Nectar and oleiferous trichomes as floral attractants in Bulbophyllum saltatorium Lindl. (Orchidaceae)

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Protoplasma(2018)255:565–

574DOI10.1007/s00709-017- 1170-4

ORIGINALARTICLE

Nectarandoleiferoustrichomesasfloralattractantsin BulbophyllumsaltatoriumLindl.(Orchidaceae)

MałgorzataStpiczyńska¹ ^&BartoszJ.Płachno²^&^KevinL.Davies³

Received:28March2017/Accepted:6September2017/Publishedonline:24September2017

#TheAuthor(s)2017.Thisarticleisanopenaccesspublication

AbstractA l thoughmanyOrchidaceaehavedeceitflowerst hatproducenoreward,themostcommonreward,whenpres- ent,isnectar.Bulbophyllum,however,isunusualinthatthelabe llarsecretionsofmostspeciesinvestigatedtodatelacksugars,a nd,therefore,cannotbeconsideredtruenectar.TheAfricanspec iesBulbophyllumsaltatoriumisanexceptioninthatitproducesn otonlynectarbutalsopossessesspecialized,capitateoleiferous trichomes.ThenectaryofB.saltatoriumisborneonthelabellum andisrepresentedbyadeep,narrow,medianlongitudinalgroov e,havingasmallaperture,andflankedbytrichomes.Isodiametr icepidermalcellsliningthisgroovesecretenectarwhichcollect sbothinthegrooveandonthesurfaceofthelabellum.Aswellasan ectary,thelabellumofB.saltatoriumalsobearsthreetypesofu nicellulartri-

chomes:thelongesttrichomesarebornedistallyandabaxial ly;themarginalonesformarimaroundtheentirelabellum,a ndfinally,massive,capitatetrichomesoccurprox-

imallyandadaxially.Theseareoleiferous,containinglargequ antitiesofoilwhichmightfunctionasprecursorsofvolatilecomp onentsoffragranceorprovideafood-

reward.Tothebestofourknowledge,thisisthefirsttimefor sucholeiferoustrichomestobedescribedforBulbophyllum.T herefore,apartfromtheircolorandmarkings,flowersofthisspe ciesareable

toattractpollinatorsinatleasttwo,possiblythreeways:food- rewardintheformofnectar;fragrance;andpossiblyfood- rewardsintheformoffood-hairs.

KeywordsAfricanBulbophyllum.Cellwallingrowths.Flowe rmorphology.Labellarmicromorphology.Nectaries.Oleifer oustrichomes.SectionPtiloglossum.Ultrastructure

Introduction

Consideredtobethelargestorchidgenus(Pridgeonetal.

2014),BulbophyllumThouarscomprisessome2200speciesoc curringthroughouttropicalAfrica,theComoros,Madag ascar,theSeychelles,ReunionandMauritius,Asia,theP hilippines,NewGuinea,thetropicalPacificislands,Australia, NewZealandandtheNeotropics,themaincentersofdistribution beingMadagascarandNewGuinea(Pridgeonetal.2014).Itisent omophilous,withmorphologicallydiverseandintricateflower sdisplayingsomeofthemostvariedandcomplexpollinationstr ategiestobefoundamongstorchids,andispollinatedbyawide rangeofinsects,mostnotably,

Coleoptera,Hymenoptera(mainlyinAfrica),suchaswasps

H

a n d l in g E d i t o r : H a n n s H . K a s s e m e y e r

*MałgorzataStpiczyńskastpiczyns ka_mal@biol.uw.edu.pl

1 FacultyofBiology,UniversityofWarsaw,BotanicGardenAl.

Ujazdowskie4,00-478Warsaw,Poland

2 DepartmentofPlantCytologyandEmbryology,JagiellonianUnivers ityinKraków,9GronostajowaSt.,30-387Kraków,Poland

3 SchoolofEarthandOceanSciences,CardiffUniversity,MainBuil ding,ParkPlace,CardiffCF103AT,UK

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andbees,includingstinglessbeesandctenuchidwasps(Joha nsson1974—

citedinvanderCingel2001;Dressler1990,1993;vanderCin gel2001;ChenandGao2011),butmainlyDiptera,includingfr uit-

flies,blowflies,fleshfliesandsignalfliesorPlatystomatidae(

vanderCingel2001;TanandNishida2000,2005;Tanetal.200 2;Humeauetal.2011;Ong2011;Ongetal.2011andreferenc estherein;OngandTan2011,2012;Liuetal.2010;Stewartetal .2014).Assuch,onewouldexpectthisgenustobeapotentiallyr ichsourceofnewinformationaboutplantadaptation(Graven deeletal.2004;Fischeretal.2007).

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566 M.Stpiczyńskaetal.

Therewardlessconditionisfrequentamongstorchids(Jers ákováetal.2006),butwhenfloralrewardsarepresent,theymain lyoccurasnectar(Dressler1990,1993;DaviesandStpiczyńska 2008).Truenectar(i.e.asugar-rich,floralsecre-

tionattractivetopollinators)isuncommoninBulbophyllum(pe rsonalobservation).Indeed,bynow,almostacenturyhaspassed sincePohl(1935)reportedtheoccurrenceofasugar-andoil- richsecretionatthebaseofthelabellumandadjacentflowerparts ofBulbophyllumlobbiiLindl.

(sect.Sestochilos(Breda)Benth.&Hook.f.)andBulbophyllu mmacranthumLindl.

(sect.StenochilusJ.J.Sm.),yetsincethen,relativelyfewstu dieshaveconsideredfood-

rewardsinBulbophyllum.Fieldstudieshavereportedfood- rewardsinanumberofspe-

ciessuchasBulbophyllumalticolaSchltr.

(sect.HapalochilusSchltr.),Bulbophyllumauratum(Lindl.) Rchb.f.

(sect.RecurvaeBenth.&Hook.f.),B.lobbiiandB.macranthu m(Pohl1935;Jongejan1994;vanderCingel2001),butana- tomicalstudiesoftheflowerwithultrastructuralandhisto- chemicalinvestigationsofthesecretoryprocessarerare,inpar ticularforAfricanspecies.Todate,thefloralanatomyofreprese ntativesoftheNeotropicalsectionsDidactyle(Lindl.)Cogn.,N apelliiRchb.f.andMicranthaeBarb.Rodr.

(Nunesetal.2014,2015,2017)hasbeenstudiedatSEMandlig htmicroscopylevel,includinghistochemicaltests,andcom- binedhistochemical,micromorphologicalandultrastructurals tudieshavebeenperformedonmembersofAsiansectionsthati ncludeCirrhopetaloidesGaray,Hamer& Siegerist,Cirrhopet alum(Lindl.)Rchb.f.

(Kowalkowskaetal.2015,2016)andRacemosaeBenth.&

Hook.f.

(DaviesandStpiczyńska2014;StpiczyńskaandDavies2016) ,andonAfricansectionsMegacliniumG.A.Fischer&J.J.Ver m.,OreonastesG. A.Fi sc her&J.J.Verm.and Ptiloglossum Lindl.(Stpiczyńskaetal.2015).Ofthese,histochemistryre- vealedthatlipidswereabundantinthelabellarcellsofmostspeci es,exceptforthoseofsect.Racemosae,wherethesecre- tionconsistedpredominantlyofprotein-

ladenmucilage(DaviesandStpiczyńska2014;Stpiczyńskaa ndDavies2016).Inmostcases,thesesecretionswereproduced bypalisade-

likeepidermalcellsliningthemedian,longitudinallabellargro ove.Traditionally,butmisleadingly(consideringtheyhadnotb eenshowntoproduceasugaryliquid),thesegrooveshaveoften beenreferredtoasnectaries.Oneexcep-

tion,however,isBulbophyllumschinzianumKraenzl.

(sect.Ptiloglossum),whosefloralsecretion,onsubjectingtorefr ac-tometry,gaveatotalsugarvalueof61.7%

(Stpiczyńskaetal.2015),stronglyindicating,giventhatit alsoproducesfra-

grance,thatitisnotpseudocopulatory,ashadpreviouslybeenpro posed(Jongejan1994;vanderCingel2001).Indeed,basedont

heformanddepthofthelabellargrooveandthepresenceoffragra nceandnectar,itispossiblethatthisspeciesispollinatedbyHymen optera(Stpiczyńskaetal.2015),espe-

ciallyastheseinsectshavebeenreportedtovisitsuchflowers(Joha nsson1974).Conversely,Stewartetal.(2014)reported

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NectarandoleiferoustrichomesasfloralattractantsinBulbophyllumsaltatoriumLindl.(Orchidaceae) 56 thatflowersofB.schinzianumvar.schinzianumarealsovis- 7

itedbyflies,andtherefore,furtherfieldstudiesarerequired.

Here,weextendourinvestigationstoafurtherAfrica nspecies,namelyBulbophyllumsaltatoriumLindl.Thehairy flowersofthisspecies,insomerespects,resemblethoseof B.schinzianum,andbotharemembersofthesamesection,na mely,Ptiloglossum.Preliminarystudies,however,indicatedt hatbothspeciesdifferinthefloralrewardsthattheyproduce.Th eaimofourpresentpaper,therefore,istoinvestigatethemicro morphologyandhistochemistryofthehirsutelabellumofB.s altatorium,tocheckforthepresenceoffloralfood-

rewardsandtodescribethetissuesinvolvedintheirproduc tion.

Materialandmethod s

TheplantsofBulbophyllumsaltatoriumusedinthisstudy werecultivatedattheBotanicGardenoftheJagiellonianUniv ersityinKraków(livingcollectionsaccessionnumberO/20 14/1185).VouchermaterialwasdepositedattheHerbariu mofJagiellonianUniversityinKraków(accessionnumberK RA464137).

Microscopyinvestigatio ns

Inthepresentstudy,weemployedmicroscopytechniques describedindetailbyStpiczyńskaandDavies(2016,andref erencestherein).Inordertodeterminethepresenceofse- cretorytissuesandsurfacesecretions,intactflowerswereim- mersedinsolutionsofthefollowingstains:SudanIIIforlipid s;rutheniumred(RR)formucilage;andCoomassieBrilliant BlueR250(CBB)forproteins.Thestainedpartsofflowerswer eexaminedbymeansofaNikonSZM1000ste-

reoscopicmicroscope.Sectionsofsecretorytissuesfrompro ximalandmedianpartsofthelabellumweresubsequentlyexa minedusingbrightfieldandfluorescencelightmicroscopy(L M).Semi-

thinsectionswerestainedusingmethyleneblue/azureII(MB /AII)forgeneralhistologyandtheperiodicacid-

Schiffreaction(PAS)forinsolublepolysaccharides.Hand- cutsectionsofbothfreshandfixedmaterialwerestainedwithS udanIII,CBB,Lugol’siodinesolution(IKI),andRRforlipids, proteins,starchandmucilage,respectively.Furthermore,a nepifluorescencemicroscopeequippedwithPrior200Wlam p(PriorScientificInstrumentsLtd.)andUV-

2Bfilterwasemployedfortheinvestigationofcellwalls,andin particular,thecuticle,inunstainedmaterialfixedin70%eth anol,sinceobservationsofcellslocateddeepinthelabellargro oveweredifficultusingSEM.Thestructureofthecuticlewasf urtherinvestigatedbystainingparadermalsec-

tionswithauramineO(Ruzin1999)andviewingthemusingblue light(FITC-Nikoncubefilter).

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Forscanningelectronmicroscopy(SEM),partsofthela- bellumweredehydratedandsubjectedtocritical-

pointdryingusingliquidCO2.Theywerethensputter- coatedwithgoldandexaminedusingaHitachiS- 4700scanningelectronmi-

croscope(Hitachi,Tokyo,Japan),atanacceleratingvoltageof20 kV.

Piecesoflabellumfrombothproximalandmedianpartswer efixedin2.5%(v/v)glutaraldehyde/4%(v/v)formalde- hydein0.1Msodiumcacodylatebuffer(pH7.0),washedinthesa mebufferandpost-fixedin1.5%

(w/v)osmiumtetroxidesolution.Theyweredehydratedusinga gradedacetoneseriesandembeddedbymeansofaSpurrEmbed dingKit(Sigma).Followingpolymerization,sectionswerec utat70nmfortransmissionelectronmicroscopy(TEM)using aLKBultra-

microtome,stainedwithuranylacetateandleadcitrate(Reyn olds1963),andexaminedusingaJEM1400(JEOLCo.,Japa n,2008)transmissionelectronmicroscope,atanac-

celeratingvoltageof80kV.

Micrometryandphotomicrographywereaccomplishedusi ngNIS-ElementsBRsoftwareandDS-

Fi1digitalcamera(Nikon,Japan),andahigh- resolutiondigitalcamera(CCDMORADA,SiS-

Olympus,Germany)forLMandTEMim-ages,respectively.

Results

Bulbophyllumsaltatoriumflowersareborneonasimplera- ceme,andeach issubtendedbyaprominentpaperybract(

Fig.1a).Thebasesofthesepalsandpetals(includingthelabell um)aremoredarklypigmentedthantheremainingparts,andthe flowershaveafaint,sweetfragrance.

Thelabellumisdenselyhirsute(Fig.1a–d).Thedistribu- tionandsurfacefeaturesofthevariouskindsoflabellartri- chomesareshownintheSEMimages(Fig.2a–

f).Thelongesttrichomes,whichmeasureasmuchas3.5m minlength×18μmindiameter,arepresentonthedistal,abaxials urfaceofthelabellum(Figs.1a,band2a).Thesetrichomesareuni cellularwithcellulosiccellwallsandanirregularlyorhelicallysc ulpturedcuticle(Fig.2d).Intheparietalcyto-

plasm,smallplastidsoccur,andthevacuolescontainantho- cyanins.Thesetrichomesdonotcontainanystoragematerials.

Marginaltrichomesformadenseborderorrimaroundthelab ellum.Theyareshorterandnarrowerthanthosedescribedprevi ously,measuringonlysome300μminlength ×10μmindiamete r,andhaveacuteapices(Figs.1cand2a,c,e).Thecuticleoverlyi ngthecellulosiccellwallsissmoothandofregularthickness.

Likethelongtrichomes,thesehairsdonotaccumulateanysubst antialstoragematerials.Adaxially,ontheproximalpartofthe labellum,andespeciallynearthetwoproximallabellarproject ions,massivecapitatetrichomesarepresent,measuring450μm

inlength ×30μmindiameter,withcapitatetips(Figs.1dand2e,f).

Thecuticleoverlyingthe

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trichomeisirregularlystriate,withhorizontallyorientatedstri -

aelocatedclosetotheapex,whichissmooth(Fig.2f)and,asvie wedusingSEM,lacksporesandcracks.Thesetrichomesconta inlargelipiddroplets(Fig.1c,d).Similarlipiddropletsalsoocc urintheepidermalandsubepidermalparenchymacells(F ig.1e–

h).Neithertheintravacuolarcontentsofthethreetypesof trichomementionedpreviously,northoseoftheadaxialepid ermalcellsdisplayedspecificautofluores-

cencewhenexposedtoUVlight.

Themedianpartofthelabellumwascoatedwithsweet- tastingnectar.UnderSEM,residuesofnectarwerevisibleasare ticulum,orasafinefibrousweftoverlyingshorttrichomesandc onicalpapillaedistributedalongsidethemedian,deep,longit udinalgroove,andthismaterialwasparticularlyabun- dantuponthecallus(Fig.2g,h).Marginalpartsofthelabel- lumwereglabrousandlackednectar.Transversesectionsofth elabellumrevealedthatthemedian,longitudinalgrooveofthel abellumwaslinedwithepidermalcellsofmeandimen- sions18.7×12.5μm.These werenotpalisade-

like,asinmanyotherBulbophyllumspp.,buteachcontaine dalargenucleusanddensecytoplasm(Fig.3a).Thesubepider malpa-

renchymacomprisedseverallayersofcompactlyarranged,s mallisodiametriccells.Adjacentepidermalcells(located morelaterally)werelarger,havingmeandimensionsof 23.3×30.5μm,andcontainedthinparietalcytoplasmanda largevacuole.Deeplylocatedparenchymacellswerelarge,w ithextensiveintercellularspaces,andresembledspongyme- sophyll(Fig.3b).Occasionally,largeidioblastswithraphides alsooccurinthisregion.Generally,starchwaslackingfromepi dermalcells(thoseliningthemediangroove,aswellasthoset hataremoremarginallylocated),butwaspresentinparenchy ma(Fig.3b,c),particularlyinthevicinityofvascularbundles.

Sectionsofthelabellumthroughthemedianlongitudinal grooveregiontestedwithRR(Fig.3d),andSudanIII,re- vealedthatmucilageandlipids,respectively,wereabsentfro mthesurfacesecretion.TestingwithCBBalsoshowedthatthe surfacesecretionlackedproteins.Stainingsectionswithaura mineO(Fig.3a)andobservationsmadeusingUVlight(Fig.3f) revealedanintactcuticlewithnovisibleporesorcracks.Ho wever,cellwallsstainedfollowingtreatmentwithRR,andthe densecytoplasmofsecretorycellsstainedwithCBB.

Vacuolarcontentsofsubepidermalparenchymaau tofluorescedlightbluewhensubjectedtoUVlight(Fig.3f), whereasstainingwithauramineOrevealedthepresenceofmin utelipiddroplets(probablyplastoglobuli)inepidermalandsub epidermalparenchymacells.

Transmissionelectronmicroscopyofadaxialepidermalc ellsfromtheproximalpartofthelabellumrevealedaprom- inent,centrallylocatednucleusanddensecytoplasmcontain- ingarelativelylargevacuole(Fig.4a).Lipidcompletelyfilledb oththisandnumeroussmallervacuoles,whereassomesmall

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Fig.1Grossfloralmorphologyand labellaranatomy,LM.aHabitofflo wer,scalebar=1mm.bWholelabel lumstainedwithSudanIII.Thepos itionofnectarygroove(visiblethro ughtheabaxialsurface)ismarkedb yanarrow,scalebar=1mm.cShort, pointedtrichomesandbasesofmas sivetrichomescontaininglipiddro plets(arrows)followingstaining withSudanIII,scale

bar=50μm.dMassivecapitate trichomescontaininglipiddrop- letsstainedwithSudanIII,scalebar

=20μm.eAsimilarprepa- rationshowingthepresenceoflipid sintheepidermalandparen- chymacellsoftheproximalpartoft helabellum,scale

bar=50μm.fSectionstainedwith MB/ABshowinglarge,graylipidd ropletsinepidermalcells,scalebar

=10μm.gSparse,minutestarchgra ins(PAS)andthincellulosiccellw allsoccurinthelipid-

secretingregion.Notegraylipiddr oplets,scale

bar=20μm.hParadermalsec- tionstainedwithauramineO.

Notetheabundant,fluorescent greenlipiddroplets,scale bar=10μm.Rraphides

vacuolesandvesiclesscatteredthroughoutthecytoplasmconta inedelectron-densematerial(Fig.4a–

c).Thecytoplasmcontainedabundantsmooth(SER)andro ughendoplasmicreticulum(RER),andcloseexaminatio nshowedthatbothSERandRERproducedsmallvesicles bybudding.Dictyosomeswerealsofrequentlyseen.Thenume rousmito-

chondriaweremainlylocatedintheparietalcytoplasm(Fig.4a, d).Plastidswererarelyobserved,butwhenpresent,theycontain edanelectron-

densestroma,fewinternaltubulesandplastoglobuli,butstarch wasabsent(Fig.4b–

e).Bycontrast,plastidsinthesubepidermalparenchymacontai nedsmallstarchgrains(Fig.4f).Plasmodesmataconnectedadj

oiningepidermalandsubepidermalcells(Fig.4b,f).Theplasmale m-mawasoftenveryconvoluted,resultingintheformationofan

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extensiveperiplasmicspace.Adistinctivefeatureofepider- malcells,andtoalesserdegreesubepidermalparenchymace lls,wasthepresenceofcellwallingrowths(Fig.4a,b)d istributedfairlyregularlyalongthelengthofthecellwalls.

Thesewerealsopresentinnectar-

secretingcellsliningthemediangroove(Fig.5a–

f).Nectarycellshadthick,outercel-

lulosicwallsandathin,smoothcuticlewithoutvisiblemicro- channels.Thesurfacesecretionthatcollectedonthesurfaceoft hecellwall(Fig.5a,b)wasoftenfibrousorweft-

like,butsometimesithada moresolidappearance.Asinlipid -containingcells,theplasmalemmawasconvoluted(Fig.5a–

f),andtheperiplasmicspaceformedcontainedmanysecreto- ryvesicles(Fig.5b–f).Furthermore,secretoryvesiclesofvar- ioussizesgatheredintheparietalcytoplasm,alongsidethe

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Fig.2Micromorphologyofthelabe llum,SEM.aPartoflabellumshowi ngmedianlongitudinalgrooveand hairycallus.Oneoftwovisiblebasa lprojectionsmarkedbyanarrow.Al sonotetheborderofshorttrichomes

—

blackasterisks,andthelong,margi naltrichomes—

whiteasterisks,scalebar=500μm.

bProximalpartoflabellumwithtw oprojections(arrows),scalebar=1 50μm.cShorttrichomes(blackaste risk)andsmoothcuticle,andlongm arginaltrichomes(whiteasterisk)o fthelabellum,scale

bar=60μm.dDetailofthestriatecut icleoflongtrichomesfromapicalp artofthelabellum.Notethehelical arrangementofcuticularstriae,sca lebar=20μm.eGlabrousproximal, adaxialsurfaceoflabellumwithri mofmassive,capitate,oleiferoustr ichomes(arrows).Marginalrimof shorttrichomesmarkedbyasterisk ,scalebar=200μm.fDetailofswoll enapexofmassive,capitatehair,sc ale

bar=10μm.gMediangroove andhairycalluscoatedwithn ectar(asterisks),scale bar=500μm.hReticulumofne ctarresiduesbetweencallus trichomes,scalebar=15μm

plasmalemma,andfusedwiththelatter(Fig.5d–

f).Abundantdictyosomeswerepresentintheparietalcytoplas m,togetherwithprofilesofSER,RERandmitochondria,aswell assmall,multivesicularbodies(MVBs)

(Fig.5a).Vacuoleshereweresmallandelectron-

translucentorcontainedflocculentmaterial(Fig.5a,c).Theygat heredpredominantlyintheapicalpartsofpapillae.Occasionally ,smallvacuolescontaininglipidwerealsoobserved.Vacuoles presentinthesubepidermalparen-

chymawerelargeandpredominantlyelectron-

translucent,ortheycontainedmembranousorfibrousmaterial .Epidermalandsubepidermalcellswereinterconnectedvianu merousplasmodesmata(Fig.5a–

c),thelatterbeingparticularlydense-

lydistributedontheadjoiningwallsofmesophyll-likeparenchymacells.

Discussion

OfthespeciesofBulbophyllumstudiedtodate,themedi- anlabellargrooveofB.saltatoriummostcloselyresem- blesthatofB.schinzianuminthatitisrelativelydeep,withanarr owaperture,anddenselyflankedbyunicellularpapillaeandtri chomes(Stpiczyńskaetal.2015).Sincethecellsliningthegro ovesecreteasugaryliquid,likethatofB.schinzianum,itcanals obereferredtoasanectary.LikethenectarofB.schinzianum,th atofB.saltatoriumalsolackslipid,proteinandmucilage,ascon firmedbyhistochemicaltests,unlikethatofAsianB.lobbii,wh ichisreportedalsotocontainoil(Porsch1905;Stpiczyńskaetal .unpublished).

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570 M.Stpiczyńskaetal.

Fig.3Nectarsecretingregionofthe labellum,LM.a

Sectionshowingsecretoryepider misliningthemediangrooveandu nderlyingsubepidermalparenchy ma(MB/AB),secretoryresidues markedbyanasterisk,scalebar=2 0μm.bEpidermisandsubepiderm alparenchymainthemarginalpart ofthelabellum.Starchispresentint hemesophyll-

likeparenchymacells(PAS),scale bar=20μm.cSection(asina)staine dwithPASrevealstheabsenceofst arch,butrelativelythickcellulosic tangen-

tialcellwallsstainwiththisre- agent,scalebar=20μm.dStaining withRRrevealstheab-

senceofmucilagefromidioblastsa ndthesurfacesecretion,scalebar=

20μm.eStainingwithauramineOr evealsanintactcu-

ticleoverlyingthenectary,scaleba r=10μm.fSectionofcallusregionv iewedwithUV.Noteau- tofluorescenceofvacuolarcon- tentsofsubepidermalcells,scaleb ar=20μm.Ididioblast

ThesurfacesecretionofB.saltatorium(basedonSEMandT EMobservations),likethatofB.schinzianum(basedonTEM), isheterogeneousandhasafibrouscomponent.UnlikeB.schin zianum,however,wherethegrooveislinedwithpalisade- likecells,thatofB.saltatoriumislinedwithmoreorlessisodia metriccells,indicating,perhaps,that

B.schinzianumdisplaysagreaterdegreeofsecretorytissueorg anization.Remarkably,thecellulosiccellwallsofthelabellum ofB.saltatoriumhavepronouncedwallingrowthsthatareabse ntfromthoseofB.schinzianum.Cellwallpro-

tuberancesarefrequentlypresentinvarioustypesofspe- cializedplantsecretorycells(transfercells)engagedinhighrat esofsolutetransport(Offleretal.2002).Theyhavealsobeenre portedforthesecretorytissuesofsomeorchidflowers,sucha selaiophorecellsofZygostatesgrandiflora(Lindl.)Mansf.

(subtribeOncidiinae;Ornithocephalusclade—

Paceketal.2012),theepidermalcellsofpetalsandthelabellar grooveofBulbophyllumweberiAmes,whosecytoplasmand surfacesecretioncontainlipids,pro-

teinsandpectinacids/mucilage(Kowalkowskaetal.2016),as wellasthenectariesofEpipactisatropurpureaRaf.

(PaisandFigueiredo1994).InB.saltatorium,cellwallingrowt hswerepresentbothincellsthataccumulatelipids,andin

nectar-

secretingcells,indicatingthattheyarebothinvolvedinactivese cretion.Transportofsecretionacrosstheplas-

malemmaofsecretorycellswithcellwallingrowthscanoccur eitherbyeccrinesecretion(Płachnoetal.2017)orbygranulocri nesecretion,wherenumeroussecretoryvesi-

clescongregateneartoandfusewiththeplasmalemma(Nepi2 007).InB.saltatorium,thecytoplasmofnectarycellscontaine dabundantvesiclesderivedfromdictyosomesandER(GERL orGolgi–EndoplasmicReticulum–

Lysosomesystem),andthesefusedwiththeplasmalemma.Sm allvesicleswerealsopresentintheperiplasmicspace,andcellw allingrowthssubstantiallyenlargethesurfaceareaoftheplas malemma.Numerousdictyosomesanddictyosome- orGolgi-

derivedvesiclesarealsoacommonfeatureofnectarycells,an doccurinorchids,suchasPlatantheraRich.

(Stpiczyńska1997)andAeridinae(Stpiczyńskaetal.2011) .Incontrasttonectarycells,SERprofilespredominateincellsth ataccumulateoils,andthesearefrequentlylocatedclosetoplast ids,asthosethatoccurinotherlipid-

producingcells,suchasthoseoftheelaiophoresofOrnithoph oraradicans(Rchb.f.)Garay& Pabst(StpiczyńskaandDa

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NectarandoleiferoustrichomesasfloralattractantsinBulbophyllumsaltatoriumLindl.(Orchidaceae) 57 vies2008),Z.grandiflora(Paceketal.2012)andLockhartia 1

Hook.(Blancoetal.2013).

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Fig.4Ultrastructureofoil- secretingcells,TEM.aEpidermala ndsubepidermalparenchymacells .Largevacuolecontainslip- id;thepresenceofcellwallin- growthsinepidermalcellmarkedb yarrows.Mitochondriaoccurinthe parietalcytoplasm,scale bar=5μm.bDetailofepidermal cellwithlargevacuoleandsmallve sicles,bothcontaininglipids.Num erousERprofilesoccurinthecytop lasm.Cellwallin-

growthsaremarkedbyarrows,scal ebar=2μm.cDetailofse- cretoryvesicles,someofwhichcon tainelectron-

densebodies,scalebar=1μm.dSta rchlessplastidswithdensestromaa ndinternalmembranes,scale bar=1μm.eDetailofparietalcytop lasmshowingRERandplastidwit hplastoglobuli,scalebar=0.5μm.f Subepidermalpa-

renchymacellswithplastidsenclo singfew,minutestarchgrains,scal ebar=2μm.Llipid,Pplastid

ThecellwallsofthesecretorycellsofB.saltatorium(par- ticularlythepericlinalcellwallsintheregionofthenectary)aret hickandcellulosic,resemblingthosereportedforthenectar iesofotherdistantlyrelatedorchidtaxa,suchasMaxillariaRui z&Pav.orHexiseaLindl.

(Stpiczyńskaetal.2004,2005,respectively),severalspecieso fAeridinae(Stpiczyńskaetal.2011)andBrassavolaR.Br.

(Stpiczyńskaetal.2010).Thickcellulosiccellwallsinthenectar yofsomeepiphyticorchidsprobablyserveasaroutefornectartra nsportontothenectarysurface.Sincenectarisfundamen tallyanaqueoussugarysolution,itmaybetransportedwithi nthepolysaccharidematrixthatconstitutesthecellwallandisth usprobablybetterprotectedagainstevaporation,especiallysin cedesiccationisa keystressfactorforepiphyticplants.Ther efore,thepresenceofthickcellulosiccellwallsinunre-

latedepiphyticorchidsisprobablyduetoconvergence.Despit ethis,thecuticlecoveringthethickoutercellwallsof

B.saltatoriumisverythin,lackingthetypicalcuticularblis tersandmicro-

channelsseeninB.schinzianum(Stpiczyńskaetal.2015)and someotherAfricanandAsianspeciesofBulbophylluminvesti gatedtodate(DaviesandStpiczyńska2014;Stpiczyńskaetal.

2015;Kowalkowska

etal.2015,2016).Therefore,bycomparison,thethincuticleofB.

saltatoriumappearstobemorepermeablethanthatof B.schinzianum,andthismayaidnectarsecretion.

Again,inbothB.saltatoriumandB.schinzianum,theun- derlyingsubepidermalcellsareisodiametricandofsimilarsize,a ndtheparenchyma,whichcontainscollateralvascularbundlesa ndidioblastswithraphides,resemblesaerialparenchymaorspon gymesophyll(Stpiczyńskaetal.2015,Stpiczynskaetal.unpubli shed).Thepresenceofaerialparenchymainlabellaisnotexclusiv elyfoundinsectionPtiloglossum,butitoccursalsoinotherAfrica n,AsianandNeotropicalBulbophyllumspecies,andaccordingto Teixeiraetal.(2004),itspresenceaidsmove-

mentofthelabellumduringpollination.

Threediversekindsoftrichomewerepresentonthelabel- lumofB.saltatorium:densebutdelicatetrichomeswithsmooth cellwalls;longtrichomeshavingirregularlysculp-

turedcellwalls,andmassivecapitatetrichomesthataccumu- latelargedropletsoflipid.Moreover,unicellulartrichomesan dpapillaecoatthelabellarcallus.Thefirsttwoclassesoftrichom e,byincreasingthesurfaceareaofthelabellum,mayalsobeinvo lvedinfloraldisplayandthus,theattractionofpollinators.

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Fig.5Ultrastructureofcellsfromn ectaryregion,TEM.aEpidermalc ellswiththick,outertangentialwal landthinradialwalls,interconnect edbyplasmodesmata.Surfacesecr etion(nectarresidue)markedbyan asterisk,scalebar=5μm.bDetailof outercellwallwiththincuticleand parietalcytoplasm.Noteconvolut edoutlineofplasmalemmaandves iclesinperiplasmicspace;surfaces ecretionmarkedbyanasterisk,scal ebar=2μm.cParietalcytoplasmwi thnumerousvesicles,ERanddicty osomes.Cellwallshowssmallingr owths(arrows),scalebar=2μm.d Electron-

denseparietalcytoplasmdemonst ratingtheGERLsystemcomprisin gdictyosomesorGolgibodies,ER profilesandthenu-

meroussmallvacuolesandsecre- toryvesiclesderivedfromit,scaleb ar=1μm.eSmallvacuolesandvesi clescontainingosmiophilicmateri al.Numerousdictyosomesoccuri ntheparietalcytoplasm,scalebar=

1μm.fParietalcyto-

plasmwithdictyosomes,ERpro- filesandsecretoryvesicles.Notec ellwallingrowths(arrows)withlo osearrangementofcellulosicmicr ofibrils,scalebar=1μm.Mbmultiv esicularbody

ThemassivetrichomesofB.saltatoriumareunusualinse veralways.Theycontainlargedropletsoflipid,yetthismate rialisabsentfromthelabellarsurfacesecretion.Therefore,it ispossiblethatthesecellsareengagedinscentproductionsince ,inosmophores,thesurfacesecretion(fragrance)tendstovol atilizerapidlyandthusdisappear.Starchaccumulation,howev er,whichistypicalofosmophorecells(Antońetal.2012,andrefe rencestherein),wasnotob-

servedhere,butthisisnotsurprising,sincethestarchmayhave beenusedasasourceofenergyforthehighlymetabolicprocessof fragranceproduction.Unliketheepidermalcellsofmanyorchid sthatproducebothfragranceandfood-

rewards,here,thereisspecializationresultingindivisionoflab orcoupledwithstructuralmodification,withtrichomesproduc- inglipidmaterials,whereasthecellsliningthegroovesecretene ctar.Althoughthemassivetrichomesareladenwithlipids,theyd ifferfromtheelaiophoretrichomesoforchidsinvesti-

gatedsofar(Paceketal.2012;Blancoetal.2013)inthatsurface secretionisabsent.Todate,theproductionofasurfacelipid- richsecretionbyBulbophyllumhasbeeninvestigatedforanum berofspecies(DaviesandStpiczyńska2014;

StpiczyńskaandDavies2016,andreferencestherein;Kow alkowskaetal.2015,2016),butoleiferoustrichomeswer enotpresentinanyofthesetaxa.Alternatively,therealsoremain sthepossibilitythattheoilfromsuchtrichomes,inAfricansp ecies,mightbegatheredbyspecializedoil-

collectingbees(vanderCingel2001),suchasspeciesofRedivi va(Melittidae).However,atpresent,thereisnoevi-

denceforthisinBulbophyllum.

Conclusion

InconsideringthespeciesofBulbophylluminvestigatedtodat e,itispossibletoidentifyseveralmaingradesoflabellarorganiz ation.Thesediffermainlyinthetypeofreceptacleinvolve dinthesecretionandpresentationoffood-

rewards,rangingfromasimple,shallowdepressiontoamorehi ghlyorganized,deeplabellargroovethatisabletosecretelipid- richfood-rewards,protein-

ladenmucilageor,asinB.schinzianumandB.saltatorium,nectar .Betweentheseextremesoccursanintermediategradecomprisi ngashallowandwideconcavity,

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asfoundinB.lobbiiandB.macranthum(Stpiczyńskaetal.unpu blished).Bulbophyllumlobbii,whichisfragrant,nectarife rousandfly-

pollinated,isconsideredbyChristensen(1994)tohavereta inedseveralfeaturescharac-teristicofbee-pollinatedspecies.

Thegrossmorphologyandrangeofelaborateandhighlyspe cificpollinationstrategiesisalreadywellknownforasmallper centageofBulbophyllumspecies,butwithmanystillbeingdisco vered,andsomanymoreremainingtobeinvesti-

gatedfully,wecanonlyspeculateaboutthefulldiversityoftheg enus,notjustaboutthefloralmorphologyofitsmem-

bers,butalsohowtheymightdifferatthemicromorphologi- cal,anatomical,ultrastructuralandbiochemicallevels,andthes ignificanceofeachoftheseoftenslightandcryptic,butnone- thelessveryrealdifferenceswithregardtopollinatorselectionan dtotheevolutionandsurvivaloftheseremarkableplants.

AcknowledgmentsTransmissionel e ct r onmic ro scopystud ieswere performedattheLaboratoryofElectronMicroscopy,NenckiInstituteof ExperimentalBiology,Warsaw,Poland.TEMfacilitieswereinstalledfort heprojectandsponsoredbytheEUStructuralFunds(SPOWWKP_1/1.4.3/

2/2005/102/222/562/U).SEMstudieswereundertakenattheScanning MicroscopyLaboratoryoftheDepartmentofBiologicalandGeologicalSc iences,JagiellonianUniversityinKraków.LMmicroscopeequipmentuse dinthisprojectwasfundedbytheNationalScienceCentrePoland,grantNo2 014/15/B/NZ8/00210toMS.

Compliancewithethicalstandards

Conflicto f in te restT h e au th o rsd e cl aret h a t th is wo r k p r es entsn o conflictofinterest.

OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo m mo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativecommo ns.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andr eproductioninanymedium,providedyougiveappro-

priatecredittotheoriginalauthor(s)andthesource,providealinktotheCreat iveCommonslicense,andindicateifchangesweremade.

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