Anatomical and histochemical characterization of Dipteryx odorata and Taralea oppositifolia, two native Amazonian species

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

Original

Article

Anatomical

and

histochemical

characterization

of

Dipteryx

odorata

and

Taralea

oppositifolia,

two

native

Amazonian

species

Paulo

Marcos

Ferreira

da

Silva

a

_,

_Eduardo

_Oliveira

_Silva

b,c,∗

_,

_Marleide

_de

_Sousa

_Chaves

_Rêgo

a

_,

Laísa

Maria

de

Resende

Castro

d

_,

_Advanio

_Inácio

_{Siqueira-Silva}

e

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_Biológicas,_Universidade_Federal_Rural_da_{Amazônia/Museu}_Paraense_Emílio_Goeldi,_Belém,_PA,_Brazil b_Coordenac¸_ão_de_Licenciatura_em_Ciências_Naturais,_Universidade_Federal_do_Maranhão,_Codó,_MA,_Brazil

c_Programa_de_{Pós-graduac¸}_ão_em_Botânica_Aplicada,_Universidade_Federal_de_Lavras,_Lavras,_MG,_Brazil d_Programa_de_{Pós-graduac¸}_ão_em_Botânica,_Universidade_de_Brasília,_DF,_Brazil

e_Coordenac¸_ão_de_Agronomia,_Universidade_Federal_do_Oeste_do_Pará,_Juriti,_PA,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4February2019 Accepted15May2019 Availableonline3July2019 Keywords: Fabaceae Idioblasts Leaﬂetblade Medicinalplants Secretorystructures

a

b

s

t

r

a

c

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Dipteryxodorata(Aubl.)Willd.andTaraleaoppositifoliaAubl.,Fabaceae:Dipterygeae,aretwo Amazo-nianspeciesofgreateconomicandpharmacologicalpotential.Theanatomyofthesespecies,however, remainspoorlystudied.TheaimofthisworkwastoinventoryleafanatomicalcharacteristicsofD. odor-ataandT.oppositifoliaandtolocateandidentifysecretorystructuresanddeterminethemainclassesof metabolitestheystore.VegetativebrancheswerecollectedinParqueEcológicodeGunma,Belém,state ofPará,Brazil.Someofthebranchesweredestinedforherborizationwhiletheremainderwas submit-tedtostandardprotocolsforanatomicalanalysisandhistochemicaltests.Bothspecieswerefoundto possessanunstratiﬁedepidermis,withD.odoratabeingamphistomaticandT.oppositifoliabeing hypos-tomatic,anddorsiventralmesophyllwithspongyparenchymaandwidecellularspace.Thetwospecies werealsofoundtopossessidioblastsandsecretorycavitiesthatproduceaheterogeneousexudate con-sistingofpolysaccharides,lipids,alkaloidsandphenoliccompounds.Thespeciespresenteddifferences inleafanatomyandchemicalcompositionofthesecretorystructures,whichmaybeusefulintheir differentiation.

Introduction

Dipterygeae (Leguminosae, Papilionoideae) is an exclusively

Neotropicalcladeofapproximately25speciesofwoodylegumes

distributedamongfourgenerawhoserepresentativesoccurin

phy-togeographical domains of the Amazon: Monopteryx Spruce ex

Benth,PterodonVogel,DipteryxSchreb.andTaraleaAubl.(Cardoso

etal.,2013).Amongthesegenera,DipteryxandTaraleastandout

duetotheeconomicpotentialoftheirspecies.Althoughthese

gen-erashareanumberofvegetativecharacters,suchasthickleaves,

wingedrachises,terminalappendicesprobablearisingfromthe

reduction of theterminal leaflet, 4–18alternateleaflets, leaflet

apexes ranging from cuspidate to obtuse and the presence of

translucentpunctuations,theirfruitshavedistinctive

morphologi-calcharacteristics,includingdrupe-typefruitwithlatedehiscence

∗ Correspondingauthor.

E-mail:silva.eduardo@ufma.br(E.O.Silva).

in Dipteryx and legumefruit withelastic dehiscence in Taralea

(Barrosoetal.,1999;Francisco,2010).

Dipteryx odorata(Aubl.) Willdand Taralea oppositifoliaAubl.

are popularly known in Brazil as “cumaru” or “cumaru-ferro”

(Sousaetal.,2007;Carvalho,2009)and“cumarurana”or

“cumaru-amarelo”(FariaandLima,2002;Sousaetal.,2007),respectively.

Bothspecieshavemultipleuses,andhavebeenusedinmedicine,

pharmacology, and the perfumery and cosmetics industries

(UchidaandCampos,2000;Bessaetal.,2001;Takemotoetal.,2001; Pesce,2009;Breitbachetal.,2013),aswellasinthetimber sec-tor(Sousaetal.,2007;Herrero-Jáureguietal.,2012;Sorianoetal.,

2012).AlmondsofDipteryxodoratahavealsobeenusedasaraw

materialintheproductionofbiodiesel(Ramalingametal.,2018).

Studies related to theanatomy of vegetative and

reproduc-tiveorgansinvolvinggeneraofLeguminosaehaveprovidednew

characters tosupportboth taxonomic(Lackey,1978;Leelavathi

etal.,1980;Coutinhoetal.,2016),andphylogeneticinvestigations

suchas,forexample,morphologicaldatacombinedwithmolecular

markersforthereconstructionofphylogeniesandtheindication

ofpossiblesynapomorphiesforgenera(Francisco,2010).In

gen-https://doi.org/10.1016/j.bjp.2019.05.004

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426 P.M.Silvaetal./RevistaBrasileiradeFarmacognosia29(2019)425–433

eral,anatomicalstudiesandstudiesofthechemicalcompounds

producedbythesespecieshavemainlyemphasizewood(Gasson,

1999)andseeds(Bessaetal.,2001;Takemotoetal.,2001;Pesce,

2009),leavinglittleknownabouttheanatomicaland

histochemi-calcharacterizationoftheleaves(Palermoetal.,2017;Silvaetal.,

2018).Nonetheless,anatomicalandhistochemicalcharacterization

isnecessaryforidentifyingpotentiallyusefulspeciesandfor

deter-miningsitesofaccumulationand/orsecretionofbiologicallyactive

products(Thadeoetal.,2009;Palermoetal.,2017),sincespeciesof

DipteryxandTaraleapossessconsiderablepharmacologicalvalue.

Inviewoftheaboveconsiderations,theaimofthisstudywas

tocharacterizetheanatomy andhistochemistryoftheleavesof

D.odorataandT.oppositifolia,lookingfordiagnosticcharactersto

delimitthetaxasincetheoccurrenceanddistributionofsecretory

spacesvariesintheDipterygeaecladeandthespeciesare

morpho-logicallysimilar.

Materialsandmethods

Studyarea

MaterialofthetwospecieswascollectedinParqueEcológico

de Gunma – PEG (1◦1386S; 48◦1741.18W), located in the

municipalityof SantaBárbara, about48km fromBelém,in the

northeasternregionofthestateofPará,Brazil.Theparkpossesses

approximately580hawithvegetationcomposedof“terra-ﬁrme”

forestalong withenvironmentsof igapósand várzeas(Almeida

etal.,2009).TheclimateoftheregionisAﬁ–tropicalhumid

(Köep-penclassiﬁcation),witha meanannual temperature of around

26◦C,aminimumof22◦Candamaximumof31◦C.Annualrainfall

variesfrom2500to3000mm,withrelativeairhumidityreaching

about85%(Sudam,1984).

Plantmaterial

Dipteryx odorata (Aubl.) Willd., Fabaceae, leaves composite,

alternate, rachis wingedand protruding withoutleaﬂets in the

apical zone; leaﬂets sub-opposite, 3–4 pairs per pinna,

ovate-lanceolate, marginentire, apex acuminateand base round, and

translucentpunctuationspresent.

TaraleaoppositifoliaAubl.,Fabaceae,leavescomposite,alternate,

opposite,rachis winged,appendix terminal;leaﬂetsalternating,

sub-opposed,rarelyopposite,4–8pairsperpinna,elliptic,

lance-olate,ovate to oval-elliptical,marginentire, apexacuminate to

cuspidate,andtranslucentpunctuationspresent.

FloweringbranchesofD.odorataandT.oppositifoliawere

col-lectedinParqueEcológicodoGunmawithvouchersforthetwo

speciesbeingdeposited inHerbariumMG,withtheregistration

numbersMG20.0490andMG20.1435,respectively.

Structuralandhistochemicalcharacterization

For anatomical and histochemical characterization, fully

expanded leaﬂets and petiolules were collectedform the third

tothefourthnodeofthemedialportionsofvegetativebranches

(includingthemainrib,marginandregionbetweenthemargin

andmainrib)andpetiolulesandﬁxedinFAA(formaldehyde,glacial

aceticacidand70%ethylalcohol;1:1:18v/v;Johansen,1940)for

24h.Portionsofthesesamples(3–4)wereﬁxedinneutral-buffered

formalin[NBF;Lillie(1965)]orferroussulphateinformalin[FSF;

Johansen(1940)]for48hforthedetectionoflipophilicand

phe-nolic substances, respectively. After ﬁxation, thesamples were

washedin distilled water, dehydratedin a tertiary butylseries

andembeddedinparafﬁn(Johansen,1940).Transverseand

lon-gitudinalsections(12–14␮m)weremadeusingasemiautomatic

rotarymicrotome (Leica RM 2245), stained with1.5% alcoholic

safraninand1%aqueousastrablue(Bukatsch,1972),andmounted

inPermount®syntheticresin.

Theepidermisofleaﬂetswasdissociatedusingthesolutionof

Franklin(1945),followedbywashinginwater,stainingwithastra

blueand1%safranin(Bukatsch,1972),and mountingin1:1v/v

water/glycerintemporarymedium(Purvisetal.,1964).

HistochemicaltestsoffreshsampleswereperformedusingPAS

(PeriodicAcidSchiffreagent)fortotalpolysaccharides(McManus,

1946); Ruthenium red for the detection of acidic mucilages

(Johansen, 1940); tannic acid/ferric chloride for mucilages (Pizzolato and Lillie, 1973); Lugol for starch (Johansen, 1940);

SudanblackBfortotallipids(Pearse,1980);Nileblueforacidic

and neutral lipids (Cain, 1947); NADI reagentfor essential oils

andoilresin(DavidandCarde,1964);Dragendorff(Sverdsenand

Verpoorte,1983)andWagnerreagents(FurrandMahlberg,1981)

for alkaloids, and ferricchloride for total phenolic compounds

(Johansen,1940).

Forthecontrolfortestsforlipophilicsubstances,sampleswere

stored in extraction solution (methanol/chloroform/water/HCl;

High,1985)for48h,ﬁxedinneutralbufferedformalin(NBF)and

submittedtotheaforementionedreagentsanddyes.Thecontrol

forthetestsforhydrophilicsubstanceswasperformedaccording

totherespectivetechniques.Sampleswerealsoanalyzedwithout

anytreatment(blank),forvisualizationofcolorinnatura.Thetests

wereappliedtosectionscutbyfreehandandthenmountedin

glycerinjelly(Kaiser,1880).

PhotographicdocumentationwasperformedusinganAxiolab

ZeissmicroscopecoupledtoaCanonPowershotA640digital

cam-era.

Scanningelectronmicroscopy(SEM)

ForSEManalyses,leaﬂetssampleswereﬁxedinFAA,dehydrated

inanethanolicseriesandcriticalpointdriedwithCO2(Bozzola

andRussel,1991).Theleaffragmentswerethengluedtoametallic

support,metalizedwithgoldandexaminedunderaLEO1450VP

scanningelectronmicroscope.

Results

Anatomicalcharacterizationofleaves

In frontalview,theadaxial leafsurface of D. odorataand T.

oppositifoliaexhibitedepidermalcellswithanticlinal(Fig.1A)and

sinuous(Fig.1B)walls,respectively.Theadaxialleafsurfaceofboth

speciesexhibitedcellswithsinuousanticlinalwalls(Fig.1CandD).

TheleavesofD.odorataandT.oppositifoliawerefoundtobe

amphis-tomaticandhypostomatic,respectively.Bothanomocytic(Fig.1C)

and paracyticstomataltypes werepresent in D.odorata,while

onlyparacyticstomatawerepresentinT.opositifolia(Fig.1D).

Stel-latenon-glandulartrichomeswereobservedinD.odorata(Fig.1G)

whilesimplenon-glandulartrichomeswereobservedinT.

opposi-tifolia(Fig.1H).Incrosssection,bothspecieswerefoundtopossess

uniseriateepidermisonbothfaces,alongwithathickcuticle(Fig.1E

andF).

Bothspecies exhibiteddorsiventralmesophyllwithstratiﬁed

palisadeparenchymaandstratiﬁedspongyparenchymawith

con-spicuousintercellularspaces(Fig.2C–F).

Theleafbladeofthetwospeciesexhibitedsmall-calibervascular

bundlessurroundedbyﬁbersforminganextensivevascularbundle

sheath(Figs.2A,C,andD).Thevascularsystemofthecentralribof

bothspecieswasfoundtobecomposedofcollateral-typevascular

bundlessurroundedbyﬁbersformingaclosedarchinD.odorata

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Fig.1. LeafanatomyofDipteryxodorata(A,C,EandG)andTaraleaoppositifolia(B,D,FandH)underlight(A–F)andscanningelectron(GandH)microscopy.A.Adaxialface withstraightepidermalcellsandstomata;B.adaxialfacewithsinuousepidermalcells;C.abaxialfacewithsinuousepidermalcellsandstomata;D.abaxialfacewithsinuous epidermalcellsandstomata;EandF.detailoftheepidermisandthepalisadeparenchyma;G.stellatenon-glandulartrichomes;H.simplenon-glandulartrichomes.Bars: 50m(A,andC–F);150m(B);13m(G);20m(H).

secretorycavitiesinthecorticalparenchymaandonlyidioblastsin

themedullarparenchyma(Fig.2GandH).

LeafmarginswerecurvedinD.odorata(Fig.2A)andstraight

inT.oppositifolia(Fig.2B).Leafmarginsofthelatterspecieswere

observedtobecompletelysclerenchymaticandpossessing

secre-torycavities.

The petioleof both species revealed a circular outline with

fundamentaltissuecomposedofseverallayersofparenchyma

con-tainingidioblastsandsecretorycavities(Fig.3AandB).Allofthese

anatomicalcharacteristicsaresummarizedinTable1.

Histochemistryofleaﬂetsecretorystructures

Thesecretorystructuresoftheleavesofbothspecieswerefound

tocompriseidioblastsandcavities,whicharelocatedinthecortical

parenchymaofthecentralribandthepetiole(Figs.2G,Hand3A,

B).ThemesophyllofT.oppositifoliaalsoexhibitedsecretorycavities

intheadaxialface(Fig.2D).Idioblastswererevealedinredwhen

stainedbysafranin(Figs.2Gand3B).

Spherical epithelial cells were observed to delimit an

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Fig.2.LeafanatomyofDipteryxodorata(A,C,EandG)andTaraleaoppositifolia(B,D,FandH)underlight(A–D,GandH)andscanningelectron(EandF)microscopy.A. Curveddownwardleafmargin;B.straightleafmarginwithsecretorycavity(SC);C.generalviewofmesophyll;D.generalviewofmesophyllwithsecretorycavity;EandF. detailofspongyparenchyma(SP);GandH.generalviewofcentralrib.Legend:(PP)palisadeparenchyma;(VB)vascularbundle.Bars:150m(A,CandD);50␮m(B);60m (EandF);300m(GandH).

idioblastsandsecretorycavitiesrevealedthattheycontainaseries

of chemical compounds, namely: total lipids (acidic and

neu-tral),polysaccharides,alkaloidsandphenoliccompounds(Table2).

Secretionswereseenpreservedinidioblastsandcavities,withthe

latterbeingpresentbothinepithelialcellsandinsidethelumen

(Figs.4A–Fand5A–F).Theexudatespresentintheidioblasts

pos-sesseda darkbrowncoloration in both species,evidencing the

presenceofphenoliccompounds(Fig.5F).

Discussion

AmongtaxaoftheDipterygeaeclade,theoutlineofthe

epider-malcells ofbothleafsurfacesvariesfromrectilineartosinuous

(Silva etal.,2018), asobservedinthepresent study.Cells with

straight and sinuous walls have been widely reported among

others species of Fabaceae, such as Aldina heterophylla Spruce

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Fig.3. PetioleanatomyofDipteryxodorata(A,CandE)andTaraleaoppositifolia(B,DandF)underlight(A–D)andscanningelectron(EandF)microscopy.AandB.overall viewofpetiole;CandD.detailofsecretorycavity(SC);EandF.longitudinalsectionofsecretorycavities.Legend:(Xy)xylem;(Ph)phloem;(Fi)ﬁbers.Bars:300m(AandB); 50m(CandD);130m(E);230m(F).

Table2

HistochemistryoftheidioblastsandsecretorycavitiesofleavesofDipteryxodorataandTaraleaoppositifolia.

Groupofcompounds Test Color Dipteryxodorata Taraleaoppositifolia

Idioblasts Secretorycavities Idioblasts Secretorycavities

Epithelium Lumen Epithelium Lumen

Polysaccharides Periodicacid/Schiffreagent(PAS) Pink + – – + – –

Rutheniumred Pink + – – + – –

Tannicacid/ferricchloride3% Black – – – – – –

LugolReagent Purpleorbrown + – – – – –

Lipids SudanblackB Black + + + + + +

Nilebluesulphate Pinkorblue – + + + – –

Terpenoids NADIReagent Red – – – – – –

Alkaloids Dragendorff’sReagent Reddish-brown + – – + – –

Wagner’sReagent Reddish-brown + – – + – –

Phenolic compounds

Ferricchloride Black + – – + – –

Fixative(SFF) Black + – – + – –

(Raddi) J.F. Macbr. (Duarte and Debur, 2003), Hymenaea

stilbo-carpaHayne(Moreira-ConeglianandOliveira,2006),Glycinemax

(L.) Merr. (Leal-Costa et al., 2008), and Senna alata (L.) Roxb.

(Rodriguesetal.,2009),aswellasforvariousspeciesof

Chamae-crista(L.)Moench(Coutinhoetal.,2016).Sinuosityofcellwalls,

however,mayberelatedtotheenvironmentwhereaplantoccurs

(Esau, 1997), as observed by Araújo and Mendonc¸a (1998) for

the walls of the epidermal cells of Aldina heterophylla, which

canbestraitinthemajorityofsunleavesandsinuousinshade

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Fig.4. LeafhistochemistryofDipteryxodorata(A–F).A–E.idioblasts;F.cavities;AandB.WagnerandDragendorffreactionfor“alkaloids”;CandD.ferricchloridereaction for“tannins”;E.Lugolreactionfor“starch”;F.Nilebluesulphatereactionfor“acidicandneutrallipids”.Bars:50m(A–F).

Table1

LeafanatomyofDipteryxodorataandTaraleaoppositifolia.

Characteristics Dipteryx odorata Taralea oppositifolia Amphistomaticleaf x – Hypoestomaticleaf – x Anomocyticstomata x – Paracyticstomata x x

Simplenon-glandulartrichomes – x

Stellatenon-glandulartrichomes x –

Sinuousepidermisonbothfaces – x

Leafwithcollateralvascularbundle x x

Presenceofsecretorycavity x x

Presenceofsecretoryidioblast x x

Curveddownwardleafmargin x –

Fibrousleafmargin – x

Petiolewithan“openarch”-shaped vascularbundle

– x

Theamphistomatic patternwithparacyticstomata observed

hereforleavesofD.odorata,aswasalsoobservedbySilvaetal. (2018),inSennaalata(L.)Roxb.byRodriguesetal.(2009),andin

speciesofBauhiniaL.byPereiraetal.(2018).Hypostomaticleaves,

asobservedinT.oppositifolia,aremorecommoninthesubfamily

Mimosoideae,asreportedbyMetcalfeandChalk(1950).

Amphis-tomaticleaveshavealsobeenobservedinSchnellaRaddi(Pereira

etal.,2018),aswellasinthegeneraAbaremaPittierandIngaMill.

(Silvaetal.,2012).

Theoccurrenceofdifferenttypesofstomatainthesamespecies,

asobservedforD.odorataandT.oppositifolia,corroboratethat,in

general,speciesofFabaceaedonothaveaspecies-speciﬁcstomatal

type,aspreviouslyreportedbySilvaetal.(2018).Theoccurrence

ofanomocyticandparacyticstomatainFabaceaeiscommon,with

thelatterbeingmostfrequentinthesubfamiliesCaesalpinoideae

andPapilionoideae,asmentionedbySolereder(1908); Metcalfe

andChalk(1979)andAraújoandMendonc¸a(1998).

The patterns of theepidermis and stomata observed in the

presentstudyhavealsobeenobservedinotherspeciesofFabaceae,

suchasspeciesofAlbiziaDurazz.(Simõesetal.,2003),andBauhinia

forﬁcataLink and B. variegataLinn.(Lusaand Bona, 2009), and

IndigoferamicrocarpaDesv.(Limaetal.,2003),aswellasamong

speciesoftheDipterygeaeclade(Silvaetal.,2018).

Differentfromtheresultsofthepresentstudy,theﬁnecuticle

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Fig.5. LeafhistochemistryofTaraleaoppositifolia(A–F).A–D.idioblasts;EandF.cavities;A.PASreactionfor“polysaccharides”;B.Rutheniumredreactionfor“mucilage”;C andE.SudanblackBreactionfor“totallipids”;D.Nilebluesulphatereactionfor“acidicandneutrallipids”;F.ironsulphateinformalinreactionfor“phenoliccompounds”. Bars:50␮m(A–E);500m(F).

useofherborizedmaterialfromdifferentcollectionsites.Thethick

cuticleobservedinthepresentstudyforT.oppositifoliawasalso

observedbytheseauthors,aswellasinotherspeciesofTaralea.

Non-glandulartrichomeshavebeenfrequentlyreportedamong

representativesof PapilionoideaeandCaesalpinoideae(Metcalfe

andChalk,1950).Thistrichometypehasbeenobservedinseveral

tribesof Leguminosae,suchas Dalbergieae,Hedysareae,

Phase-oleae,SophoreaeandSwartzeae(Solereder,1908),includingthe

Dipterygeaeclade(Silvaetal.,2018).Thetaxonomicimportance

of trichomesinLeguminosae hasalso beendiscussed forother

angiospermfamilies (Cowan,1950; Carlquist, 1961; Tomlinson,

1969).In addition,analysesof thesigniﬁcanceof trichomesfor

thetaxonomyofPhaseoleaeprovedessentialfordelimitinggroups

(Lackey,1978).Incontrasttotheﬁndingsreportedhere,Silvaetal. (2018)foundglabrousleavesinT.oppositifolia,whichwas

proba-blyduetothedifferentoriginsoftheplantmaterialusedandthe

methodologiesemployed.

Thedorsiventralmesophyllrecordedinthetwospecies

stud-iedhereconformswithwhathasbeenreportedforothergenera

ofFabaceaebyMetcalfeandChalk(1950)andSilvaetal.(2012),

andwhoreportedthesamecharacteristicinBauhiniamicrostachya

(DuarteandDebur,2003).AccordingtoSilvaetal.(2018),

dorsiven-tralmesophyllprevailsintheDipterygeaeclade,althoughthese

authorsobservedisolateralmesophyllinD.odorata,incontrastto

theﬁndingsreportedhere.Inadditiontothedorsiventral

orga-nizationofthemesophyll,theoccurrenceofbistratiﬁedpalisade

parenchymaanddevelopedspongyparenchymahavebeenwidely

reportedamongspeciesofFabaceae(AraújoandMendonc¸a,1998;

DuarteandDebur,2003;Rodriguesetal.,2009).

Small-calibervascularbundlessurroundedbyﬁbersforminga

sheathhavealsobeenreportedtooccurintheDipterygeaecladeby

Silvaetal.(2018).Accordingtheseauthors,theshapeoftheleaﬂet

marginiscurveddownwardinD.odorataandT.oppositifolia,which

wasconﬁrmedhereonlyfortheformer.

Secretory structures, such as idioblasts and cavities, have

beenpreviouslyreportedforspeciesofPapilionoideae and

Cae-salpinoideae (Solereder, 1908; Metcalfeand Chalk, 1950; Fahn,

1985;TeixeiraandGabrielli,2000;LusaandBona,2009;Leiteetal., 2014;Palermoetal.,2017),includingtheDipterygeaeclade(Silva

et al.,2018).Althoughthepresentstudy didnotﬁndsecretory

canalsinthecorticalparenchymaofthemainribandpetioleofT.

oppositifolia,aspreviouslyreportedbyPalermoetal.(2017),their

presenceinthisspecieswouldserveasavaluabletaxonomic

char-acterindistinguishingitfromD.odorata,forwhichthereareno

recordsofthesestructures.Exceptforstarchandacidicand

neu-trallipids,thesecretorysystemsofD.odorataandT.oppositifolia

producedsecretionsofthesamechemicalnature.Thissimilarity

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larphytogeographicdomains,suchastheAmazon.Histochemical

testsperformedbyPalermo etal.(2017)onleavesofT.

opposi-tifoliadetectedthepresenceoftotallipidsintheepithelialcells

and/orlumenofsecretoryspaces,aswasfoundinthepresentstudy.

Idioblastsandsecretorycavitiesarestructuresthatarespecialized

inthesecretionofcompoundssuchasmucilageand/orgum,

phe-noliccompounds,andlipophilicmaterial,includingheterogeneous

secretions,amongothers(Fahn,1985),whichhavedeterrentand

antimicrobialeffects(Langenheim,2003).Thepresenceofphenolic

compoundsintheleavesofD.odorataandT.oppositifolia,aswellas

theabsenceofmucilage,observedinthepresentstudy,was

sim-ilartothatreportedbySilvaetal.(2018).However,theresultsof

thisauthordivergedinregardstotheothercompounds(starch,

lipidsandpectins),probablyduetothefactthattheyworkedwith

herbariamaterial.

The categories of chemical compounds inventoried in the

idioblastsand cavities in thetwo studiedspecies represent an

importantseriesforplantsingeneral.Forexample,

polysaccha-ridesareonewaythatplantsstoresugarandbuildstructures,such

asthecellwall. Starch,also detectedinthepresent study,is a

polysaccharidewithgreatfoodvalue,andhasalsobeenreportedto

occurinlargequantitiesinthecotyledonsofD.odorata,fromwhich

coumarin,asubstancewithproventherapeuticaction,isextracted

(Bessaetal.,2001).

Mucilage comprises complex polymers of acidic or neutral

polysaccharides of hydrophilicnature and withhighmolecular

weights (Mastrobertiand Mariath, 2008), conferring important

rolesinplantdevelopment,suchasprotectionofstructuresand

organs,waterretention,carbohydratereserve,transpiration

reduc-tion,protectionfromsolarradiationandherbivory,rootlubrication,

insect trapping, seed dispersal and regulation of germination

(Langenheim,2003;Pimenteletal.,2011;Rochaetal.,2011;Alves etal.,2012).

Alkaloids,whichwerefoundinthepresentwork,havealsobeen

foundinotherspeciesofDipterygeae,suchasDipteryxalataVogel

andPterodonpubescens(Benth.)Benth.(Palermoetal.,2017).They

arenitrogencompoundsofvaryingstructureandareamongthe

mosttoxicclassesofcompounds.Pyrrolizidine-likealkaloidsare

foundinseveralplantfamilies,butaremostcommoninAsteraceae,

BoraginaceaeandFabaceae.Ingeneral,theyfunctionindefense

againstpredatorsandareknowntocauseintoxicationinanimals,

includinghumans(Silvaetal.,2006;Lucenaetal.,2010;Matosetal.,

2011).

Bragaetal.(2007)attributedthebiologicalactivityofextracts

ofvariousmedicinalplantswithanti-leishmaniasisandanti-fungal

actionstosecondarymetabolites,suchasﬂavonoids,alkaloidsand

coumarin.Itgeneral,theusesoftheoilthatthesespeciesprovide

arewidelyknown,bothinfolkmedicineandincosmetics,which

hasresultedingreatcommercialvalueandreinforcesthe

impor-tanceofstudiescharacterizingspeciesthathavethesesubstances

toenhancetheireconomicpotential.

AlthoughD.odorataandT.oppositifoliaproducesecretionswith

similarclassesofchemicalcompounds,theanatomicalstructureof

theirleavescanaidindistinguishingthem(thespeciesare

mor-phologicallysimilar,whichmakesdistinguishingthemintheﬁeld

difﬁcultwhenfruitisabsent),andsupporttheirinclusioninthe

Dipterygeaeclade.

In conclusion, the occurrence and chemical composition of

idioblastsandsecretorycavitiesin leaves,thelocationof

stom-ata,thetypesoftrichomes,thecurvatureandligniﬁcationofthe

leafmarginandtheshapeofthevascularbundleofthepetioleare

allanatomicalcharacterswithpotentialdiagnosticvalueforthe

Dipterygeaeclade,andespeciallyforthespeciesD.odorataandT.

oppositifolia.

Conﬂictofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Authors’contributions

PMFScontributedincollectingplantsamples,andconducted

thelaboratorywork.PMFS,EOSandAISSwrotethetext.MSCRand

LMRCcontributedtocriticalreadingofthemanuscript.Allofthe

authorshavereadtheﬁnalmanuscriptandapprovedits

submis-sion.

Acknowledgments

TheauthorsthanktheLaboratóriodeAnatomiaVegetalofthe

Coordenac¸ãodeBotânica,MuseuParaenseEmílioGoeldifor

pro-vidinginfrastructure,andCAPESforprovidingthescholarshipto

theﬁrstauthor;andthereviewersandeditorfortheircomments

andsuggestionstoimprovethemanuscript.

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