Onsilverwings:afragilestructuralmechanismincreasesplumage conspicuousness

IsmaelGalva´n,Juan J. Negro,GaryR.Bortolotti andAntoni Margalida

I. Galva´n (correspondence), Dept. of Evol.Ecol., Museo Nacionalde CienciasNaturales (CSIC), Jose´ Gutie´rrez Abascal2, E-28006Madrid,Spain,E-mail: — J.J.Negro,Dept.of Evol.Ecol.,Estacio´n Biol.deDon˜ana(CSIC),Avda. AmericoVespucios/n,E-41092Sevilla,Spain— G.R.Bortolotti,Dept.ofBiol., Univ.ofSaskatchewan, 112SciencePl,SaskatoonSKS7N

5E2,Canada—A. Margalida, BeardedVultureStud.andProt.Gr.,Apdo.43,E-25520ElPontdeSuert(Lleida),Spain—Presentaddressof

I.G.:Dept.ofEcol., Univ.deAlcala´,EdificiodeCiencias,Ctra.deBarcelonaKm.33,600,E-28871Alcala´deHenares,Spain.

Wereportforthefirsttimetheexistenceofastructuralmechanismoffeathersdifferentfromiridescencethatmakes plumageconspicuous.Byusingelectronandlightmicroscopy,weshowthatthemechanismconsistsofspeciallengthened andtwisteddistalbarbulesthatareverysusceptibletodamage.Thedorsalsideofthesebarbulesistranslucent,which createsadistinctivesheencolourationtofeathersthatotherwisewouldbe dark.Whendistalsheenbarbulesarebroken, theblackproximalbarbulesareexposed,thusgeneratingaconspicuousdifferencebetweenabraded andnon-abraded areas.Totalandultravioletreflectanceofsheen (non-abraded)areasarestrikinglyhigherthaninabradedareas.We proposethatthismechanismrepresents acaseofconvergentevolutioninspeciesthatarelimitedindevelopingcolourful plumagepatches.Futurestudiesshouldexplorethepotentialofthiscolourmechanismtoactasasignalofindividual qualityoridentity.

Highlyreflectivesilverystructures standout fromtheir surroundings, ascanbeseenfromthenighttimeshinefrom theeyesofcatsinthebeamofaflashlightandthebright silvergleamofbaitfishastheytwistandturntoavoida predator. The brightnessof a silvery,reflectivesurface allowsforbothextremeconspicuousnessandvisibilityfrom aconsiderabledistance.Whilewethusmight expectthisto beexploitedbyanimalsforcommunicationorsomeother function,examplesarerelativelyfew.Silverystructureshave beendescribedin theexoskeletonofsomeinvertebrates (e.g.,silverfishinsectsLepismasaccharina) andinthescaly integumentofsomevertebrates(fish;Parker2005).How- ever,therearefewexamples of silvery structuresin terrestrialvertebrates,withtheexceptionofthereflective eyesofanimalswithtapetumlucidum,atissuelayerinthe eyethatreflectsthelightbackthroughtheretina.Here wepropose thatcertainmodifiedandsheen coloured featherspresentinseveralaviantaxahavepropertiesthat are similarto reflectivesurfaces:when illuminatedby sunlight,perceivedbrightnessinthesefeathersisenhanced comparedtomoretypicalfeathers(Fig.1).Thisisanovel typeofstructuralcolourationthatappearstohavebeen overlookedeventhough the production mechanisms of structuralcolourin the integumentofbirdshavebeen extensivelyandintensivelyinvestigatedandreviewedinthe pastcentury(LucasandStettenheim1972,Fox1976),and manyrecentadvancesontheirphysicsandevolutionhave beenpublished(Prum2006).Sheenfeathersdifferfrom

those with iridescence,definedasthe phenomenon of glitteringofdifferentcoloursthatchangeaccordingtothe anglewithwhichtheobjectisseenorilluminated(Lucas andStettenheim1972,Prum2006).Iridescentstructural coloursareproducedbycoherentscatteringfromarraysof melaningranules (Prum2006).Thecolourofsheen feathersalsodiffersfrombrightwhite,whichisproduced bylargeairvacuolesinthebarbulesoffeathers,as described, forinstance,byDyck(1976)forthewinterplumageofthe rock ptarmiganLagopusmuta.

Our aim is to describethe structure and optical propertiesofsheenfeatherstostimulatefutureworkon

the ecologyand evolutionof this overlookedtype of plumagecolouration.Wefocusonthenoticeableexample

ofsheenstructureonthedorsalsurfaceofadultbearded

vultures Gypaetusbarbatus, a species whose plumage characteristicssome of us havethoroughlystudied, as thesebirds applycosmeticsoilstotheirfeathers(Negro

etal.1999,2002)andshowfine-tunedplumageadapta-

tions(Margalidaetal.2008).

Methods

Juvenile beardedvulturessportamattedarkbrown plumagesimilartothatofotherOldWorldvulturesand most other birds of prey (order Falconiformes).The numberofsheenfeathers increasesannuallywithmoult

Figure1.Examplesofbirdsshowingfeatherswithsheenstructures.(a)Adultbeardedvulture, (b)Immature(left),andadult(right) beardedvulturesinflight.Notethebrightappearanceoftheadultbirdcomparedwiththeimmatureevenonasnowy(i.e.dark)day, (c)Adult,whitemorphred-footedboobyinflight.Notehow thesheenstructuresofflightfeathersshinemuchmoreintheleftwing, whichisundersunnylight,thanin therightwing,whichisintheshadow.(d)AdultAndeancondorVulturgryphusshowing conspicuous whitishfeathersonthewing,(e)AdultEgyptianvultureNeophronpercnopterusshowingsheenstructuresonremigesthatgivethema whitishappearance.(f)AdultmarabouLeptoptiloscrumeniferus.Thewhitelinesonsecondarycoverts,whichacquirethatcolourbecause ofsheenstructures,whenfresharevisibleallalongthewing.

(seephotosinAdamsandLlopisDell2003),suchthat adultbeardedvultures oversixyearsoldhaveabright sheenappearanceontheupperparts(Fig.1a),fromheadto tail,andonremigesandrectricesparticularlywhenflying (Fig.1b).

Weusedlight(LM),andscanningelectronmicroscopes

(SEM)todescribethemorphological characteristicsofthe sheenfeathers.Wealsotookphotographsofthesestructures

withtheaimofillustrating themorphologicalcharacter-

isticsoffeathersthatgeneratethesheeneffect.Toobtain SEMimages oftheultrastructureoffeathers,wecut portions offeathers atdifferentpointsthat appearedto theunaidedeyeashavingdifferentlevelsofabrasion.The sampleswereanalysedwithaFEIQUANTA200SEM

operatinginlowvacuummodeand25kVandusinga

wavelengthdispersiveX-raydetector(WDS).Thesamples weremountedonstainless-steelpegs byusingdouble- sidedgraphitetapebeforebeingtransferredtotheSEM chamber.ThelightmicroscopewasanOlympusBX51with

amountedDP70cameraforimagecapture.

Tocharacterizetheopticalpropertiesofsheenfeathers, weusedan OceanOpticsUSB2000spectrophotometer

(range 250—800 nm) with ultraviolet (deuterium) and

visible(tungsten-halogen)lamps and a bifurcated400 micrometerfiber-optic probe(Dunedin,Florida).The fibre-opticprobeprovidedbothilluminationandobtained lightreflectedfromthesample,providingareadingarea ofc.1mm2.Themeasurementsweretakenata908angle to feathersamples.Allmeasurements wererelative to a white‘‘Spectralon’’ tablet(WS-1-SS,Dunedin, Florida). Measurementsweretakenatdifferentpointsoffeathers differinginthelevelofabrasion(seeFig.4),exceptfor

theabrasionexperiment (seebelow)forwhichthree measurementsweretakenat the samepoint, removing theprobeaftereachmeasure.Toconfirmthattheoptical propertiescanbeenvironmentallymodified,weexperi- mentallyremovedthesheenstructurein thedistalarea ofabeardedvulturefeatherbylightlyscrapingthesurface withthebladeofapairofscissors,andthenmeasuredthe reflectancespectrumatthesamepoint afterwards.Asa control,weperformedthesameexperimentalmanipula- tion on thedistalblackareaofaprimaryfeatherofa EuropeanmagpiePicapicaasthisspeciesdoesnotpresent sheenstructuresonitsfeathers.

Inadditiontotheresultsobtainedwiththemethods describedaboveforthebeardedvulture,wealsodescribe

thesheenstructureobservedinotherspecies suchasthe snowgooseAnser caerulescens and thered-footedbooby Sulasula,asthisstructurecan beeasilydetectedwithouta

microscopebecauseat closerangetheyhavea unique

‘dusty’or‘fuzzy’appearance thatisperceivedasgreyishor whitish.

Results

Structure ofsheenfeathers

ObservationsunderLMand SEM(Fig.3) ofsheenfeathers of bearded vulturesrevealedthat distal barbuleswere flattenedand twisted,in asimilarwayasdescribedin someiridescentfeathers(Lucasand Stettenheim1972, BrinkandvanderBerg2004),exceptthatdistalbarbules responsiblefor iridescencecontainedlargeamounts of

Figure2.Detailedviewsofsheenstructuresinfeathers.(a)Primaryfeatherofanadultbeardedvulture.(b)Flightandcovertfeathersof anadultbrownmorphred-footedbooby.Inthisspecies,variationintheregularityofthepatterngeneratedbysheenbarbulesamong feathersisduetowear,whichallowsonetoidentifyfeathersofdifferentages(indicatedbyarrows).

melaninwhereassheenbarbulesweretranslucent.Proximal barbules,however,wereflatand dark,astheycontain melaningranules.

Sheenfeathersofthebeardedvulturewerespeckledwith darkspotsinan apparentlyhaphazardfashion(Fig.2a).All individualsexamined(i.e.,15captiveand20free-ranging

individuals)showedthesespots,andatleastsomeofthe spotswerepresentin growingfeathers.The darkspots dottingsheenfeathersareplaceswheredistalbarbuleswere broken, revealingdark proximalbarbules of the barb

underneath.

The tip ofthedistalbarbule(i.e.thepennulum)insheen feathersis lengthenedwith respectto a ‘conventional’

barbule,sothatitswhitish dorsalsideisexposedalonga greaterdistancefrom the point at whichit istwisted (Fig.3; seeShawkeyet al.2005 for additionalexam-

plesshowing theultrastructureofconventionalfeathers). Sheenfeathersarealsodifferentcomparedtootherfeather typeswithlengthenedbutnottwistedbarbules(e.g.owls;

Bachmannetal.2007).Thislengtheningandtwistingof distalbarbulescausesthem tobreakat thepointwherethey twistbecausetheycannotgetcaughtonthebarbulesofthe adjacentbarbsfromthat point on, indicatingthat this

morphologyprobablyincreasesthelikelihoodoffeather abrasion(Figs.2and3a,b,Fitzpatrick1998).Asforthe darkfeatherspots,theirsizedependsonthenumberof

adjacentdistalbarbulesthatarebroken(Fig.3a).

Opticalpropertiesofsheenfeathers

Whenwemeasuredthereflectance ofabeardedvulture featheratapointwherenobrokenbarbuleswereobserved (i.e.thesheencolourwasuniform),theresultingreflectance spectrumshowedadefinedpeakintheultraviolet(UV) range,followedbyadecreaseandasubsequentincreasein reflectance atlongerwavelengths(Fig.4a).Thespectrum obtainedforthesampletakenatapoint wheresomebroken barbuleswere observedshowedasimilarshapebutaslightly lowerUVpeak. However,thespectrumforapointatthe darkestpartofthefeather,withahighernumber ofbroken barbules,alsoshowedalowerUVpeakbutwithconsider- ablyhigherand increasingreflectancevalues at longer wavelengths, similarto the typicalreflectancespectrum

offeatherswithmelanin-basedcolours (McGraw2006). Similarresults wereobtainedwhenabeardedvulture featherwasexperimentallyabraded(Fig.4c). The fact that differences in UVreflectancebetweenabradedand non-abradedparts of beardedvulturefeathersis more markedafteranexperimentalabrasionthaninanaturally abradedfeather,indicatesthatdifferences betweencurves obtained(fromabradedandnon-abradedpoints)couldbe observedonlyaftertheexperimentalabrasionoffeathers (Fig.4c).

Wear-induceddamagewasalsoevidentintheplumage

oftheotherspecieswithsheenfeathersthatwereexamined (Fig.2b).Indeed,differencesinshapeofspectrabetween abraded and non-abraded parts of featherswereeven moremarkedinthesnowgoosefeather(Fig.4b)thanin thebeardedvulturefeather(Fig.4a).Therefore,itappears

thatforsomespecies, physicalabrasionshavetobevery severe before UVreflectancechangeswiththedisappear- anceof the silverysheen,or, alternatively, the silvery sheen isassociatedwithbutnotfullycoupledtotheUV

reflectanceofthesefeathers.Incontrast,reflectancecurves forabradedandnon-abradedareasofaEuropeanmagpie featheroverlappedalongalargeproportionofthespectral range,and the total reflectanceafterthe experimental

abrasionhadtheoppositeeffectandevenincreasedafter manipulation(Fig.4d).

Speciesthathaveevolvedsheenfeathers

In additionto the speciesusedin thisstudy,wehave observedsheenstructures in severalother species.We confirmedthatthese structureswerethesameasthosein thecaseofthebeardedvulturebyusinglightmicroscopy. Inparticular,wehavenotedthepresenceofthistypeof colourationinpelicans(Pelecaniformes),ducksandgeese (Anseriformes),storksand ibises (Ciconiiformes),New Worldvultures,kites,harriers, Egyptianandbearded vultures (Falconiformes), cranes(Gruiformes)and sand- grouse(Pterocliformes;seeFig.1,videoclips1and2,and Fig.S1—S6insuppl.materials).Eventhoughweexamined numerousbirdsintheorderPasseriformes,wefailedtofind sheenfeathers.

Figure3.Ultrastructureoffeatherscontainingsheen barbules.(a)SEMmicrographofaprimaryfeatherofanadultbeardedvulturewith sheenbarbuleswithdifferentlevelsofwear.Distalbarbules(DBb),whichcovertheproximalbarbules(PBb)oftheadjacentbarbs(b), twistatthemiddleoftheirlength(Tp)followinganapparentlyregularpattern.Aftertwisting,DBbshowa translucentareathatcreates thesheencolourthatcontrastsagainstdarkPBb.NotethatDBbalwaysbreakattheTppoint,notatthepointofinsertioninthebarb, hencenotlosingtheircapacityofbindingadjacentbarbs.(b)SEMimageshowingthedetailofdistalbarbulesasin:(a)atTp.(c)SEM imageofanadultmagpieprimaryfeather.Thepicturewastakenat thejunctionofthe blackandwhiteareasofthe feather,butthereare nomorphologicaldifferencecausingthedifferenthuesasthereisinsheenfeathers.Inthistype of‘conventional’feather,barbulesare almostcompletelystraight.

Discussion

Structuralcharacteristics

Weconsidersheencolourationuniqueinbeingfragile,asit ishighlysusceptibletodamage.Asweshowed(Fig.4b), onecaneasilytakeabladeorabrasivemedium(evena fingernail)and removethe barbules to revealthe dark feather underneath. The loss of feather elementshas

sometimesbeenreportedasamechanismtocreateorna- mentsinbirds,suchastheimpressiveracketedtailofthe turquoise-browedmotmot Eumomota superciliosa,dueto theselectivelossofbarbsintherectrices(Murphy2007), and theblackbibofmalesparrows(e.g.,Boglianiand Brangi1990).

Insomespecieswherethedensityofbarbulesis high,the appearanceofsheencanbepurewhite,asintheflight

feathersoftheAndeancondorVulturgryphus(Fig.1dand

Figure4.Reflectancespectraobtained atanareawithoutbrokenbarbules(squares),atotallyabraded(i.e.black/brown)area(circles) andanareawithanintermediatedegreeofbrokenbarbules(triangles)inabeardedvulture(a)andasnowgoose(b)feather.Asimilar result(c)wasobtainedwhenabeardedvulturefeatherwasexperimentallyabraded(circles:reflectance spectrumfromanareaafter barbuleswere experimentallyabraded;squares:reflectancespectrumatthe sameareabeforebarbuleswere abraded).(d)Theresultofthe sameexperimentalabrasiononaEuropeanmagpiefeatherwithoutsheenstructure.Notehowreflectancecurvesforabraded(circles)and non-abraded(squares)areaslargelyoverlap,and,contrarytotheeffectin(b),thereflectancefortheabradedareaslightlyincreased comparedtothenon-abradedarea.

seevideoclip1insuppl.materials).Thiskindofwhite, however,canbeeasilydistinguishedfrom‘conventional’ whitelikethatdescribedbyDyck(1976),asthesefeathers haveamattewhiteanda‘dusty’ appearance. The particularlystrikingexampleoftheAndeancondorillus- trateshowthismechanismofspecialbarbulescangenerate brightnessinfeathersthatareinfactdark.Thissuggeststhat originallyblackfeathers,mayhaveevolvedassuchbecause ofselectivepressuresactingonmelanization(e.g.environ- mentalwearorneedforcrypsis;LucasandStettenheim

1972,Bortolotti2006,Margalidaetal.2008andreferences therein),andthen amechanismdevelopedtochangecolour byincreasingthe brightness and thus the generalcon- spicuousnessofindividuals.

Whendistalbarbuleswereintact,theUVpeakinthe reflectancespectrumoffeathersalsostoodoutagainstthe

remainingportionofthespectrum.Thistypeofspectral

shapeforsheen-whitefeathers,withaninitialdefinedpeak attheUVrangefollowedbyadecreaseandstabilizationin

reflectance values,hasalsobeenobserveedforthewhite plumage of other species with ‘conventional’ feathers

without the sheenstructure discussedhere (Penteriani etal.2006, McGlothlinetal.2007).Atthisstagewe

cannotdeterminewhethersheenfeathershavetheeffect ofincreasingoverall plumagebrightnessoronlyreflectance

at UV wavelengths, but it isnoticeablethat signalling mechanismstostandoutagainstdarkenvironmentsoften

involvethereflectanceofUVlight.Thishasbeenshown fornestlingskin,mouth and carotenoid-basedplumage

colour(Heebetal.2003,Huntetal.2003,Jourdie etal.

2004,Galva´netal.2008),andforthecolourofblue-green eggsincavitynestingbirds (Avile´setal.2006).Asimilar

signallingdeviceinvolvingthe production of startling

flashesofUVlighthasbeenrecentlydiscoveredinfish livinginhabitatswithlimitedenvironmentallight(Novales

Flamariqueetal.2007).Therefore,itislikelythatsheen feathersincreasetheconspicuousnessofbirdsbyincreasing

theirUVreflectance,asUVlightstronglycontrastsagainst certaindarkbackgrounds (seereferencesabove).Ofcourse,

thiswouldnotnecessarilyexcludethepossibilitythatsheen feathersincreaseconspicuousness byincreasing brightness

atallspectralregions.Furtherstudiesshouldsearchfor associationsbetweenprevalenceof sheen feathersand

detailedhabitatcharacteristics.

Possiblefunctionsofsheenstructures

Afirstpossibilityisthatthebreakagesusceptibilityofthe pennulumofsheenbarbulesmakethemasignallingdevice withoutlosingtheirmechanical functionofbinding adjacentbarbs,asthedarkbasesofthedistalbarbulesare not easilydestroyedby abrasion. Alternatively,inter- individualdifferences inthepatternofabrasionofthese barbules iscausedbybehaviouralattributesofbirdsand thussignals,forexample,dominancestatusifthatpatternis modifiedbyagonisticinteractionswithconspecifics.Lastly, it ispossiblethat sheenstructuresserveassignalsfor individualrecognition,iftheydonotconfersignificantand differentialcostswithrespecttoqualitytobearersandshow high(i.e.multimodal)variation(TibbettsandDale 2007). Futurestudiesshouldaddressthesepossibilities.

Giventheoccurrenceofsheenfeathersindifferentavian orders,the structurecreatingthis typeofplumagehas surprisinglybeenoverlooked.Selectionpressuresarestrong infeathertypesthatarecriticalforflightandforprotecting certainpartsofthebodyfromabrasion,andtheincreased resistancetowearmayfrequentlyhavebeenanimportant factorfavouringmelanizationofthistypeoffeather(Lucas andStettenheim1972,Bortolotti2006,McGraw2006, Schreiberetal.2006).Theplumagesofallspeciesinwhich wehaveobserved sheenfeatherstructures is mostly composedofblack,brown,grey andwhitecolours, suggestingalackofotherpigmentsresponsibleforbright colours,suchascarotenoids. Thus,sheenstructuresmay representanalternative,andperhapsinexpensive,wayto becomemorebrightlycoloured,or at leastto increase

‘achromatic conspicuousness’ byincreasingtotalplumage brightness,inthosespecies thatarelimitedingenerating

brightcoloursthroughthepresenceofotherpigments.

Acknowledgements—Theauthorscontributedequallytothispaper, sotheorderofauthorshipisnot meaningful.The authorsof thephotographsshowninfiguresareJ.J.N.(Fig.1d,Fig.S1, Fig.S2andFig.S5),G.R.B.(Fig.1b,c,f,Fig.2a,bandFig.S1), A.M. (Fig.1a),Jose´JuanHerna´ndez(Fig.1e)andFranc¸ois Mougeot(Fig.S6).WethankRichardO.Prum,BeaArroyoand Franc¸oisMougeotforcommentsonthemanuscript,andJudit Smitsforaccessto,andassistanceoperatinghermicroscope.An editorandtwoanonymousrefereesimprovedpreviousversions ofthemanuscript.FinancialsupportforI.G.wasobtainedfrom theprojectCGL2007-61251andaFPIgrantfromtheSpanish MinistryofScience and Innovation(formerlyMinistryof Education and Science).J. J. N. wassupported by project CGL2006-07481on themechanismsofcolourevolutionand funded by the SpanishMinistryof Scienceand Innovation. G.R.B.wassupportedbyagrantfromtheNaturalSciences andEngineeringResearchCouncilofCanada,andtheStuartand MaryHoustonProf.inOrnithology.A.M.wassupportedby MinisteriodeMedioAmbiente, MedioRuralyMarinoand Departament de Medi Ambient i Habitatge of Generalitat deCatalunya.

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Appendix