, Rodriguésia 61(3):505-518.2010 http://rodriguesia.jbri.gov.br Leaf anatomy and micromorphology of six Posoquería Aublet species (Rubiaceae) AnatomiaemicromorfologiafoliardeseisespéciesdePosoqueriaAublet(Rubiaceael RosanidoCarmode OliveiraArruda Dona MariaSaiterGomes2 Aline CarvalhodeAzevedo'3 MichelleLima Magalhães ' & Mario Gomes4 , Abstract ThepresentstudydealswiththeleafanatomyandleafsurfaceofPosoqueríaaaitifoliaMart.,P. latifoliaMart.,P. longiflora Aublet,P. macropusMait,P.palustrís(Rudge) Roem. and Posoqueriasp„ collectcd in ffagments of Atlanticrainforest,RiodeJaneiro,Brazil.Theepicuticularwaxmayoccurintheformoffilaments,granulesorcrusts. Theleavesarecoveredbyathickcuticularlayerthatmaybesmoothorstriatcd.Paracyticstomata,andnon-glandular trichomesarelimitedtotheabaxialsurface;thelattcrarenumerousinP.palustrís,andrareinP. longifloraandP. latifolia. Leaveshaveadorsiventralstructure,withonlyonelayerofpalisadeparenchymaandvaricdamountsof spongyparenchyma.IdioblasLscontainingcrystalliferoussandwcreobserved,andweremoreabundantinP.latifolia. Theleafbladevascularsystemisformedbycollateralbundlcswithaparenchymatoussheath,associatedwithfibers. Thevascularsystemofthepetioleandtheleafbladefomtsanarch.SomeofÜtcanatomicalfcaturesobscrvcdcanbe uscdtodistinguishthespeciesstudied.Anatomicalleafcharactcrscouldbeusedintherccognitionofsixspeciesof Posoqueriastudied,suchasanticlinalwallofepidemia!cells,waxdeposition,trichomesandshapeoftheleafntargin. Kcy words: leafanatomy, scanningclcctron microscopy,taxonomy, Cinchonoidcae. Resumo S3oapresentadasinfomtaçòcssobreaanatomiacasuperfíciefoliardePosoqtvriaaaitifoliaMart.,P.latifoliaMart.,P. longifloraAublet,P.macropusMart.P.palustrís(Rudge)RoemePosoqueriasp..espéciesocorrentesanfragmentosda FlorestaPluvialAtlântica,RiodeJaneiro,Brasil.Aceraepicuticularpodeocotrernaformadefilamentos,grânulosou crostas Asfolhassãorecobertasporespessacamadaarticular,quepodeserlisaouestriada.Nafaceabaxialacutícula apresenta-seestriadaemP. latifoliaePpalustrís.Osestômatosestãorestritosàfaceabaxialc sãodotipoparacítico. TricomastectoresocorremapenasnestafacesendonumerososcmP.palustrís rarosemP.longifloraeP. latifolia.As folhastêmestruturadorsiventral,comapenastunacamadadeparènquimapaliçádicocvariadonúmeronoparênquima esponjoso Nomesofiloforamobservadosidioblastoscontendoareiacristalífera,maisabundantescmP.latifolia.Na lâmina foliaro sistema vascularé formado por feixesdo tipocolateral, envolvidosporbainhaparenquimâtica, e apresentamfibrasassociadas.Nopcciolocnanervuraprincipalosistemavascularapresenta-secmarco.Características anatômicasfoliarespodemserusadasnadistinçãodasseisespéciesdePosoqueriaestudadas,taiscomo:aparede anticlinaldascélulasepidérmicas,deposiçãodecera.tricomaseaformadamargemdafolha. Palavra-chave: anatomia foliar, microscopia eletrônica de varredura, taxonomia. Cinchonoidcae. recently divided into two subfamilies, lhe Introduction & Cinchonoideae and the Rubioidcac (Robbrecht Rubiaceae (Gentianales) is one of the four Manen2006). iargestfamiliesofangiosperms,withrepresentatives Belonging to Cinchonoidcae subfamily, hawainbddietlisynacdnliudsdtelrisifebaufbtoeordumtsw.o1rT0l.hd7ew0i0fdaesm,pielpcyrieeissse,mnotwinhnoigpchhdyilvweeetrrisece aPnodsoiqsuceortínapAousbeldeotfisapfporuonxdiimnattheelyNco14trsoppeicciersegtihoant 1UniversidadeFederaldoEstadodoRiodejaneiro(UNIRJO).InstitutodeBioeiíncias.Dep.o,Boliiniea.Av.Pasteur458,22290-540.RiodeJaneiro.RJ.Autorapara correspondência:rosaniarrudaáTgmail.com :UniversidadcFederalRuraldoRiodeJaneiro(UFRJU).InstitutodeBiologia.DeptO.Botânica.Rod.465.km7,25890-000.Scropídica.RJ. IniciavàoCientífica. 4ExtractaMoléculasNaturaisS.A.,Av.CarlosChagasFilho791.CidadeUniversitária,21941-904,RiodeJaneiro.RJ. SciELO/ JBRJ cm .2 13 14 15 16 17 506 Arruda.R.C.O. etaL aredifficulttoberecognizedinvegetativeStateand (Bidegain & Michael 2003). In this site, the plants presentgreatmoiphologicalsimilarity(Macias 1988). are submitted to seasonal flooding in the rainy ToBrazil,sixspeciesaredescribed(Macias 1988); season, when the water table rises considerably. mostofthemareendangered(Leonhardtetal.2008). Posoqueria acutifolia was collected at the Parque Some speciesofthis group are used as omaments, Estadual da Serra da Tiririca, Niterói, RJ because of their decorative leaves and perfumed (22°48’S,42°57’W),afragmentoftheAtlanticRain flowers. Most species of Posoqueria display the Forest.Inthisregiontheannualaveragetemperature characteristic pollen catapult mechanism, which is is24 C,withannualrainfaliof1300mmconcentred thetrademarkfeatureofthePosoquerieae(Delprete in the summer. Herborized leaves ofP. macropus 2009). According to Ariza et al. (2007), indolic (Sucre7678)andPosoqueriasp.(Martinelli8804), alkaloids,iridoids,andacetatesofhexanileandhexile, obtained from the Instituto de Pesquisas Jardim among other compounds, have been identified as Botânico do Riode Janeiro (RB), were rehydrated perfume sources in Posoqueria latifolia (Rudge) accordingtoGomes(2002). Roem.,popularlyknownas“açucena-do-mato”.For The fresh leaves were fixed in F.A.A. P. acutifolia Mart., laboratory tests have identified (formalin, acetic acid, ethanol 70%) for48h and analgesic and anti-inflammatory activities of the preserved in 70% ethanol (Johansen 1940). For methanolic extracts, which may be related to this light microscopy, sections of the leafblade and speciesuseinpopularmedicine(Souzaetal.2007). Petioleweredehydratedingradedethanol-butanol Among the organs that can be useful to series, embedded in paraffin, sectioned taxonomic propositions, the leaf is the most transversely and longitudinally in rotatory examined onebecause ofits internai architecture, microtomeat athicknessof 12 mm. The sections which is a classic source ofuseful information in were stained with 1% safranin - 1% astra blue in the systematics, specially the epidermis and the tartaricacid(Luqueetal. 1996). cuticle(Stuessy 1990;Juddetal. 2009). Theepidermis wasstudied usingsectionsof MantoAvbanoiut&mViiecirroam(o19r9p3h/o1l9o9g7)yatnhdeKosctusdiiseestaolf. tpheerolxeiadfeb(lFardaenkdliisnso1c9i4a5t)edThieneapceitdiecrmaicsidw/ahsysdtraoigneend (2004) stand out and are source of important with 1% safranin, alcian blue, or 0.5% fuchsin informationabouttheleafsurfaceofdiversespecies (modificationsinKraus&Arduin 1997).Inorderto ofthisfamily.Descriptiveanatômica]studiesofthe remove wax and to confirm its presence, sections vegetative or reproductive parts also have been ofherborizedleaveswereboiledinchlorofonmthree &c(aMrGarnoitemTodehvsioasun1ti9s9etw6tui.adtlyhD.ed1oet9Tsh9oce5nrr;iibGB&erosaMmzaetirhlsieiaaetlntehaalr2f.u0b0a2i80no)0ai.0td;osmApyrercauindedas ewtlieemrceetsrcforonirtoimcniaeclr-mpoiosniucntotepd,yroi(nedSaEthhMort)opulaganhtael.aysFgiorsra,dtehldeeassfcearpinienesicneogsf micromorphology of six Brazilian Posoqueria ethyl alcohols. Leaf fragments were mounted on rtMRsaoeporecetcoim.igd,e.nesiPn:&.ztipPiSna.fclgyhuautsclthfutreet.isia,seftPoMus.larpirleeatocs.niM,geatisarh.fntal.dot,rPaomPs.Aaouqyblault.eib,rfeioPla.iuamsspa.(ecRfaruuioldmpgiuenis)gn JcfsotrSuaoMbtm-se5dhFc3ewoori1rab0tttahehsrdegcioahuwlnimidnstitwbhnoeagcfsghoeoerlalmeeldcisbtcoeraaomilnnnoditgucesnoomtbbtsiss,eceerdfrrorvovseenecedodhppaireinnon.dtphMseaeaertcJSestEErtiiuoOManb.Llss of leaf blade were treated with the following Materials and Methods reagents: starch grains were localized by Lugol staining(Johansen 1940), lipidiccompoundswere Adult leaves of Posoqueria latifolia and P. recognized by Sudan Red 7B in glicerol/ethanol longiflora were collected from plants found at the (Kraus & Arduin 1997), pectin substances with ParqueNacionaldaFlorestadaTijuca(22°57’Sand 0.02% ruthenium red (Kraus & Arduin 1997); 43°18’W),inRiodeJaneiro,(RJ).Inthisregion,the primary walls were identified with zinc iodine annualavaragetemperaturevariesbetween 19.3°Cin chloride(Jensen 1962)andthesecondarywallswith thewinteranmdm25.5°Cinthesummer,andtherainfallis acidphloroglucinforlignifiedcells(Foster 1949); about2000 annually(Vieira 1994).Posoqueria phenolic compounds were confirmed with ferric palustriswascollectedinRiodasOstras(RJ,22°31S'- chloride(Johansen 1940).Testswithhydrochloric 41°55'W)wheretheannualaveragetemperatureis acid were used to detect calcium oxalate crystals 22°Candtheannualrainfallbetween1500and2000mm (Jensen 1962). Rodríguèsià 61(3):505-518.2010 SciELO/JBRJ 2 13 14 15 16 17 18 507 Leafanatomyandmicromorphology ofPosoqueriaAublet (Rubiaceae) cuticular-wax strata (Fig 5 a-d). On the abaxial Results surface, above the stomatal pore, the cuticle layer Inmostofthestudiedspccies,theepidemiis, mayformconspicuousledges(Fig.5c).Thestomata inadaxialsurfaceiscomposedofordinaryepidcrmal arelocatedon thesame leveiorslightlybelow the cells,withsinuousanticlinalwalls(Fig. 1 a-f).and other epidermal cell and the subsidiary cells can thick, notably in Posoqueria macropus(Fig. 1 d). form projections in the rcgion ofthe substomatal Overvascularbundles,suchasintheabaxialsurface chamber(Fig.5c).Thetrichomeshavethickwalls ofPosoqueria sp. the anticlinal walls can present (Fig. 5 b), and in Posoqueriapalustris, the basal themselvesstraightorcurved(Fig. 1 f).Theabaxial regionoftheseptatetrichomc ispitted(Fig. 5d). surface is composed by ordinary epidermal cells, All the species studied show dorsiventral stomataandtrichomes(Fig. 2a-f)- Theepidermal mesophyll, with one layerofcells in the palisade anticlinalcellswallsaresinuousinallspecies(Fig. parenchymaandabout 10to 14layersinthespongy 2a-e),withtheexceptionofPosoqueriaspinwhich parenchyma(Fig.6a-e).Thepalisadecellsarewidcr theyarestraight. P. acutifoliahasglabrousleaves andshorterinPosoqueriaacutifoliaandP.latifolia (Fig.2a),inP.macropusnumerous,non-glandular, (Fig.6a-b)andlongerandnarrowerinthercmaining unicellulartrichomes,withthicklignifiedwallswere species(Fig.6c-e).Idioblastscontainingtinypolyhedral observed (Fig. 2 d). In P. palustris there are crystalsofcalciumoxalate(crystallifcroussand)are pluricellularuniseriatedtrichomes,long,nonlignified observedontheentiremesophyllinallspeciesstudied (Fig. 2e). Posoqueria sp is markedby presenceof (Fig.6c,0-TheyaremoreabundantinP. latifolia numerouspapillae(Fig. 2f). Rare shortunicellular and P. palustris. These cells occur in a greater non-glandulartrichomesareobservedontheabaxial frequcncybeneaththepalisadeparenchyma,ornear surface ofthe leafofP. latifolia and P. longiflora, theepidermisoftheabaxial surface,isolatedorin restricted to midvein; in P. palustris, numerous groups, especially in P. latifolia and P. palustris pluricellulartrichomesareobserved. (Fig. 6 c-f). The histochemical tests showed that Thestomatabelongtotheparacytictypeand the crystallifcrous cells also accumulate phenolic arearrangedrandomly(Fig2).Thesubsidiarycells compounds. may bc of different sizes; some stomata may be The vascular system of the leaf blade is flankedinparallelbymorethanonesubsidiarycell. formedbybundlesofthecollateral type,ofvaried The six species investigated present some large calibers (Fig. 6 a, d). The vascular bundles are stomata, around which the epidermal cells are surrounded by a parenchymatous sheath that may organized radially, and the cuticle layer forms a contain chloroplasts, crystalliferous sand, and radiate pattem (Fig. 2 b). In P.macropus and P. phenolic compounds. Internally to this sheath palustris the stomata are partly covered by lignified fibers occur in variable proportions. In numeroustrichomes(Fig.2d-e). Posoqueriaacutifolia,P. latifoliaandP.macropus Theepicuticularwaxcanoccurwithoutdefincd thesclcrenchymaoccursinalargerproportionthan omamentation,intheformoffilaments,granulesand thevasculartissues (Fig. 6a,d). incrusts(Figs.3a-h,4a-g).Thegranulescanbcof Thecross scction showedthattheoutline of variedsizesandarepresentinPosoqueriaacutifolia leaf margin is truncatc-revolutc in Posoqueria (Fig. 4a), P. longiflora (Fig. 3 d,4d), P. macropus longiflora and slightly revolute in the othcrs (Fig.4e), P. palustris(Fig.4 0 andPosoqueriasp species(Fig.7a-d).Intheepidermis,characteristics (tFriigc.h4omge),si(nFcigl.ud4ien)g.oTvheerctrhuesststoomcactuar(iFnigP..4mad,crg)opaunsd osibmsielravretdo,thnoosteicoifntghetrheasttothfethceutleiacfulbalradelacyaenrbies (Fig. 3 0.P.palustris(Fig. 3g) andare thick in P. stronglythickcned, formingconspiciouscuticular oltahfteiPfa.odlaliaaxti(iFafilogl.siu3arb(f)Fa;ictgeh.eo4ffiacll)al.mseTpnethsceioeccscutu(irFciilgne.ai3bsaaxs-imha)loosautnrhdfaoocnne thflhaaevnnegieans.ptahTpeihrleelsoctsoeofmaptmhpoeenalreaeanfpcibedl.eardIemnataelnrndcaelilnllysP,a.trlheoensgmmiaafrllgloiernar the abaxial surfaceofP. longiflora (Fig.4d), and isfilledwithparenchymatouscellswiththickcned striatedonthe abaxial surfaceoftheotherspecies walls; among them idioblasts withcrystals occur. (Fig.4b,e-g),speciallyinP. latifolia(Fig.4c)and Lignified fibers were observed associated to the inPosoqueriasp(Fig.4g). vascular system. Inall speciesinvestigatedthe leafepidcrmis In transversal scction the outline oí petiole cisovoenree-dlabyyreadlfayoerrmeofdcbuytictlheicakn-dwawlellcld-dceevlelsloapnedd c8aan-cb)e.sOunlcatthee,ppeltaino-lceoannvdexaolrocngonocfatvhce-cmoindvvecixn(,Fiogn. Rodriguèsia 61(3):505-518.2010 SciELO/JBRJ cm 2 13 14 15 16 17 18 508 Arruda.R.C.O.etal cF.iPg.urleon1gi-flLoeraaf\edp.iPd.ermmaicsroopfuPso-soeq.uPe.rpiaaluAsutbrlies;tfi.nPf.rolnotnaglivjiloerwa,,aidnaxtihealmsiudrvfeaicne-reag.iPo.na(c{u)t.ifolia;b P latifolia Rodriguésia61 3 505-518.2010 ( ): SciELO/JBRJ cm .2 13 14 15 16 17 509 Lealanatomyand micromorphologyof Posoqueria Aublet (Rubiaceae) — co , Anhletinfrontalview,ab*axia*lswurifnaBce,-hca.,Ph.eacutifolia; b. P°.rlsa“tijol’ia; ' trichomes (d, e) andpapillae (0- Rodríguèsia61(3):505-518.2010 SciELO/JBRJ 2 13 14 15 16 17 lí 510 Arruda. R.C.O.etal. Figure3-ScanningelectronmicroscopyofleafbladeofPosoqueriaAublet-a.P. acutifolia;b-c.P. latifolia; d-e.P. longijlora; f.P. macropus; g. P.palustris; h. Posoqueria sp. Adaxial surface showingwax in granules (d), in crusts(f) and inthickcrusts (b). Rodriguésia 61(31:505-518.2010 SciELO/JBRJ cm i 0 Leafanatomyandmicromorphology ofPosoqueria Aublet (Rubiaceae) Figure4-ScanningelectronmicroscopyofleafbladeofPosoqueriaAublet-a P. acutífolia;b.P.acutifoliaboiled; teriPchloamtiefsoliinaP4.dmPaclroongpiufslo(rea);aen.dP.P.mapcalruosptursis;(f.0P.anpdalpuasptriilsl;aeg.inPoPsoosqouqeureinaasps.p.Ab(ag)x..aTlhesusrufrafeaceschcoawn.nbgesstmoomoat.ha(a-go)r, striated(b-c,e-g). Epicu.icularwax(arrow)in filamcnts(c)andgranules(e). Rodriguésia 61(3):505-518.201 SciELO/JBRJ cm l 12 13 14 15 16 17 . 1 8 0 512 Arruda,R.C.O.etal. Figure5-CrosssectionsofPosoqueriaAubletleaf,detailsofepidermis-a.P. latifolia withathickcuticularlayer (arrow); b.P.longiflora,trichomeinmidveinregion;c.P. longiflora,stomatawithdevelopedrimsandsubsidirycells inthesubstomatalchamber;d.P.palustris,lignifiedtrichomewithpittedwall(arrow). both surfaces of the leaf, the epidermal cells are palisade parenchymaare shorterand widerthan the organizedinrowsandhavethicker,nearlystraight remainingpartsoftheleafblade.Inthecorticalportion walls. Stomata can be found in this region ofthe belowtheabaxialsurfacethereisaparenchymawhich leafblade.Inthecorticalregionofthepetiolethere shows progressively more-voluminous cells with are two to five layers of angular collenchyma thinnerwallstowardsthevascularsystem. beneaththeepidermis.Afundamentalparenchyma In this study the most relevant leaf containing idioblasts with crystals, phenols, or characteristics tothe distinguishing ofthe species somesclereidsarelocatedintemally(Fig.8d,h).In arerelatedintheTable 1 all species, the vascular system is arranged in an arch(Fig.8a-c,e),withthexylemcellsarrangedin Discussion rows andthephloem in the externai position, like Fromalltheanatômica]featuresexamined.the thecollateraltype.Additionalbundlesareobserved ones that showed ahigherpotencial ofutilization attheextremitiesofthevascularsystem. to identify the studied species were: cuticularand In the region of the midvein, beneath the wax sculpturing, aspect ofthe anticlinal walls of epidermis,thecorticalregionisoccupiedbythreeto epidermal cells and presence and types of five layers ofcollenchyma that varies between the trichomes. The variation on the thickness em angularandlamellartypes,andundemeaththereisa sinuosityfromtheanticlinalwallsisquotedfotthe palisadeparenchymaconnectingthetwopartsofthe RubiaceaebyMetcalfe&Chalk(1950)andGomes leafblade(Fig. 8f-g). Inthisportion,thecellsofthe etal. (2000). Harberlandt (1928)believesthat the Rodriguésia 6 3 505-5 20 ( ): 1 . 1 SciELO/JBRJ L2 13 14 15 16 17 513 Leafanatomyandmicromorphology ofPosoqueria Aublet (Rubiaceae) Rodriguésia61(3):505-518.2010 SciELO/JBRJ 514 Arruda,R.C.O.etal. Figure7 CrosssectionsofPosoqueriaAubletshowingvariationoutlineoftheleafmargin-a.P. acutifolia-. b.P. latifolia\c.P. longiflora\ d. P.palustris. Table 1 DistmctivecharacterestotheidentificationofsixPosoqueriaAublet(Rubiaceae)species. SCpheacriaecsters/ Posoqueria Posoqueria Posoqueria Posoqueria Posoqueria Posoqueria acutifolia latifolia longiflora macropus palustris sp. Epidermis, adaxial surface Ouwtallilne of anticlinal sinuous sinuous sinuous sinuous sinuous straight Cuticular surface smooth smooth smooth smooth smooth striated Epicuticular wax smooth crusts granules crusts crusts smooth Epidermis, abaxial surface Ouatnltiincleinoafl wall sinuous sinuous sinuous sinuous straight straight to curves Cuticular surface striated striated smooth striated striated striated Epicuticular wax granules filaments granules granules granules granules Fofortmriacnhdomdeisstribution absent unicellular, long, long, long, papillae, restrict to unicellular, unicelular, pluricelular, intercoastal midvein restrict to intercoastal intercoastal region region midvein region region region Ouitnlitnreanosfvetshael lseeacftimoanrgin revolute revolute trreuvnoclauttee/ revolute revolute revolute Rodriguésia 61(3):505-518.2010 SciELO/JBRJ, cm 13 14 15 ..