Rev. bras. farmacogn. vol.26 número5

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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

The

structural

studies

on

the

medicinal

plant

Haplophyllum

telephioides

Mehmet

Tekin

a,_∗

_,

_Nuraniye

_Eruygur

b

a_Department_of_{Pharmaceutical}_Botany,_Faculty_of_Pharmacy,_Cumhuriyet_University,_Sivas,_Turkey b_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_Cumhuriyet_University,_Sivas,_Turkey

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t

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Articlehistory: Received11March2016 Accepted9May2016 Availableonline17June2016

Keywords:

Haplophyllumtelephioides Medicinalplant Morphology Anatomy Rutaceae Turkey

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b

s

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HaplophyllumtelephioidesBoiss.,Rutaceae,isanendemicherbwhichgenerallygrowsatthestepperegion inCentralTurkey.Itsaerialpartsinfloweringstageareusedbylocalpeopleagainstinfluenzavirusdue toitsantimicrobialeffect.Themainpurposeofthisstudywastoexaminethemorphology,anatomy andhistologyofthevegetativeandreproductiveorgansoftheH.telephioides,whichareusedin popu-larmedicine,thereby,contributingtothepharmacognosticevaluationofthespecies.Thespecieswas studiedmorphologicallyindetail.Newmorphologicalfeaturesweredescribedforthem,suchasthe dimensionofleaf,sepalandfruit,sizeoffilament,antherandpistil,anddiameteroftheovary.In addi-tion,somedeviatingfeatureswerefoundinrelationtopreviouspublisheddescriptionsforthespecies, suchasplantheightandpetallength.Theanatomyofplantpartssuchasstem,leaf,sepal,petal,filament andpistil,werestudiedusinglightmicroscopyandscanningelectronmicroscopy.Stemhasincipient secondarygrowth.Theleafisamphistomaticandthemesophyllisequifacial.Stomataareanomocytic andsunken.Inmicromorphologicalstudies,itisdeterminedthattheleafiscoatedbyathickcuticleand aboveepicuticularwax.Schizogenousglandswerefoundinallvegetativeandreproductiveorgans.The structuralfeatureshereinfoundcanassistthediagnosisofH.telephioides.

Introduction

TheRutaceaeisalargefamilyrepresentedby161generaand about1900speciesintheworld(Simpson,2006).Rutaceae rep-resentativesoccurintropicalandsubtropical regions,especially inSouthernAfricaandinAustralia(Heywood,1978;Kubitzkietal., 2011).Thefamilyhaseconomicallyimportantspeciesformedicine (Gözleretal.,1996;Sa˘glametal.,1999),food,gardeningand orna-mentalspecies(Kubitzkietal.,2011).Themostdistinctivefeature ofthefamilyisthepresenceofsecretorycavities,whichoriginate schizogenous,lysigenous,orschizolysigenous.Theseoccur partic-ularlyintheleavesandreproductiveorgans(MetcalfeandChalk, 1950;ZeybekandZeybek,1994;Groppoetal.,2008).These secre-torycavitiescontainvolatileoils,alkaloids,resin,hesperidinand someotherchemicalcompounds inleaf,stem, flowerand fruit ofthespecies(Metcalfeand Chalk,1950;Kubitzkiet al.,2011).

HaplophyllumA.Juss.isagenusofRutaceae,withca.70species

(Townsend,1986;SoltaniandKhosravi,2005)showingthelargest diversity in Irano-Turanian region, particularly in Turkey, Iran

∗ _{Corresponding}_author.

E-mail:mtekin@cumhuriyet.edu.tr(M.Tekin).

and CentralAsia, andMediterranean phytogeographicalregions (Navarroetal.,2004;Salvoetal.,2011;Manafzadehetal.,2014).

Haplophyllumhasfourteenspeciesinwhicheightofthemare

endemictotheFloraofTurkeyandEastAegeanIslands(Townsend, 1967).AccordingtotherecentstudyconductedbyGüneretal. (2012),Haplophyllumisrepresentedninespeciesinwhichsixof themareendemicfor Turkey.Many studieshaveexaminedthe phytochemistryand medicinalattributes ofsome of Haylophyl-lum,analyzingtheircontentsforalkaloids,lignans,glycosidesetc. (Patraetal.,1984;Cantrelletal.,2005;UlubelenandÖztürk,2008; Parhoodehetal.,2012).Forexample,phytochemicalcomposition

ofHaylophyllumbuxbaumii(Poir.)G.Donwasstudiedand

com-pounds suchas quinolinealkaloids, kokusaginine,skimmianine and␥-fagarinewereisolatedfromthisspecies(Ulubelen,1985). Inadditiontoalkaloids,therearereportsonlignans(Gözleretal., 1984),haplomyrtin,(−)haplomyrfolin(Evciumetal.,1986)and

justicidin-A(KhalidandWaterman,1981)forsomeHaplophyllum. Duetothese chemical compounds,someHaplophyllumsuchas

Haplophyllumpatavinum(L.)G.Don,areusedinfolkmedicineas

antimicrobial,antimalarial,insecticidalagent.Haplophyllum

tuber-culatumJuss.,whichnaturallygrowsinSaudiArabia,isusedforthe

cureofrheumatoidarthritis,malariaandinthecureofsome gyne-cologicalproblems (UlubelenandÖztürk, 2008;Al-Yahyaetal.,

http://dx.doi.org/10.1016/j.bjp.2016.05.007

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1992).HaplophyllumhispanicumSpachisanendemictotheEastof Spain,andisknownforitshighskinreactivity,beingemployed clin-icallyagainstvitiligowithsomesuccess(MassmanianandPrieto,

1996).HaplophyllumsuaveolensG.DonandH.buxbaumii,which

naturallygrows in Turkey, are alsoknown as medicinalplants (Cetinetal.,2012).

Duringfieldworks,byinterviewingwithlocalpeople,we rec-ognizedthattheaerialpartsofHaplophyllumtelephioidesBoiss.in floweringstageareusedasamicrobicideagainstinfluenza,insome districtsofSivasprovince(Turkey).Phytochemicalinvestigationsof

H.telephioideswerestudiedbyUlubelenetal.(1994)andUlubelen

andÖztürk(2008).Thesestudiesfoundthepresenceofsecondary metabolitessuchasalkaloids7-hydroxy-9-methoxyflindersineand lignans,4-acetyldyphyllin,inadditiontohaplomyrtin,dyphyllin andvanillicacid.Oneofthesesecondarymetabolites,dyphyllin,a newnaturalcompound,hasbeennotedtopotentlyinhibit vacular-ATPase,and thereby lysosomal acidification in osteoclasts.This leadstoabrogationofboneresorption(Sorensenetal.,2007)and reducev-ATPaseexpressioningastricadenocarcinomacells(Shen etal.,2011).Dyphyllinisalsoreportedtodisplaysignificant anti-tumoractivity(FukamiyaandLee,1986;Jansenetal.,2006),and showsantiviral effectsonvariousviralstrainsincludingthatof knowndrug-resistantstrainsbyalteringcellularsusceptibilityto influenzavirusesthroughtheinhibitionofendosomalacidification, thusinterferingwithdownstream virusreplication(Chenetal., 2013).Inaddition,somedyphyllinderivatives,isolatedfrom

Hap-lophyllumlinifolium(L.)G.Donwhichhaveactivitiesagainsttopical

anti-inflammatoryandantitrypanosome(Schinellaetal.,2008). In thisstudy, weinvestigatethe structureof vegetative and reproductiveorgansofH.telephioides.Thisspeciesisendemictothe regionlimitedbyCentralAnatoliawithKayseri,Nevs¸ehir,Yozgat andSivasprovinceinTurkey.AccordingtoIUCNthreadedcategory,

H.telephioideshasbeenplacedunderLR-nt(lowerrisk-near

threat-ened)byEkimetal.(2000).SuchasH.telephioides,manyspecies

ofHaplophyllumaredistributedinnarrowgeographicareas,which

makesthemmorevulnerabletotheriskofextinction(Salvoetal., 2011).

ThoughH.telephioidesiswellcharacterizedbyphytochemical studies,theknowledgeonthestructures ofplantpartsusedas medicinalisincomplete.Infact,therearenodataonanatomyand micromorphologyforthisspecies.Onlysuperficialmorphological descriptionsareavaliableinFloraofTurkeyandEastAegeanIslands (Townsend,1967).Consideringthesestudies,presenceofdyphyllin

inH. telephioides,its usageasantimicrobialplantby local

peo-pleandusageofrelatedspeciesasmedicinalplantincreasesthe importanceofthepresentstudy.Thereforetheaimofthisstudy

istodetaileddescribethestructureofvegetativeand reproduc-tiveorgansofH.telephioides,usingmorphologicalandanatomical procedures.Thiswillprovidenewstructuralinformationforthis species ofwell-known importance infolk medicineand phyto-chemicalproperties.Thisstudymayalsoprovidenewdiagnostic featuresforidentificationofH.telephioides.

Materialsandmethods

Plantmaterial

HaplophyllumtelephioidesBoiss.,Rutaceae,werecollected

dur-ing the flowering and fruiting period from different natural populationsinSivasprovinceofTurkey.Fieldworkswerecarried outin2015.Locality1:B6Sivas:Ulas¸district,Ziyarettepe,1411m, 39◦₃₃′_08.1′′_N;₃₇◦₀₁′_12.5′′_E,_22.05.2015;_Locality_2:_B6_Sivas:_Hafik

district,Topc¸uyeniköyvillage,1383m,39◦₄₆′_59.3′′_N;₃₇◦₃₅′_15.9′′_E,

26.06.2015;Locality3:B6Sivas:Sivas-Kangal-Gürünroad inter-section,roadside,1545m,39◦₀₇′_52.7′′_N;₃₇◦₁₄′_32.6′′_E,_30.06.2015.

Morphologicaldescriptionwasmadeforspecimensofeach local-ity.Foranatomicalprocedures,thespecimensoffirstlocalitywere used. Taxonomical identificationwas madeby first author and somematerialswerepreparedasherbariumvouchers.Thesewere registeredundercollectornumbersM.Tekin1671,M.Tekin1689, M.Tekin1699andareconservedattheCumhuriyetUniversity, Fac-ultyofScienceHerbarium(CUFH),DepartmentofBiology,Sivas, Turkey(Fig.1).

Structuralanalysis

ThemorphologicalfeaturesofH.telephioideswerestudiedboth infreshandherbariummaterials.Partsofthefreshmaterialwere storedin70%alcohol–watersolutionforlateranatomical proce-dures.Theseconsistedinhand-madecrosssectionofstem,leaf, sepal,petal,filament,ovaryandstyle.Alsoparadermalhand-made sectionsofadaxialandabaxialsurfaceoftheleafweretakenwith razorblade.Thesectionswerestainedin1%Alcianblue(Sigma)and 1%SafranineO(Sigma),inaratio3/2(DavisandBarnett,1997). Thesectionswerekeptabout5mininthedye.Semi-permanent slidesweremoundedusingglycerin–gelatine(Jensen,1962).The structuralinvestigationsofvegetativeandreproductivepartsofH.

telephioidesweremadeusingOlympusBX22lightmicroscopy.

Pho-tomicrographsweretakenusingOlympusBX51lightmicroscopy coupledwithOlympusDP70digitalcamera.Stomatalindexwas calculatedaccordingtothestudyofMeidnerandMansifield(1968).

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Somepartsofthematerialwereanalyzedunderscanning elec-tronmicroscopy(SEM).Forthis,theplantpartsweremountedon aluminumstubsandcoatedwithgold.Themicromorphologicaland anatomicalobservationsweremade,andmicrographsweretaken withaLEO440SEMatdifferentmagnifications.

Results

Morphologicalaspects

Perennial herb, 4–18cm. One or generally numerous stems, erectorascendens,paledark-brownatbase,green atthe mid-dleandupperparts(Fig.1).Thestemiscoveredwithdenselyfine whitehairs.Leavesaresimple,dense,alternate,sessileandovate toellipticorsubovate,andcoveredbyshortsimplenon-glandular hairs. Leaves are 2–18mm×1–9mm and containing numerous

tinyblackdotswhichcorrespondstothesecretorycavities.The inflorescences are terminal, with 2–16 flowers, or occasionally withasolitary flower.Inflorescenceis cymose,small and com-pact, andthe branchesare covered whitewooly non-glandular hairs. Flowers are usually pentamerous, and occasionally hex-amerous.Sepalsare2–3.5mm×0.7–1.5mm,linear-lanceolateor

triangular-shaped,darkgreenwhenfreshandblackishgreenwhen dry,outersidedenselywhitishhairy,innersideglabrous.Petals are6–9mm×3–4.5mm,usuallyovatetolanceolate,occasionally

elliptic-shaped, slightly concave, entire, bright yellow. There is medianlongitudinalstripwhichispaletodark-greencoloredand ismoredistinctwithwhitishhairsabaxiallyandglabrous adaxi-ally.Filamentsare4–6mmlongandthick,graduallyattenuatefrom basetoapexandarecoveredwithwhitishhairsatbase.Anthers are1–2mmlong,dorsifixedandlongitudinallyopening.Pistilsare 5–6mmlong,ovariesareca.2mmindiameter.Ovarycomposed offivecarpels,andeach carpelbearing fourovules. Carpelsare withaprominentandsharplypointedconicalapicaltubercles. Cap-sulesare5lobed,3–4mm×4–5mm,palegreentoyellowcolored.

Surfaceofcapsuleiscoveredshorthairswithpointedtubercles andconvexortuberculateglands atbelow.Seedsarereniform, 1.1–1.5mm×1–1.3mm,coal-blackcoloredandcoveredby

trans-verseridges,onlateralanddorsalside(Fig.1andBox1).

Anatomicalandhistologicalaspects

Stem

The cross section taken from middle area of the stem was observed.Theshapeofsectionsiscircular (Fig.2A,D).The epi-dermisisuniseriateanditscellsaresmall,rectangularorsquarish inshape.Theoutercellwalloftheepidermisisthickbut,the cuti-clelayeronthesecellsisthin.Thereareunicellularnon-glandular hairsonstemepidermis(Fig.2A,D).Cortexconsistsof8–13cell layers.Cortexcellsareovalorcircular-shapedandtherearesome intercellularspacesbetweenthem.Thecells intheoutercortex layershavechloroplastandarethusphotosynthetic.Thesecells aresmallerthanthenon-photosyntheticinnercortexcells.There arealsofewschizogenousglandsinthecortexlayer,justbelowthe epidermis(Fig.2B,E;Fig.3).Innerside,thereareisolatedpatchesof fibers,with1–3celllayers,betweeninnercortexlayersandphloem, thusprobablyofphloematicorigin(Fig.2B,E).Thephloemconsists of3–5celllayers,cambiumisdistinctandconsistsof1–3cell lay-ers.Thexylemhasvesselelements, sclerenchymaticcellsand a uniseriateparenchymatousrays.Thepithislargeanditscellsare ovalorcircular-shapedandtheyhaveintercellularspaces(Box1,

Fig.2C,F).

Leaf

Thereareuniseriateepidermisesonbothsurfacesofleaf. Epi-dermalcellsareovalorrectangular-shaped.Thereareunicellular hairsonadaxialandabaxialepidermises(Figs.4A,Band5).The outercellwallofthebothepidermalcellsisthick.Epidermalcells havearugosecuticleandapparentlycoveredbywax(Fig.5C,D). Theleafisamphistomaticandthemesophyllisequifacial.Adaxial andabaxialpalisadeparenchymashave2–3celllayers.Thecellsof palisadeparenchymaaregenerallycylindricalorrectangular, occa-sionallyovoid-shaped.Thespongyparenchymahas1–3celllayers, positionedbetweenthetwopalisades.Spongyparenchymacells arecircular,ovoidorirregular-shaped.Intheleafmesophyll,there arelargeschizogenousglandsclosetobothepidermises(Fig.4A,C). Themidribisoval-shaped(Fig.4B).Thereisonemaincentral vas-cularbundle,whichissurroundedbyauniseriateparenchymatous bundlesheath.Thestomataareanomocyticandsunken,occurring inbothsidesoftheleaf(Figs.4Dand5).Stomatalindexandstoma

Box1

ComparisonofsomediagnosticmorphologicalandanatomicalfeaturesofHaplophyllumtelephioidesandthreeHaplophyllumspecies(H.myrtifoliumBoiss.,H.vulcanicum Boiss.etHeld.andH.megalanthumBornm.)instudyofUlukıs¸etal.(2016).

H.telephioides H.myrtifolium H.vulcanicum H.megalanthum

Morphologicalfeatures

Leavesshape Ovate,ellipticor

subovate Lanceolate, lanceolate-elliptic, rarelyovate Lanceolate, ovate-lanceolate Lower lanceolate-elliptic,the upperdistinctly rhomboid-lanceolate

Leavessize(mm) 2–18×1–9 8–23×3–7 2–17×2–9 7–20×2–8

Sepalsshape Linear-lanceolateor

triangular Lanceolate-oblong, oblanceolate-ovate Deltoid-ovateto lanceolate Lanceolate-elliptic

Sepalsize(mm) 2–3.5×0.7–1.5 1–3.5×0.5–1.5 2–4×1.5–2 3–4×1.25–1.75

Petalshape Ovatetolanceolate,

occasionallyelliptic

Oblong-ovate,ovate Oblong-ovate,clavate Oblong-ovateor

ellipticlanceolate

Petalcolor Brightyellow Whiteto

sulphur-yellow

Creamy-white Whiteorcreamy-white

Petalsize(mm) 6–9×3–4.5 6–8.5×3–4.5 8–11×4–5 7–14×4–5

Filamentslength(mm) 4–6 4.5–5 4.5–5.5 4–6

Capsulesize(mm) 3–4×4–5 2–3×4–5 3–3.5×5–6 3–3.5×5–6

Anatomicalfeaturesofstem

Epidermiscellshape Rectangularorsquarish Rectangularandoval Rectangular Rectangular

Cortexcelllayers 8–13 7–9 6–12 7–12

Shapeofcortexcells Ovalorcircular Oval,cylindricalor

rectangular

Ovalorrectangular Ovalorrectangular

Phloemsclerenchymacelllayer 1–3 2–4 5–9 1–4

Cambiumcelllayer 1–3 1–3 1–4 3–5

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Fig.2. PhotomicrographsofstemcrosssectionofHaplophyllumtelephioides.Generalappearance(AandD),detailofstem(BandE),detailofvascularsystem(CandF).A,B andCunderlightmicroscopy(LM);D,EandFunderscanningelectronmicroscopy(SEM).Abbreviations:cambium(ca),cortex(cx),non-glandularhair(eh),epidermis(ep), fibers(fi),phloem(ph),pith(pt),schizogenousgland(sg),vesselelement(ve),xylem(xy).

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Fig.4.MicrophotographsoftheleafcrosssectionsofHaplophyllumtelephioides.Generalview(A),detailofmidribregion(B),detailofmesophyllandschizogenousgland(C), detailofsunkenstomatainadaxialepidermis(D).Abbreviations:cuticle(cu),epidermalhair(eh),guardcell(gc),abaxialepidermis(le),phloem(ph),palisadeparenchyma (pp),schizogenousglandsac(sg),spongyparenchyma(sp),stoma(st),adaxialepidermis(ue),adaxialepidermiscell(uec),adaxialepidermiscellwall(uew),vascularbundle (vb),xylem(xy).

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Fig.6.MicrophotographsofperianthpartscrosssectionofHaplophyllumtelephioides.Generalviewofsepal(A)anddetailofsepalmidribregion(B),generalviewofpetal(C) anddetailofpetalmidribregion(D).Abbreviations:non-glandularhair(eh),abaxialepidermis(le),parenchymatouscell(pc),phloem(ph),secretorycell(sc),schizogenous gland(sg),stoma(st),adaxialepidermis(ue),vascularbundle(vb),xylem(xy).

densityfoundwere10.57and264stomaper1mm2_for_abaxial_side ofleafand11.67and292stomaper1mm2_for_adaxial_side_of_leaf, respectively(Figs.4and5).

Sepal

Sepals areslightly concave in adaxialsurface and convexin abaxialsurface.Epidermalcellsarerectangular,ovalor squarish-shapedinbothsurfaces.Therearenon-glandularhairssolelyon abaxialepidermis.Mesophylliscomposedof2–4parenchymatous celllayerswhichpresentedconspicuousintercellularspaces.There areoccasionallyschizogenousglandsinsepalmesophyll. Vascu-larizationismadebysmallcollateralvascularstrands,positioned closetotheadaxialepidermis.Themainvascularbundleis circular-shaped(Fig.6A,B).

Petal

Petalsare“V”shapedincrosssection.Epidermalcellsare rectan-gular,ovalorsquarish-shaped.Themesophyllconsistsof4–8layers ofoval-shaped,parenchymatouscells.Inthemesophyll,thereare occasionallyschizogenousglandslocatedclosetoabaxial epider-mis.Vascularizationismadebyawidecentralcollateralstrandand byanumberofsmalleroneswhichpositionedclosetotheadaxial epidermis(Fig.6C,D).

Pistil

Thestyleepidermisisuniseriate,withrectangularor squarish-shapedcells.Thestylehave5(6)stylarcanals,eachwithasecretory epidermisandanarrowlumenfilledwithsecretion(Fig.7A,B). Therearelotsofvascularbundlesthatformsaringcloseto epider-mis.Occasionallyschizogenousglandsoccurlaterallytothestylar canals,especiallythepartofstylewhichclosetoovaryside(Fig.7B, C).Theovarieshave5locules,andbeneaththeouterepidermiseach loculewallhas2–3schizogenousglands(Fig.7D).

Filament

Filaments have an elliptical shape, a seen in cross sections. Theirepidermalcellsarerectangularorsquarish-shaped.Filaments havesparsenon-glandularhairsandschizogenousglandscloserto theepidermis.Vascularizationismadebya widesingle amphi-cribralbundlewhichlocatedatthecenterofeachfilamentincross section.Therearecircularandoval-shapedparenchymatouscells withintercellularspacesbetweenepidermisandvascularbundle (Fig.7E,F).

Discussion

Despiteofitsusageinfolkmedicineduetoitsantimicrobial effect,themorphologyofH.telephioidesisnotknownentirelyand thisstudyisthemostcomprehensiveinvestigationon morphol-ogyofthespeciesafterTownsend(1967).However,thisisthefirst studyconductedtodeterminetheanatomical andsome micro-morphologicalcharacteristicsofaerialpartsinfloweringtimeof

H.telephioides.

Inthisstudy,somedifferentmorphologicalcharacteristicshave beendeterminedfromitsdescriptiongivenforH.telephioidesin FloraofTurkeyand EastAegeanIslands(Townsend,1967).The heightofplantandlengthofpetalweredeterminedas4–18cm and 6–9mm,respectively while these measurementsare given as 7–12cm and 7–9mmin thestudy ofTownsend (1967).The leafshape wasdeterminedas ovate,elliptic orsubovateinthis study,whilethisfeaturewasspecifiedasonlyovateinthestudyof

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Fig.7.MicrophotographsofthereproductivepartscrosssectionofflowerofHaplophyllumtelephioides.Generalviewofstyle(A),detailofstyle(B)anddetailofschizogenous glandsinstyle(C),generalviewofovariumlocule(D),generalviewoffilament(E),detailoffilament(F).Abbreviations:carpelwall(cp),cuticle(cu),non-glandularhair(eh), epidermis(ep),parenchymatouscell(pc),phloem(ph),ovulum(ov),secretorycell(sc),schizogenousgland(sg),stylarcanal(sy),vascularbundle(vb),xylem(xy).

alliedHaplophyllumspecies(Haplophyllummyrtifolium,

Haplophyl-lumvulcanicumandHaplophyllummegalanthum)instudyofUlukıs¸

etal.(2016)weregiveninBox1.

TherearesomeanatomicalstudiesonRutaceae(Metcalfeand Chalk, 1950; Saunders, 1934; Moore, 1936; Souza et al., 2004; Muntoreanuetal.,2011;Cruzetal.,2015),butanatomicalstudies

onHaplophyllumaredeficient.

InRutaceae,stomataareconfinedtotheabaxialsideinmost species(MetcalfeandChalk,1950).Similarly,oneoftheothergenus ofRutaceae,PilocarpusVahlandrelatedgenerahave hypostoma-tousleaves,withstomataatthesameleveloftheepidermiscells (Duval,1903;Marquete,1981;Spegazzinietal.,2002;Gallardoand Jiménez,2002).Conversely,inH.telephioides,stomataareexistboth sides,soleavesareamphistomatic.Furthermore,stomataofH.

tele-phioidesareatthelowleveloftheepidermiscellswhichknownas

sunkentype.AccordingtoMetcalfeandChalk(1979),intheleaf,the anticlinalwallsofepidermalcellsmaybestraightorwavyinsome membersofRutaceae.IntheleavesofH.telephiodes,weobserved thatsolelystraightanticlinalwallsinadaxialandabaxialsurfaces (Fig.5A,B).

ForTurkeyflora,H.megalanthumisanotherrelatedendemicto

H.telephioidesandthemorphological,anatomicaland

palynologi-calpropertiesofH.megalanthumwereinvestigatedbyAkyoletal. (2012).WhenwecomparetheresultsonH.megalanthumwithour resultsonH.telephioides,theanatomicalcharacteristicsof both species,there aresomeconsiderabledifferencesbetweenthem. Suchas,instemcrosssection,thecambiumiscomposedof1–3cell layers,phloemisnarrowandpithregioniswideinH.telephioides. Whereas,thecambiumiscomposedof3–5celllayers,phloemis wideandpithregionisnarrowinH.megalanthum.Atthemore out-sideofstem,thereareunicellularnonglandularhairs,andincortex layer,thereareoccasionallyschizogenousglandsinH.telephioides, whereasforthesepropertiesthereisnoreportinH.megalanthum

(Akyoletal.,2012).Intheleafcrosssection,theshapeofepidermis cellsisovalorrectangularinH.telephioides,whileitissolely rect-angularinH.megalanthum.Thereissignificantepicuticularwax layerandsunkentypestomataonadaxialandabaxialsurfacesof

theleafinH.telephioides,whilethereisnoreportforthese

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regionforH.telephioides.Recently,Ulukıs¸etal.(2016)have stud-iedcomparativelydetailed morphology,stem and leafanatomy andpollenfeaturesofmorphologicallyrelatedTurkishendemics,

H.myrtifolium,H.vulcanicumandH.megalanthumwithsystematic

aspect.ThemorphologicalandanatomicalcharacteristicsofH.

tele-phioidesandthreespeciesofUlukıs¸etal.(2016)’sstudyweregiven

comparativelyinBox1.Ulukıs¸etal.(2016)werefoundthatthe numberofcortexlayersandsclerenchymaticcelllayersonphloem instemanatomyaretobeimportantasspeciesspecific charac-tersfortheirstudiedthreespecies.Whenwecomparethestem anatomyresultsofH.telephioideswiththeresultsofH.myrtifolium,

H.vulcanicumandH.megalanthum,wefoundthatthesignificant

differenceisthenumberofsclerenchymaticcelllayeronphloem, justasbetweenthreespeciesinstudyofUlukıs¸etal.(2016)(Box1). InleafanatomyofH.telephioides,thenumbersofupperandlower palisadeandspongyparenchymacelllayersareidenticalwithH.

myrtifolium,H.vulcanicum,andH.megalanthum.Therearesome

differencesinmidribanatomyofH.telephioidesandbetweenthese threespecies.Incomparisontheformandsizeofvascularbundlein themidribregion,themaindifferenceismidribshapewhichisoval

inH.telephioideswhereas,itisadaxiallyconcaveandabaxially

con-vexinthreespeciesofthestudyofUlukıs¸etal.(2016).Additionally,

H.telephioideshascompactandsmallvascularbundle,whereas,H.

myrtifolium,H.vulcanicum,andH.megalanthumhavelargemidrib

andinallthreespeciesitformsaprojectingparts.

LiuandHu(1998)studiedmorphologyandanatomyofsecretory cavitiesintheleavesof22 genera,40speciesand2varietiesof Rutaceae.Asaresultofthisstudy,authorssuggestedthatthereare threetypesofsecretorycavitiesinRutaceae:anotchtype(between leafteeth),amesophylltypeandamixedtype(withbothnotch andmesophyllcavities),suggestingthatthemixedtypeofcavities developedfromthenotchandmesophylltypes.Consideringthe studyofLiuandHu(1998),schizogenousglandsofH.telephioides

leavescouldbeclassifiedasmesophylltype(Fig.4A,C).

FlowersofRutaceaespeciesarenotwell-knownintheir mor-phologicalandanatomicalaspects(Souzaetal.,2004).Thereare somestudieswhichfocusedonfloralbuddevelopmentonsome membersofRutaceae(Souzaetal.,2003;ElOttraetal.,2013).Souza etal.(2004)werestudiedmorphologyandanatomyoftheflower

ofMetrodoreanigraSt.Hill.fromRutaceae.Theywereindicated

secretoryglandswhichimmersedintheparenchymaticmesophyll ofsepals,petalsandovaries.ThisfeatureissimilartoH.telephioides. Inaddition,Souzaetal.(2003)studiedfloweranatomyofanother Rutaceae,PilocarpuspennatifoliusLem.andtheyindicated secre-toryglandsinpetal,ovaryandstylecrosssection,asH.telephioides. Furthermore,in cross section of petalof P.pennatifolius,Souza etal.(2003)determineddrusecrystalswhicharenotobservedany organsofH.telephioidesasinpetals.InRutaceae,thickfilamentwas citedbyLersten(2004).ThefilamentsarethickinH.telephioidesand almostequalwithstyleswidth.Inaddition,thefilamenthassingle andlargeamphicribralvascularbundleinmiddlearea(Fig.7E,F). Conversely,stylehaslotsofsmallvascularbundleswhichlocated betweenepidermisandstylarcanals(Fig.7A).

Asaconclusion,ascanbeseenfromtheliteraturestudies,this studyseemstobethefirstandextensiveresearchonmorphology andanatomyofthestem,leaf,sepal,petal,filamentandpistil,in additiontothemicromorphologyofH.telephioides.Inthisstudy, somedifferent morphological resultsand newcharacters were determined,andbyusingthesecharacters,morphological

descrip-tionoftheH.telephioideswaspossiblyextended.Allanatomical

characteristicswhichbelongtothepartsusedinfolkmedicinewere investigatedindetail.Thepresenceofschizogenousglandswhich producebiologicallyactivesubstanceswereshownanatomically invegetativeandreproductiveorgans.Inaddition, micromorpho-logicalpropertiesofsomeoftheseorganswerestudied.Therefore, someresultsgiveninthisresearchwerecomparedwiththeresults

ofothermembersofHaplophyllumintheliteraturerecordsand dif-ferencesbetweenthemwerestatedindetail.Alltheseresultscan beusedtoevaluateofthediagnosticfeaturesofH.telephioidesas wellasthegenusHaplophyllum.

Authors’contributions

MT contributed to collecting and identifying plant samples, preparing the slides, data analysis and drafting the paper. NE contributed to writing and critical reading of the manuscript. Bothauthors have read thefinal manuscript and approved the submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthorsthanktotwoanonymousreviewerswhomade con-structivesuggestionsthatimprovedthefinaltext.

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