.microptera(Fig.5E),andM. sessili-folia(Fig.5F).ThehypodermiswasonlyobservedinM.glomerata (Fig.5C,C′_)._{Additionally,}_it_was_only_in_M._campanulata_that_the

upper epidermiscells had a very conspicuous lenticular shape (Figs.3A,E,H,4Aand5A).Inallthestudiedspecies,the epider-miswascoveredbyathincuticleandthestomatawerelocatedat thesamelevelastheotherepidermalcells.

InMikania,theepidermalsystemisoftenformedbyasingle epidermallayer,althoughthehypodermisappearsinafewspecies suchasM.confertissima,M.glomerata,M.hatschbachii,M. hooke-riana(Oliveiraetal.,1994),andM.laevigata(Budeletal.,2009).

Oliveiraetal.(1994)reportedthatthepresenceofthisstratumcan beusedtoclassifytheMikaniaspeciesintotwogroups.

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Fig.2. Mikaniaspp.Viewoftheleafsurface,showingstomata(st)anduniseriateglandulartrichome(ut)–Abaxialside–A.M.campanulata,B.M.cordifolia,C.M.glomerata, G.M.hastato-cordata,H.M.microptera,I.M.sessilifolia.Adaxialside–D.M.campanulata,E.M.cordifolia,F.M.glomerata,J.M.hastato-cordata,K.M.microptera,L.M.sessilifolia. Scalebar=20␮m.

immersedinthemesophyllandsurroundedbytheparenchymatic sheath(Fig.5B–F).Secretoryductswereobservednearthevascular bundles.

This arrangement of the chlorenchyma was also confirmed in several Mikania (Rodrigues et al., 1996; Budel et al., 2009; Amorinet al., 2014;Araújo etal., 2015).However,Milanet al. (2006)foundthatthearrangementofthemesophyllofM. glom-eratavariedinaccordance withthedifferentregions oftheleaf blade.

Someanatomicaldifferenceswereobservedintheshapeofthe midrib.M.campanulatahadabiconvexshapewithaobtuse pro-jectionontheadaxialsideandprominentandroundedconvexity

ontheabaxialside(Fig.6A).M.cordifolia(Fig.6B)andM. glomer-ata(Fig.6C)hadabiconvexshapeandwereslightlyconvexonthe adaxialsideandroundedontheabaxialside.M.hastato-cordata (Fig.6D)andM.sessilifolia(Fig.6F)hadaslightlyflatconvexshape andM.micropterahadabiconvexshapewithanobtuseprojection ontheadaxialsideandprominentandangularconvexityonthe abaxialside(Fig.6E).

Theuniseriateepidermisofallthetaxawascoveredbyasmooth cuticleand thestomawerepositionedatthesamelevel asthe otherepidermalcells,exceptinM.cordifolia,wherethestomata werelocatedabovetheotherepidermalcells.Oliveiraetal.(2000)

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Fig.3. Mikaniaspp.Viewoftheleafsurface–scanningelectronmicroscopy(SEM)showingstriatedcuticle(cu),capitateglandulartrichome(ct),stomatum(st),non-glandular trichome(nt),uniseriateglandulartrichome(ut).A,E,H.AdaxialsideofM.campanulata;B.AbaxialandF.AdaxialsideofM.cordifolia;CandD.AdaxialsideandG.Abaxial sideofM.glomerata;J.AbaxialandM.AdaxialsideofM.hastato-cordata;I,K,N.AbaxialsideofM.microptera,L.AdaxialsideandO.AbaxialsideofM.sessilifolia.

numberofangularcollenchymaonbothsideswasobservedinall thespecies(Fig.6A–F).

Withregardtothevascularpattern,onlyM.sessilifoliahada col-lateralvascularbundleatthecenterofthemidrib(Fig.6F).Theother studiedspeciesshowed3–5freevascularbundlesinanopenarc (Fig.6A–E).Thesevascularbundlesweresurroundedbyan endo-dermiswhichhadstarchgrains.Secretoryductswerelocatednear orbetweenthevascularbundlesandwereformedof4–12cells withauniseriateepitheliuminallthespecies.

Inthepresentstudy,thepetioleofM.cordifolia,M.glomerata, M.hastato-cordataandM.sessilifoliahadaconcave-convexshapein

transversesectionandwereroundedontheabaxialside(Fig.7B–D, F).M.campanulatahadaflat-convexshapewithtworibsonthe adaxialside(Fig.7A).M.micropterahadaflat-convexshapewith two ribsontheadaxialside and three ribsontheabaxialside (Fig.7E).

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Fig.4.Mikaniaspp.A–C,E,E,G.Trichomesincross-sectionandD,F.Trichomesinfrontalview.A.Capitateglandulartrichome(ct)inM.campanulata,B.Non-glandular trichome(nt)inM.cordifolia,C.Capitateglandulartrichome(ct)inM.glomerata,D.Uniseriateglandulartrichome(ut)inM.microptera,E.Uniseriateglandulartrichome(ut) inM.hastato-cordata,F.Capitateglandulartrichome(ct)andstomatum(st)inM.sessilifolia,F.Non-glandulartrichome(nt)inM.sessilifolia,G.Capitateglandulartrichome (ct)inM.sessilifolia.Scalebar=20␮m.

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Fig.6. Mikaniaspp.Midribincross-section,showingcollenchyma(co)epidermis(ep),groundparenchyma(gp),palisadeparenchyma(pp),spongyparenchyma(sp),vascular bundle(vb).A.M.campanulata,B.M.cordifolia,C.M.glomerata,D.M.hastato-cordata,E.M.microptera,F.M.sessilifolia.Scalebar=200␮m.

Variable strata of angular collenchyma appeared under the epidermis.

Theshape and thevascular pattern of the midriband peti-ole helpto differentiatetaxa into genusas reportedby Wosch etal.(2015)andPortoetal.(2016).In thepresentstudy,these characteristicswereoneof themostrelevantin theanatomical study.

Inthepresentstudy,thecross-sectionalstemshapewas hexago-nalinM.cordifolia(Fig.8B),M.microptera(Fig.8E)andM.sessilifolia (Fig.8F),buthadsixacutewingsinM.microptera(Figs.8E,K)andsix roundedwingsinM.sessilifolia(Figs.8Fand9I).Ontheotherhand, acircularstemshapewasobservedinM.campanulata(Fig.8A),M. glomerata(Fig.8C)andM.hastato-cordata(Fig.8D).Inaddition,M. campanulatahadtwovaleculesonoppositesides(Fig.8A).

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Fig.8.Mikaniaspp.Generalappearanceofstemincross-section,showingtheepidermis(ep),pith(pi),ribs(ri),sclerenchymatousring(sr).A.M.campanulata,B.M.cordifolia, C.M.glomerata,D.M.hastato-cordata,E.M.microptera,F.M.sessilifolia.Scalebar=200␮m.

AcircularstemshapeseemstobethepatterninMikania(Ritter andMioto,2005),althoughM.malacolepsis(Rodriguesetal.,1996) andM.micrantha(Araújoetal.,2015)hadahexagonalshape.

Thestemepidermiswasuniseriateandpossessedanomocytic stomata,aswellasathinandslightlystriatedcuticle(Fig.9A–C, G,I)which reactedpositivelywithSudanIIIinthe histochemi-caltests(Fig.9C).However,thestomatawerelocatedabovethe otherepidermalcellsinM.cordifolia(aspreviousdescribedforthe midrib).Glandularandnon-glandulartrichomes(asdescribedin theleaves)werealsoencounteredinthestem.Additionally,the stemofM.sessilifoliashowedseveralcapitateglandulartrichomes (Fig.9I,L).Undertheepidermistherewere1–3continuous lay-ersofangularcollenchymainallthestudiedspecies(Fig.9A–C, G,K).

Inthepresentstudyitwasobservedthatthestemshape,the presenceofasclerenchymatousringinthecortex,andthepresence of secretoryducts, sclerenchymatouscells, and calciumoxalate crystalsinthepithwereveryimportantmeansofdifferentiating Mikaniaspp.asseeninBox3.

In thepresentstudy, onlyM.cordifoliahad a sclerenchyma-tousring inthe cortex(Figs. 8Band 9B)and thisreacted with hydrochloricphloroglucininthehistochemicaltests(Fig.9B).The sclerenchymatousringinthecortexwasmentionedinM. hirsutis-sima(OliveiraandAkisue, 2005)andM.malacolepsis(Rodrigues etal.,1996).

Inall theMikaniaspecies,theendodermis boundthecortex internally(Fig. 9A, B, E, G) and contained starch grains, which reactedwithiodine-iodide(Fig.10E).Perivascularfibercapscould beobservednexttothephloem(Fig.9A,B,E,G,K)andthesereacted withhydrochloricphloroglucin(Fig.9B).Acambialzonewas evi-dentandformedmorexylemthanphloem(Fig.9G,K).

Inthepresentstudy,thesesecretoryductswereobservedinthe pithofM.cordifolia(Fig.8B)andM.glomerata(Figs.8Cand9F).

SecretoryductsinthepithwereobservedinM.conferta(Oliveira etal.,1999),M.glomerata(NevesandSá,1991)andinM.laevigata (Budeletal.,2009).

Inallthespecies,thepithwaswell-developedandcomprised isodiametricthin-walledparenchymaticcells(Figs.8A–Fand9J), but M.campanulata showedsclerenchymatous cells in thepith (Fig.8A)thatreactedwithhydrochloricphloroglucininthe his-tochemicaltests(Fig.9D).

Prismaticcrystalswereonlyobservedinthemedullaryregionof M.cordifolia(Fig.9H).Thepresenceorabsenceofcrystals,andtheir type,canbecharacterizedastaxonomicfeatures(Meric,2009)and crystalshavenotbeencitedintheMikaniagenus(Oliveiraetal., 1994,1999,2000;Rodriguesetal.,1996;Budeletal.,2009;Amorin etal.,2014;Araújoetal.,2015).

Inthepresentstudy,thecrystalswereanalyzedfortheir ele-mentalcompositionandthespectrashowedprominentpeaksfor calcium(26.9%),carbon,(31.4%),oxygen(25.6%)andsulfur(16.1%), ascanbeseeninFig.10,indicatingthesecrystalswerepossible complexcompoundsofwhichcalciumoxalatewasthemain com-poundandcalciumsulfatewastheminorcomponent.

Theoccurrenceofcrystalsiscommoninplants,eventhough theirsizeandnumberareresponsivetochangesinthe concentra-tionofcalciumintheenvironment(Nakata,2003).Excesscalcium isfrequentlyprecipitatedincalciumsaltssuchasoxalate, carbon-ate,silicate, sulfate,phosphate, citrateand malate (Weiner and Dove,2003).Crystalshavebeenidentifiedinseveralstudiesas cal-ciumoxalatebyusingEDS (LerstenandHorner,2011;Heetal., 2012;Ramanetal.,2014).However,asmallnumberofstudieshave indicatedcrystalsformedbycalciumsulfate(StoreyandThomson, 1994;Heetal.,2012).

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Fig.9.Mikaniaspp.A,D.M.campanulata,B,E,H.M.cordifolia,C,F.M.glomerata,G,J.M.hastato-cordata,K.M.microptera,I,L.M.campanulata.Detailsofthepartsofthe stem,showingca,cambia;ct,capitateglandulartricome;co,collenchyma;cu,cuticle;cx,cortex;cr,crystalsofcalciumoxalate;en,endodermis;ep,epidermis;pi,pith;ph, phloem;sc,sclerenchymatoussheath;scc,sclerenchymatouscells;sd,secretoryducts;sg,starchgrain;andxy,xylem.Scalebar=50␮m(D,E,G,I,J,L),100␮m(A–C,F,K).

Fig.10.EDS(energy-dispersiveX-rayspectroscopy)spectraofcrystalsofM. corid-ifolia.

stemssuppliedadditionalhelpfuldatafordifferentiatingbetween them.

Theway in which thetissues, elements and cells are orga-nizedwithina plantorganallowsthediagnosticfingerprint for

purposesofidentification(AmericanHerbalPharmacopeia,2011). Inthepresentstudy,themostimportantfeatureswereoccurrence ofhypodermisandlensshapedepidermalcells,setoftrichomes; midrib,petioleandstemshapeandvascularpattern; sclerenchy-matousringinthecortex,sclerenchymatouscells andsecretory ductsinthepith.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.

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Authors’contributions

VPA,AAH,PAR,BEMandBJassistedincarryingoutthe labo-ratorywork.VLPSand CRCFhelped inconductingthescanning electronmicroscopy(SEM)analysis.JPPandPVFprovideda crit-icalreadingofthemanuscript.JMBplannedtheproject,collected theplantmaterialandwasresponsibleforitsidentification;she alsosupervisedthelaboratorywork,andwrotethepaper.Allthe authorshavereadthefinalmanuscriptandapprovedthe submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorswouldliketoexpresstheirthankstothefollowing: theAraucariaFoundationforitsfinancialsupport;Prof.Dr.Nelson IvoMatzenbacherforthesamplesofplants,foridentifyingthem, andforpreparingFig.1B;andProf.Dra.InêsJaneteMattozoTakeda forthesamplesofM.sessilifolia.

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