Roy L.Caldwell Integrative Biology, Berkeley, California, University of CA94720,USA. 57069, USA. *These authors contributed*These equally tothiswork. authors Australia. 1TQ, UK. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3425-3431doi:10.1242/jeb.107581 Received 2May2014; Accepted9July2014 ‡ 5 1 angle. Underwater, differential sensitivitytoeitherangleordegree polarization definestheextenttowhichwavesoscillateat same which theelectricfieldoflightoscillates,whiledegree of The angleofpolarizationdescribesthepredominantdirection in 2003a; Croninetal.,2009). other visualdimensions,suchashueandbrightness(Cronin et al., communication, polarizationoftenformscompositesignals with Cronin etal.,2009;Chiou2011). Inthecontextof 1996; Croninetal.,2003a;Chiou2007;Mäthger2009; possibly asameansofconspecificcommunication(Shasharetal., cephalopod molluscsusepolarizationinformationtodetectpreyand Dacke etal.,2003),whileintheocean,somecrustaceansand (Wehner, 1976;RosselandWehner, 1986;LabhartandMeyer, 1999; many insectsusethecelestialpolarizationpatternfornavigation prevalent ininvertebrates(Wehner andLabhart,2006).Onland, evolved inbothterrestrialandaquaticanimals,isparticularly Polarization sensitivityisacommonvisualspecializationthathas Signal evolution,Sensorybias,Multi-modalsignal KEY WORDS:Stomatopod,Mantisshrimp,Polarizationvision, content-driven (ultimate)selectionpressures. these complexsignalsbasedonbothefficacy-driven (proximate)and propose ajointframeworkfortheevolutionofpolarizedform provides overintensityandhueinashallowmarineenvironment,we Combining theseresultswiththeincreaseinefficacy thatpolarization in theseanimalsincreasethesignal-to-noiseratioofsignal. and demonstratethatthepropertiesofpolarizationvisionsystem for apre-existingbiastowardshorizontallypolarizedsignalcontent of stomatopod,aswellrelatedprotosquillids.We presentevidence of polarizedsignalsinspeciesHaptosquilla, dimensional visualsignal.Inthiswork,weinvestigatetheevolution information, contentthatisoftenpartofamorecomplexmulti- molluscs, communicateusingvisualsignalsthatcontainpolarized marine species,suchasstomatopodcrustaceansandcephalopod foranumberofdifferent visuallyguidedbehaviours.Several The polarizationoflightprovidesinformationthatisusedbymany Martin J.How Haptosquilla Out oftheblue:evolutionhorizontallypolarizedsignalsin RESEARCH ARTICLE Baltimore County, 1000HilltopCircle, Baltimore, MD21250,USA. INTRODUCTION ABSTRACT Author ([email protected] for correspondence Queensland,StLucia,QLD4072, Queensland BrainInstitute, The University of Bristol,Tyndall Avenue, BiologicalSciences, University of BristolBS8 School of The termpolarizationisusedtodefineseveralpropertiesoflight. 2 Department of Biology, SouthDakota, Vermillion, SD of University of Department 3 Department of Biological Sciences, University of Maryland BiologicalSciences, University of of Department 1, *, MeganL.Porter 4 , N.JustinMarshall (Crustacea, Stomatopoda,Protosquillidae) 2, *, AndrewN.Radford 5 , ThomasW. Cronin a widespreadgenus ) 4 Department of Department 1, the contextof the signal’s polarizationcontent, wemeasurethe tweediei (Dana 1852),H.glyptocercus We comparefourrepresentativeprotosquillidspecies: to polarizedloomingstimulipresented onmodifiedLCDmonitors. We dothisbyexploitingtheanimal’s innatebehaviouralresponses different formsofpolarizationsignalcontentarespeciesspecific. use experimentstoinvestigatewhetherthebehaviouralresponses to polarization communicationinprotosquillidstomatopods.First, we is alsohorizontallypolarized(Chiouetal.,2005;Cronin2009). (e.g.H.trispinosa, glyptocercus illustrates thebluesignalinfourspecies: 1 structural reflection(Chiouetal.,2005; Cronin etal., 2009). Fig. Haptosquilla Caldwell andDingle,1975;Chiouetal.,2011). A commonfeatureof both sexualandagonisticcommunication(DingleCaldwell, 1969; Protosquillidae) isknowntousesignalsfromthefirstmaxillipeds for et al.,2009).Thestomatopodgenus or circularlypolarized(Chiouetal.,2005;Chiou2008;Cronin species ofstomatopodproducevisualsignalsthatareeitherlinearly polarization sensitivity. Opticaltechniqueshavealsoshownthatmany provided additionalinformationontheunderlyingmechanismsof molecular methods(Porteretal.,2009;Roberts2011) have Chiou etal.,2008),opticalmodeling(Roberts2009)and Chiou etal.,2008).Opticalmeasurements(Marshall1991; photoreceptor classesintheeye(KleinlogelandMarshall,2006; the spatialvariationofpolarizationsensitivityindifferent terms ofpolarizationvision.Electrophysiologicalstudieshavedetailed is stillverymuchunknown. polarization signalcontentrelativetothevisualsystemofreceivers Cronin etal.,2009).Incontrast,theevolutionarycontextof Mäthger andHanlon,2006;Chiouetal.,2007;2009; polarization and/orcoloursignalsareproduced(Chiouetal.,2005; optical mechanismsbywhichpolarizationandmulti-component reviews, seeHorváthandVarjú, 2004;Robertsetal.,2011) orthe underlying retinalmechanismsofpolarizationsensitivity(for 2011). Previousresearchinthisfieldhasfocusedoneitherthe reliable providerofinformation(Shasharetal.,2011; Johnsenetal., short ranges(Waterman, 1954;Cronin,2001),whichrendersita of lightremainsmoreconstantthanothervisualdimensionsover (Cronin etal.,2014).Insuchchangingconditions,thepolarization time ofday, andbecauseofenvironmental factorssuchasturbidity downwelling lightcanvarydramatically, bothasafunctionofthe clear marinewaters,theintensityandspectralcompositionof Cronin etal.,2009;Shashar2011). Forinstance,inshallow, of visualinformation(Croninetal.,2003a;Cronin2003b; of polarizationhasseveralfundamentaladvantagesoverotherforms 3 *, KathrynD.Feller and NicholasW. Roberts Here weexplorethepotentialevolutionarypathways of Stomatopod crustaceansaresomeofthebest-studiedspeciesin (Serène 1950)and , first maxillipedsistheproductionofaconspicuousblue H. stoliura 3 , ShelbyE.Temple H. stoliuraand and C. hystrix H. banggai.Insomespeciesofthe (Wood-Mason 1875), 1,‡ (Nobili 1899). Haptosquilla trispinosa, H. banggai),thisreflection Haptosquilla 1 , Second, andin H. trispinosa Chorisquilla (family 3425 H.

The Journal of Experimental Biology 3426 a significantlygreaterprobabilityofresponsetothehorizontally Haptosquilla trispinosa polarization propertiesofmaxillipedsignals. a phylogenyofprotosquillidspeciestoconsidertheevolution between twodifferent anglesofpolarization.Finally, weconstruct threshold atwhichH.trispinosa RESEARCH ARTICLE Responses topolarized stimuli RESULTS A Response probability C 0.25 0.50 0.75 1.00 0.25 0.50 0.75 1.00 0 0 CD AB Vertically polarized Vertically polarized Haptosquilla trispinosa stimulus stimulus Chorisquilla tweediei , H. glyptocercus Horizontally polarized Horizontally polarized stimulus stimulus are nolongerabletodiscriminate B D and Vertically polarized Vertically polarized C. tweediei Haptosquilla glyptocercus stimulus stimulus Chorisquilla hystrix all showed Horizontally polarized Horizontally polarized stimulus stimulus (Kruskal–Wallis test: tweediei significant difference between sample size( responsive tohorizontallypolarizedlight(Fig. 2A–C). Fig. trispinosa: polarized stimuluscomparedwithavertically( Z =2.42, d.f.=9,P (B) H.glyptocercus conspicuous maxillipedsignals.(A)Haptosquillatrispinosa Fig. responded withthatprobability. each probabilityrepresentthenumberofindividualsthat tweediei (A) Haptosquillatrispinosa individual totheverticallyandhorizontallypolarizedstimulii. Fig.

in theirrelativeresponsestothetwostimuli .Illustrativeexamples,shownbyarrows,ofthe 1. Wilcoxon test: The JournalofExperimentalBiology(2014)doi:10.1242/jeb.107581

.Pairedplotsoftheprobabilityresponse ofeach 2. Chorisquilla hystrixalsoappearedtobemore n =5) precludedstatisticaltesting.Therewasno and (D) =0.02; χ 2 =2.90, d.f.=2,P , (C)H.stoliura C. hystrix.Numbersofpoints(opencircles)at Z C. tweediei: =2.93, d.f.=9,P H. trispinosa, , (B)H.glyptocercus and (D) =0.24). Z=2.77, d.f.=9,P =0.002; H. glyptocercus H. banggai

2D), butthesmall , (C)Chorisquilla H. glyptocercus . =0.004; and , H. C. :

The Journal of Experimental Biology tweediei H. stoliura, have blue-reflectingfirstmaxillipeds,butonlythereflections from polarizing signals(Fig. blue signalsandnootherspeciespossesseitherorhorizontally maxilliped signaltypes.Ofthesespecies,only nine generaandfourfamilieshavebeeninspectedfor first Outside oftheProtosquillidae,sixotherstomatopodspecies from possessing blueorandhorizontallypolarizedfirstmaxillipeds. stoliura, further specieshavebeenanalyzed( horizontally polarized.Within therestof the Protosquillidae,five species (Fig. very weak,spectrallybroadreflectionsfromthe (Fig. reflections from trispinosa ipae nFg 4.Both displayed inFig. C. tweediei RESEARCH ARTICLE groups ofspecies thatcorrespondtothetwo known typesoffirst (Fig. in particularthegenus recovering theprotosquillids[bootstrap percentages(BP)=98],and previous studies(Barberand Boyce2006;Porteretal.,2010) Phylogenetic analysesofprotosquillid relationshipsrecapitulate The firstmaxillipedreflectionsfrom material Table background wasbetween31.4and20 the difference betweenthepolarizationanglesofstimulusand Haptosquilla trispinosa Fig. the anglesofpolarizationstimulusandbackground(x Phylogenetic analyses polarized signals of Presence polarized light linearly discriminationLevel between two of angles of to thestimulus. above, theydisplayedaconsistentstatisticallysignificantresponse responded tothepolarizationstimulus;atvaluesof31.4 false positives(* test wasusedtodeterminewhichresponsevaluesdiffered fromthelevelof stimulus type(whitecircles)andasanoverallmean(dottedline).McNemar’s background leveloffalsepositiveresponsesarerepresentedforeach The responsedataarefittedwithahyperbolictangent(dashedline). Visual analysesofotherspecies

.Responsesof 3. 5). Within the 4B) showbluereflectionsfromthemaxillipedscomparedwith ,

H. banggai, Response probability Echinosquilla guerinii are horizontallypolarized(Fig. and 4C,D). OftheblueHaptosquilla P H. banggai,

S1). Atanglesof20 <0.05). C. hystrix H. glyptocercus Haptosquilla, ourphylogenyrecoveredtwosub- H. trispinosa H. pulchella

5). showed littleornoresponsetostimuliwhen H. trispinosa Angular difference (deg) Haptosquilla are presentedinthemicroscopeimages H. pulchella * (black circles)todifferencesbetween and are unpolarized(Fig. , H. nefanda deg orless,theanimalsrarely H. trispinosa, Protosquilla folini (Fig. Haptosquilla C. excavata

(BP=89), asmonophyletic e Fg 3;supplementary deg (Fig. 4A) andH.glyptocercus and *** and

4A) whereasthe G. smithii H. nefanda H. glyptocercus, , H. hamifera reflections, showed that C. hystrix,

Chorisquilla 4B). ), withnone

deg and possess -axis). are all H. H. C. Furthermore, itisonlythesub-groupof protosquillids, onlythegenus the comparativephylogeneticanalyses,revealedthatofthese and polarizationpropertiesofthemaxillipeds,incombinationwith are verticallypolarized.Themeasurementsofthestructuralcolour response tohorizontallypolarizedstimulicomparedwiththosethat protosquillid speciestesteddisplayedagreaterprobabilityof to alooming,linearlypolarizedstimulus.Moreover, allthe stomatopod haveaninherent(i.e.non-trained)behaviouralresponse Our resultsprovidedirectevidencethatseveralspeciesof polarizing. maxilliped signaling,eitherblueandunpolarizedor responses tosuch polarizationsignals. investigate howthedegreeof polarizationaffects behavioural demanded oftheanimals.Further workisalsostillneededto that themeasureddiscrimination thresholdisspecifictothetask behavioural contextshouldalso notbeignored.Itisquitepossible in thedorsalandventralhemispheres. However, theprecise have measuredspecificallyrepresent apropertyofthevisualsystem et al.,2014)].Ifso,thepolarizationdiscriminationresponses we an equivalentdiscussionforcolourvision,seeThoenetal.(Thoen visual informationissimplifiedtospeedupsensoryprocessing [for information. Therefore,itisconceivablethatmuchofthe early visual scanofthetarget with themidbandtofillinremaining the target, andpresumablythis wouldbefollowedbyasubsequent the stomatopodvisualhemispheres,whichelicitasaccade to experiment isthereforemostlikelytobestimulatingresponses in motion componentoftheLCDloomingstimulususedin our on thecolourandpolarizationinformation(Landetal.,1990). The midband isthenscanned,ratherlikealine-scansensor, toexpand dimensional representationofthevisualscene,overwhich the that thetwohemispheresareprimarilyinvolvedinproducingatwo- but inturnenhancetheprocessingefficiency. Currently, itisthought amount ofinformationthatcanbeprocessedfromthevisualscene separated byaspecializedmidband)mayplacelimitationsonthe visual systemsareusedtoresolvehighlevelsofpolarizationdetail. current evidencethatothercrustaceanandcephalopodpolarization suggestive oftuningforhighcontrastsignalscomparedwiththe cuttlefish, species [fiddlercrab, threshold isanorderofmagnitudehigherthanmeasuredinother effectively low-passfilteringanyvariationinthebackground.This improve thesignal-to-noiseratioofalinearlypolarizedsignalby angles ofpolarization.Suchacoarseleveldiscriminationwould 21.4 and30 (Temple, 2012). have previouslyevolvedtoimprovecontrastdiscrimination prevalent inreflectionsfromobjectsandpreferentialsensitivitymay angle ofpolarizationmaycomefromthefactthatthisismost pre-exist? Whilstwecanonlyspeculate,thebiasforahorizontal question raisedbytheconceptofsensorybiasiswhydoes (Guilford andDawkins,1991;EndlerBasolo,1998).A common maxilliped signalstobehorizontalinthe protosquillids couldhavebiasedthepolarizationcontentoffirst predisposition towardshorizontallypolarizedstimuliseenacrossthe horizontally. Therefore,itispossible thatthecommonbehavioural In thesespecies,thepolarizationofsignalsisalwaysorientated trispinosa DISCUSSION The complexnatureofstomatopodeyedesign(twohemispheres Haptosquilla trispinosaalsodisplayedathresholdofbetween that possessestheadditionalpolarizedsignaldimension. Sepia plangon,1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.107581 deg intheirresponsetodistinguishingbetweentwo Uca vomeris,3.2 Haptosquilla deg (Temple etal.,2012)]andis Haptosquilla deg (Howetal.,2012); displays thebluesignals. H. trispinosa including clade 3427 H.

The Journal of Experimental Biology 3428 RESEARCH ARTICLE horizontally polarized lightprovidesafurther explanation forwhy dimension isthereforefairlyclear. Thebehaviouralbiastowards increase insignalefficacy bytheinclusionofthisextra visual such asthoseinwhichthese species ofstomatopodreside.The information, particularlyinspectrally variablelightenvironments, Introduction, polarizationprovides areliableformofvisual Dawkins, 1991;Hebetsand Papaj, 2005).Asdescribedinthe evolution arebothefficacy-driven andcontent-driven(Guilford and Marler, 2005).Inthiscontext,theselective pressuresonsignal as toelicitabehaviouralresponseintheintendedreceiver(Parten way thatitwillbereceivedinitsintendedform,andinterpreted relies oninformationbeingsentthroughtheenvironmentinsuch a these maxillipedsignalsinstomatopods.Successfulcommunication potential evolutionarypathwayofthepolarizationproperties of Overall, ourfindingsprovideaframeworkforunderstanding the D C B A V Chorisquilla hystrix Chorisquilla tweediei Haptosquilla glyptocercus Haptosquilla trispinosa H

Reflectivity Reflectivity Reflectivity Reflectivity 0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2 0.3 0 0 0 0 0 0 0 700 600 500 400 700 600 500 400 0 0 0 700 600 500 400 0 0 0 700 600 500 400 Wavelength (nm) Wavelength (nm) Wavelength (nm) Wavelength (nm) Vertically polarized Horizontally polarized visual systems under thecorrectenvironmental lightconditions. consider howthesedualdimensions areviewedtogetherbyreceiver polarization andcoloursignalsin otheranimalsshouldalsocarefully and Papaj,2005)?We suggestthatfuturestudies of combined multi-component signals(Hasson, 1991;Candolin,2003;Hebets of receiverresponseasisdescribed byanamplifierhypothesisof combine inafunctionalway, forexample,increasingtheaccuracy independently forpurposesofinformationredundancyordo they bias; and(2)dothespectralpolarizationdimensions act polarization contrastofthesignalandbackgroundinfluence the principles, respectively, fortheevolutionofthiscomplexsignal. nature ofthebiassuggestbothproximateandultimatedriving polarized. Together, theadditionofpolarizationtosignaland the polarizedcontentofsignalshasevolvedtobehorizontally Two questionsforthefuture are:(1)canmanipulatingtherelative The JournalofExperimentalBiology(2014)doi:10.1242/jeb.107581 respectively, relativetotheaxesof themaxillipeds. and HinA denotetheverticalandhorizontaldirections, triangles representtheverticallypolarizedreflectivity. V represent thehorizontallypolarizedreflectivityandopen circle ineachimage.Inthespectralplots,opencircles are thereflectionspectrafromareadenotedby (A) Haptosquillatrispinosa Fig. tweediei

.Microscopeimagesofthemaxillipeds. 4. and (D) C. hystrix.Accompanyingeachplot , (B)H.glyptocercus , (C)C.

The Journal of Experimental Biology beach sand.Eachindividualwasplacedinsidean8 AIRFORCE (NF)]. University ofQueensland[AEC,permitno.QBI/223/10/ARC/US All procedureswereapprovedbytheAnimalEthicsCommittees of the (24–25°C, naturaldaylightillumination,andfedpiecesoffrozenshrimp). flow-through marineaquariumfacilityattheLizardIslandResearchStation G12/35042.1). Animalsweremaintainedbeforetestinginanaturalseawater Barrier Reef,Australia,inAugust2011 (Queensland–GBRMPA permit RESEARCH ARTICLE Individual stomatopodswereplacedina30×15×15 individuals of trispinosa, 10individualsofbothH.glyptocercus behavioral responsestopolarizedstimuli,wecollected39individualsof To investigatetheinherentabilityofstomatopodstogeneratedistinct and evolutionofsignalcomplexity. represent anexcellentbehaviouralsystemtoinvestigatethefunction the combinedcolourandpolarizationsignalsinstomatopods the functionsofindividualcomponents(HebetsandPapaj,2005), Whilst itisnotalwayseasytodecomposecomplexsignalsandtest stimulus content Relationship andpolarization between behaviouralresponses Animals MATERIALS ANDMETHODS Tank A Stomatopod intube Maxilliped colourandpolarization facingLCD Not blueandunpolarized Blue andunpolarized Blue andpolarized 100 100 C. hystrix Camera 99 Odontodactylus cultrifer 98 77 Odontodactylus scyllarus Looming stimulus P from offshore reefsnearLizardIsland,Great 100 0.04 57 89 H 94 LCD 64 86 100 Taku spinosocarinatus 96 100 Haptosquilla moosai Protosquilla folini 100 72 B falcatus Chorisquilla tweediei

Stokes parameter Haptosquilla hamifera Chorisquilla trigibbosa 71 100 and Haptosquilla togianensis Haptosquilla proxima –1.0 –0.5

Chorisquilla excavata Echinosquilla guerinii smithii Haptosquilla stoliura cm tankcontaininglocal

Haptosquilla glyptocercus 0.5 1.0 mm diametercleartube oerstedii 96 Haptosquilla pulchella 0 Chorisquilla gyrosa 0 5 0 5 0 5 700 650 600 550 500 450 400 C. tweediei 100 Chorisquilla spinosissima Chorisquilla hystrix Haptosquilla nefanda Haptosquilla tuberosa Gonodactylellus annularis Wavelength (nm) Chorisquilla brooksi Chorisquilla mehtae Haptosquilla corrugata P0 Haptosquilla trispinosa , andfive P3 P2 H. P1 wavelength (Fig. computation ofthepolarizationellipseeachstimulifor any ¼ waveFresnelrhomb(EdmundOptics,York, UK),whichpermittedthe measurements (Fig. polarization propertiesoftheLCD,accuratebroadbandStokesparameter contrast betweenthebackgroundandloomingstimulus.To checkthe polarization, butmostimportantlyaninherentzeroluminanceandchromatic polarization ofthelightpermittednotonlydynamiccontrol at theair/glass/waterboundaries).Thesimpleelectro-opticcontrolof s(takingintoaccountrefraction ofthevisualfieldanglein1 deg cover 22.5 (H stimulus),respectively(Pignatellietal.,2011). Thestimuliexpandedto output polarizationcouldbecontrolledasvertical(V stimulus)orhorizontal LCD withagrayscalevalueofeither0(black)or255(white),thelocal By removingthefrontpolarizerfromLCDscreenandaddressing presented inarow. We randomlyvariedthetimebetweensuccessivestimuli, background. Nomorethanthreeinstancesofthesamestimulus were stimuli and10V stimuli, against aperpendicularlylinearlypolarized resolution at60 , wasanLCDscreen(Viglen LC552;1280×1024 presentation ofthestimuli.Onoutsidetank,andinfront a videocamera(CanonLegriaFS20)thatrecordeditsresponsetothe 6A).Directlyabovetheanimalwas forward ofthefrontendtube(Fig. Cronin etal.,1991).Theanimalwaspositionedsuchthattheeyeswere and restrainedusingasmallamountoffishingline(Landetal.,1990; All animalsreceivedabalancedpseudo-randomizedpresentationof10 H C The JournalofExperimentalBiology(2014)doi:10.1242/jeb.107581

Hz); theeyeswereatadistanceof~12

6C).

two maingeneraoftheProtosquillidae. polarized stimuli,illustratingtheoccurrenceacross in thisexperiment,allofwhichhaveabiastohorizontally Species namesinboldindicatethoseanimalsmeasured maxilllipeds hasbeenmappedontothephylogeny. blue signalsandpolarizingonthefirst respectively. Whereknown,thepresence orabsenceof family Protosquillidaeareindicatedby‘H’ and‘P’, Nodes representingthegenus Branch supportvaluesrepresentbootstrappercentages. representative speciesfromtheGonodactyloidea. protosquillid speciesrelationships,rootedusing Fig. 6B) weremadeusingGlan–Thompsonpolarizersanda

.A maximumlikelihoodphylogenyof 5. ellipses at560 the verticalandhorizontalpolarization function ofwavelength.(C)Anexample of horizontally polarizedstimulusasa Stokes parameters(P0–P3)ofthe example measureofthenormalized setup infrontoftheLCDscreen.(B)An experimental apparatus. Fig.

.Schematicdiagramofthe 6.

nm. Haptosquilla

cm fromthescreen. (A) Thetank

pixel spatial and the 3429

The Journal of Experimental Biology Video recordings weresynchronizedtothe stimulusbymeansofanaudio the wholeexperiment wasconductedwithoutexperimenter intervention. automatically usingMATLAB (r2011, MathWorks, Natick,MA,USA)and mounted onthetopedgeoffront aquariumwall.Stimuliweregenerated recorded usingadigitalvideocamera (SonyHDR-SR11, Tokyo, Japan) intensity. Stomatopod eyemovementsinresponsetothestimuluswere polarized background,withnocorresponding changesinhueorlight stimulus thatvariedintheangle ofpolarizationagainstahorizontally background andrangedbetween0255forthestimulus,resulting ina greyscale valuesaddressedtothemonitorwereset0(black)for the (taking intoaccountrefractionattheair/glass/waterboundaries). The s ofthevisualfieldanglein1 deg circle stimulusexpandedtocover27 very similarproperties,waspositionedagainstthefrontwall.A looming (How etal.,2012)forcalibrationdetails]tothatdescribedabove,but with front wall.A different polarization LCDmonitor[HP L1906;seeHowetal. response toeachstimulustype. number ofresponsesoutthe10presentations,givingaprobability illustrating thetypicalsaccadiceyemovementresponsein stomatopods. Fig. 3430 a 20×20×30 sensitivity of A similarmethodwasusedtomeasurethepolarizationangularcontrast stimulus orfrom3 eye movementswereobservedina5 foranexample).Nosuchsaccadic 7 were rapidlybroughttogether(seeFig. the stimulusasasaccadiceyemovement,inwhichoneorbotheyestalks optokinetic responseofthefocalanimal.We definedapositiveresponseto whether theanimalrespondedtotwostimulustypes,wemonitored from 20to120 RESEARCH ARTICLE polarized light linearly Discrimination between two threshold of angles as afunctionoftime. numbered framesdisplayedinA.Theredlineindicatesthestimulusdiameter polarized contraststimulus.Thenumbersandfilledpointscorrespondtothe angular separationoftheeyestalksasafunctiononsetlooming position ~0.3 eye position0.1 two imagesshowtheeyesbeforestimulus,thirdimageshows polarized background).Eachimageisasingleframe,~0.2 looming polarizedcontraststimulus(horizontallyonavertically B A

.Measurementsofthebehaviouralsaccadic responseofthe 7. Angular separation of eye 1 stalks (deg) 10 20 30 40 50 0

cm aquariumpartitioninburrowspositioned~12 s afterthestimulusonset.(B)Themeasuredchangein H. trispinosa.Individualunrestrainedanimalswerehousedin 0 (A) Time sequences ofimagesfromavideorecording

s afterthestimulusonset,andfinalimageshowseye

s, tominimizeanyeffect ofhabituation.To determine

s afteritspresentation.Animalswerescoredbytheir . . 2.4 1.6 0.8 24 1 eye separation Measured angular 2 Time (s) 3 4 3

s periodbeforetheonsetof 3.2 1 2 3 4 5

0 H. trispinosa s apart;thefirst

Stimulus diameter (cm)

cm fromthe to a independent groups(n between stimuliwasrandomized20and60 respectively). Thestimulusorderwasfullyrandomizedandtheinterval and Images ofthemaxillipeds stimuli (anglesof0,0.5,1,5,7,9and11 made ina5 microphone portofthecamera.Measuressaccadiceyemovementswere signal conveyedbyaudiocabledirectlyfromthecomputerto McNemar’s test. trispinosa Kruskal–Wallis ranksumtest.Theindividualsaccadicresponses of rank testsanddifferences betweenspecieswerecalculated usinga vertically polarizedloomingstimuli wereanalysedusingWilcoxon signed- Statistical Computing).Response probabilitiestoeitherhorizontallyor All statisticalanalyseswereconducted inR3.0.2(RFoundationfor signals withinthegenus To investigatethepotentialevolutionarypathwayofcolorandpolarization Posada 2008). according totheresultsofjModelTest v0.1.1(GuindonandGascuel 2003; GTR+gamma model,asthiswasthebest-fittingmodelavailableinRAxML, ribosomal genes(16S,18Sand28S).Allpartitionswereanalyzedwith the COI codonpositions1and2;(2)position3;(3)all of the al., 2009).ThreepartitionsweredesignatedfortheRAxML analysis:(1) (CIPRES) Portalv.2.0 (Stamatakis2006;Stamatakisetal.,2008;Miller implemented ontheCyberinfrastructureforPhylogeneticResearch Maximum Likelihood(RAxML)v.7.2.7 withrapidbootstrappingas dataset wasusedtoreconstructaphylogenyusingRandomizedAxelerated sequences. Thefourgeneregionswerethenconcatenatedandthecombined codons) andthenmanuallyalignedusingthetranslatedaminoacid for evidenceofpseudogenes(e.g.stopcodons,indelsnotcontinuouswith (Katoh etal.,2002;Katoh2005).TheCOIsequenceswereinspected the E-INS-IstrategyinMAFFT v6.0.0(http://mafft.cbrc.jp/alignment/server/) data sourcesandgenerepresentation). across species(seesupplementarymaterialTable 28S nuclearrDNA genes,althoughthenumberofsequencesavailablevaried cytochrome oxidaseI(COI)and16Smitochondrialgenes,the18S used aconcatenatedmatrixconsistingofnucleotidesequencesfromthe stability atdeepernodeswithinthephylogenyandtouseasoutgroups.We (Gonodactyloidea) wereincludedtoprovideincreasedresolutionand species fromwithinthesamefamily(Protosquillidae)andsuperfamily (supplementary materialTable sequenced followingthemethodsofPorteretal.(Porteral.,2010) GenBank, providedbyP. Barber(BarberandBoyce,2006)orwere and mitochondrialgenesforallavailablespecieswereobtainedfrom Polarization analysis of themaxillipedsignals Polarization of analysis Statistical analysis Phylogenetic analyses thorough arotatablelinearpolarizer. Gonodactyloidea havebeenassessedvisuallybyviewingthemaxillipeds representative speciesofstomatopodsacrossthesuperfamily the colourandpolarizingnatureoffirstmaxillipedsfrom17other respective polarizedreflectancespectrum.Overseveralprecedingyears, the pathofreflectedlightinsidemicroscopetocollecteach Optics). Linearhorizontalandverticalpolarizationfilterswereplacedin 1 light wascollectedatthebackfocalplaneofsecondeyepieceusinga of theeyepieceandilluminatingmaxillipedsnormally. Thereflected source (OceanOptics,Dunedin,FL,USA)mountedatthebackfocalplane species weremeasuredusinganOceanOpticshalogenHL-2000light extension onthetrinocularhead.Spectralreflectiondataofsamefour digital camera(Canon,Melville,NY, USA)mountedusingaphototube Microsystems, Wetzler, Germany)usinga10×objectiveandCanonG9

mm diameteropticfibreconnectedtoaQE65000spectrometer(Ocean Nucleotide sequencesofthe16S,18Sand28Sgeneswerealignedusing C. hystrix to different angulare-vectorcontrastswere analysed usinga s periodbothbeforeandafterthestimuluspresentation.Two The JournalofExperimentalBiology(2014)doi:10.1242/jeb.107581 were takenthoughaLeitzcompoundmicroscope(Leica =15 and14animals)weretestedusingtwosetsof Haptosquilla H. trispinosa,

S2). Additionalrepresentativestomatopod , DNA sequencesfrombothnuclear

deg and20,31,56,7074 H. glyptocercus

S2 forfulldescriptionof

s. , C. tweediei

deg, H.

The Journal of Experimental Biology ulod .adDwis .S. Guilford, T. andDawkins,M. Caldwell, R.andDingle,H. RESEARCH ARTICLE http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.107581/-/DC1 Supplementary materialavailableonlineat a recipientofYulgilbar FoundationLizardIslandPostdoctoralFellowship. (FA8655-12-1-2112 toT.W.C., N.J.M.andN.W.R.) supportedthiswork.S.E.T was BB/H01635X/1 toN.W.R.), andtheAirForceOffice ofScientificResearch Biotechnology andBiologicalSciencesResearchCouncil(BB/G022917/1 photographs oftheanimals.Allauthorshelpedwriteandeditmanuscript. designed andcarriedoutthespectralreflectionexperiments.R.L.C.provided and undertooktheanalysis.M.L.P. carriedoutallthephylogeneticstudies.K.D.F. N.W.R., A.N.R.andM.J.H.designedperformedthebehaviouralexperiments The authorsdeclarenocompetingfinancialinterests. for providingsequencedata. assistance, toInnesCuthillforhelpwiththestatisticalanalysis,andPaulBarber We areverygratefultotheLizardIslandResearchstationstaff foralltheir Guindon, S.andGascuel,O. Chiou, T. H.,Mäthger, L.M.,Hanlon, R.T. andCronin,T. W. Chiou, T. H.,Cronin,T. W., Caldwell,R.L.andMarshall,J. Candolin, U. Barber, P. andBoyce,S.L. References Supplementary material Funding Author contributions Competing interests Acknowledgements Chiou, T. H.,Marshall,N.J.,Caldwell,R.L.andCronin,T. W. Chiou, T. H.,Kleinlogel,S.,Cronin,T., Caldwell,R.,Loeffler, B.,Siddiqi,A., Cronin, T. W., Marshall,N.J.and Land,M.F. Cronin, T. W., Caldwell,R.L.and Marshall,J. Dingle, H.andCaldwell,R.L. Cronin, T. W., Shashar, N.,Caldwell,R.L.,Marshall,J.,Cheroske,A.G.and Cronin, T. W., Shashar, N.,Caldwell,R.L.,Marshall,J.,Cheroske,A.G.and Dacke, M.,Nilsson,D.-E.,Scholtz,C.H.,Byrne,M.andWarrant, E.J. Endler Cronin, T. W., Johnsen,S.,Marshall,J.andWarrant, E.J. Cronin, T. W., Chiou,T. H.,Caldwell,R.L.,Roberts,N.andMarshall,J. animal signals. estimate largephylogeniesbymaximumlikelihood. stomatopods. 2053-2061. stomatopods throughDNA barcodingofstomatopodlarvae. selection. spatial propertiesofpolarizedlightreflectionsfromthearmssquid( polarized lightreflectorsinstomatopodcrustaceans. Soc. and cuttlefish( stomatopod light-reflecting propertiesofsignallingappendagesaltermatechoicebehaviourina crustacean. Goldizen, A.andMarshall,J. 168 mantis shrimps(Crustacea,Stomatopoda,Gonodactylidae). 1-11. Comp. Biol. Chiou, T. H. 5158, 85-92. Chiou, T. H. colour visioninamantisshrimp. Animal behaviour:insectorientationtopolarizedmoonlight. Princeton, NJ:PrincetonUniversityPress. Polarization signalsinmantisshrimps. 33 , 115-136. , 233-240. , J.A.andBasolo,L. 78, 575-595. Trends Ecol.Evol. (2003). Theuseofmultiplecuesinmatechoice. Curr. Biol. 43, 549-558. (2003b). Polarizationvisionanditsroleinbiologicalsignaling. (2003a). Polarizationsignalsinthemarineenvironment. Naturwissenschaften Sepia officinalis Gonodactylus bredini Anim. Behav. 18, 429-434. Haptosquilla trispinosa (1975). Ecologyandevolutionofagonisticbehaviorin 13, 415-420. 42, 1-14. (2006). EstimatingdiversityofIndo-Pacificcoralreef (1969). Theaggressiveandterritorialbehaviourofthe L.). J.Exp.Biol. (1998). Sensoryecology (2003). A simple,fast,and accuratealgorithmto (2008). Circularpolarizationvisioninastomatopod Nature (1991). Receiverpsychologyandtheevolutionof 62, 214-222. Manning (Crustacea:Stomatopoda). Proc. SPIE 411, 547-548. 210 (1991). Optokinesisingonodactyloid . (2001). Sensoryadaptation.Tunable Mar. Freshwat.Behav. Physiol. 7461, 74610C. , 3624-3635. Syst. Biol. Proc. SPIE , receiverbiasesandsexual Nature Biol. Rev. Camb.Philos. (2014). 52, 696-704. J. Comp.Physiol.A Proc. Biol.Sci. (2007). Spectraland 5888, 58881B. (2011). Changesin (2005). Biological 424 Visual Ecology Loligo pealeii , 33. Proc. SPIE Behaviour (2003). (2009). Integr. 273 44, ) , . Wehner, R.andLabhart,T. Wehner, R. Waterman, T. H. Thoen, H.H.,How, M.J.,Chiou,T. H.andMarshall,J. Porter, M.L.,Zhang,Y., Desai, S.,Caldwell,R.L.andCronin,T. W. Shashar, N.,Johnsen,S.,Lerner, A.,Sabbah,S.,Chiao, C.C.,Mäthger, L.M.and Shashar, N.,Rutledge,P. andCronin,T. Temple, S.E.,Pignatelli,V., Cook,T., How, M.J.,Chiou,T. H.,Roberts,N.W. and Stamatakis, A.,Hoover, P. andRougemont,J. Stamatakis, A. Roberts, N.W., Chiou,T. H.,Marshall, N.J.andCronin,T. W. Hasson, O. Rossel, S.andWehner, R. Roberts, N.W., Porter, M.L.andCronin,T. W. How, M.J.,Pignatelli,V., Temple, S.E.,Marshall,N.J.andHemmi,M. Hebets, E.A.andPapaj,D.R. Katoh, K.,Kuma,Toh, H.andMiyata,T. Johnsen, S.,Marshall,N.J.andWidder, E.A. Horváth, G.andVarjú, D. Katoh, K.,Misawa,Kuma,K.andMiyata,T. Labhart, T. andMeyer, E.P. Kleinlogel, S.andMarshall,N.J. Land, M.F., Marshall,J.N.,Brownless,D.andCronin,T. W. Mäthger, L.M.andHanlon,R.T. Marshall, N.J.,Land,M.F., King,C.A.andCronin,T. W. Pignatelli, V., Temple, S.E.,Chiou, T. H.,Roberts,N.W., Collin,S.P. andMarshall, Partan, S.R.andMarler, P. Miller, M.A.,Holder, M.T., Vos, R.,Midford,P. E.,Liebowitz,T., Chan,L.,Hoover, Porter, M.L.,Bok,J.,Robinson,P. R.andCronin,T. W. Mäthger, L.M.,Shashar, N.andHanlon,R.T. stomatopod . Evolution ofanatomicalandphysiologicalspecializationinthecompoundeyes Warrant andD.-E.Nilsson),pp.291-348.Cambridge:CambridgeUniversityPress. 927-932. color visioninmantisshrimp. 22, R121-R122. 265. functions. Hanlon, R.T. concealed communicationchannel? Marshall, N.J. the RAxML Web servers. analyses withthousandsoftaxaandmixedmodels. Nat. Photonics quarter-wave retarderwithexcellentachromaticityinthevisiblewavelengthregion. J. Exp.Biol. High e-vectoracuityinthepolarisationvisionsystemoffiddlercrabUcavomeris. emphasis onfeatherdecorations. mechanisms underlyingpolarizationvision. framework oftestablehypotheses. in accuracyofmultiplesequencealignment. Res. rapid multiplesequencealignmentbasedonfastFouriertransform. transparent zooplankton. contrast enhancerinpelagicpredators:lessonsfromsitupolarizationimagingof Patterns inNature Gonodactylus chiragra polarization sensitivityinthecompoundeyesofstomatopodcrustacean Microsc. Res.Tech. survey ofommatidialspecializationsinthedorsalrimareacompoundeye. movements ofthemantisshrimp Stomatopoda). communication signals. using polarizedlightreflectionsfromtheirskin? light communicationinsquid. structure: thedetectionofpolarizedlight. eyes ofmantisshrimps(Crustacea,Hoplocarida,Stomatopoda).I.Compoundeye diversity ofvisualpigmentsinStomatopoda(Crustacea). mechanism incephalopodsandfishes. N. J. http://www.webcitation.org/5imQlJeQa. Accessed29September2010. P. andWarnow, T. 30, 3059-3066. (2011). Behaviouralrelevanceofpolarizationsensitivityasatargetdetection (1976). Polarized-lightnavigationbyinsects. Philos. Trans. R.Soc.B (1991). Sexualdisplaysasamplifiers:practicalexampleswithan 215 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.107581 (2011). Underwaterlinearpolarization:physicallimitationstobiological 3 (1954). Polarizationpatternsinsubmarineillumination.Science J. Comp.Physiol.A (2006). RAxML-VI-HPC:maximumlikelihood-basedphylogenetic , 2128-2134. (2012). High-resolutionpolarisationvisioninacuttlefish. , 641-644. . London:Springer. 47, 368-379. (2009). . J.Exp.Biol. Am. Nat. Philos. Trans. R.Soc.B Syst. Biol. J. Exp.Biol. (1986). Polarizationvisioninbees. (2006). Polarisationvision.In (2004). The CIPRESPortals. Science Biol. Lett. (1999). Detectorsforpolarizedskylightininsects:a (2005). Issuesintheclassificationofmultimodal (2006). Anatomicalbasisforcamouflagedpolarized 166 Behav. Ecol. 366 Behav. Ecol.Sociobiol. (2005). Complexsignalfunction:developinga 167 (2006). Electrophysiologicalevidenceforlinear 57, 758-771. 209 J. Exp.Biol. Polarized LightinAnimalVision: Polarization , 231-245. 343, 411-413. 213 , 649-654. , 155-166. Philos. Trans. R.Soc.B 2 , 4262-4272. , 494-496. Philos. Trans. R.Soc.B (1996). Polarizationvisionincuttlefisha , 3473-3486. Odontodactylus scyllarus Philos. Trans. R.Soc.B Nucleic AcidsRes. (2005). MAFFT version5:improvement (2009). Docephalopodscommunicate (2008). A rapidbootstrapalgorithm for 2 J. Exp.Biol. , 189-197. (2002). MAFFT: anovelmethodfor (2011). Polarizationsensitivityasa 199 366 CIPRES. ArchivedbyWebCite at (2011). Themolecularbasisof Bioinformatics , 2077-2084. , 655-670. Sci. Am. Invertebrate Vision (2014). A different formof 57, 197-214. 212 V Nature (1991). Thecompound is. Neurosci. 235 , 2133-2140. 33, 511-518. 366 (2009). A biological 334 (2009). Molecular 22, 2688-2690. (1990). Theeye- , 106-115. , 734-741. 366 323 , 33-56. Nucleic Acids , 627-637. , 128-131. (Crustacea, Curr. Biol. 26 (2010). (2012). 3431 (ed. E. , 255- 120,

The Journal of Experimental Biology

Table S1. Responses of H. trispinosa to differences between the angles of polarization of the stimulus and the background presented on a modified LCD monitor

Angular difference / Stimulus Responses to stimulus degrees set Responses before YES NO P stimulus

0.0 1 YES 0 0 ns

NO 1 14

0.5 1 YES 0 0 ns

NO 0 15

1.2 1 YES 0 1 ns

NO 1 13

5.0 1 YES 0 1 ns

NO 1 13

6.9 1 YES 0 1 ns

NO 2 12

11.5 1 YES 0 0 ns

NO 0 14

20.0 2 YES 0 2 ns

NO 3 9

31.4 2 YES 0 0 0.041

NO 6 8

56.6 2 YES 1 0 0.008

NO 9 4

69.8 2 YES 0 0 0.041

NO 6 8

74.4 2 YES 0 0 <0.005

NO 10 4 Number of responses are presented for the each of the four possible ways the animal could respond before and to the stimuli. N=15 for the 1st set of angles tested and

N=14 for second. McNemar’s test was used to compare the responses before and to the stimulus at a given angle. ns is non-significant.

The Journal of Experimental Biology | Supplementary Material Family Genus Species COI 16S 18S 28S-1 28S-2 Gonodactylidae Gonodactylaceus falcatus HM138786 HM138827 HM138871 HM180015 HM180059 Gonodactylidae Gonodactylelllus annularis HM138783 HM138824 HM138868 HM180012 HM180056 Gonodactylidae Gonodactylus chiragra HM138785 HM138826 HM138870 HM180014 HM180058 Gonodactylidae Gonodactylus smithii HM138788 HM138829 HM138873 HM180017 HM180061 Gonodactylidae Neogonodactylus oerstedii HM138796 HM138838 HM138882 HM180026 HM180070 Odontodactylidae Odontodactylus cultrifer N/A HM138839 HM138883 HM180027 HM180071 Odontodactylidae Odontodactylus scyllarus HM138798 HM138842 HM138886 HM180030 HM180074 Protosquillidae Chorisquilla brooksi Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Chorisquilla excavata HM138776 HM138816 HM138860 HM180004 HM180048 Protosquillidae Chorisquilla gyrosa Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Chorisquilla hystrix* HM138777 HM138817 HM138861 HM180005 HM180049 Protosquillidae Chorisquilla mehtae Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Chorisquilla spinosissima AF205254 N/A N/A N/A N/A Protosquillidae Chorisquilla trigibbosa N/A AF107609 N/A N/A N/A Protosquillidae Chorisquilla tweediei* HM138778 HM138818 HM138862 HM180006 HM180050 Protosquillidae Echinosquilla guerinii HM138780 HM138820 HM138864 HM180008 HM180052 Protosquillidae Haptosquilla corrugata Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Haptosquilla glyptocercus* HM138789 HM138830 HM138874 HM180018 HM180062 Protosquillidae Haptosquilla hamifera KM074037 KM074036 KM074038 KM074039 KM074040 / KM074041 Protosquillidae Haptosquilla moosai Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Haptosquilla nefanda Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Haptosquilla proxima Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Haptosquilla pulchella AF205238 N/A N/A N/A N/A Protosquillidae Haptosquilla stoliura AF205241 N/A N/A N/A N/A Protosquillidae Haptosquilla togianensis Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Haptosquilla trispinosa* HM138790 HM138831 HM138875 HM180019 HM180063 Protosquillidae Haptosquilla tuberosa Barber & Boyce 2006 N/A N/A N/A N/A Protosquillidae Protosquilla folini HM138799 HM138843 HM138887 HM180031 HM180075 Takuidae Taku spinosocarinatus HM138811 HM138855 HM138899 HM180043 HM180087

N/A = sequence not available Barber&Boyce 2006 = sequences provided by authors BOLD = new sequences from this study

Table S2. , color and polarization state of the first maxillipeds, and GenBank accession numbers for sequences for Stomatopoda species from this study. Gene sequences are either from Porter et al. (2010), provided by Barber and Boyce (2006), or new to this study (in bold). * Those species used in polarization behavioural trials.

The Journal of Experimental Biology | Supplementary Material