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Downloaded From: https://bioone.org/journals/Journal-of-Parasitology on 15 Aug 2022Terms of Use: https://bioone.org/terms-of-use ANEWGENUSOFTAPEWORM(CESTODA:ONCHOPROTEOCEPHALIDEA)FROMSAWFISH(ELASMOBRANCHII:PRISTIDAE)J.N.Caira,K.Jensen,andC.A.FylerDepartmentofEcologyandEvolutionaryBiology,75N.EaglevilleRoad,Unit3043,UniversityofConnecticut,Storrs,Connecticut06269-3043.CorrespondenceshouldbesenttoJ.N.Cairaat:janine.caira@uconn.edu:Collectionsfromthedwarfsaw“sh,Pristisclavata,nearDarwin,Australia,in1997ledtothediscoveryofthenewonchoproteocephalideangenusMatticestusn.gen.„ataxonthathasbeenreferredtoinmolecularphylogeneticanalysesinwhichithasbeenincludedasNewgenus8.Itstypespecies,Matticestusanneaen.gen.,n.sp.,andasecondspecies,Matticestuskathleenaen.sp.,aredescribed.PlacementofthistaxonintheOnchoproteocephalideaissupportedmorphologicallyinthatbothspeciesbearascolexwith4bothridiaeachwithapairofbi-prongedhooksandspinitrichesthatextendthroughoutthelengthofthebody.SequencedatafortheD1…D3regionofthe28SrDNAgenealsoplacethegenussolidlyamongtheotherelasmobranch-hostedmembersoftheorder.Thenewgenusdiffersfromtheotherelasmobranch-hostedgeneraintheorderinthatitsmemberspossessacombinationofbiloculatedbothridiawithlaterallappetsontheposteriormarginoftheanteriorloculusandapairofbi-prongedhookswithadistinctivecon“gurationoftuberclesandinternalchannels.Itsmembersarealsoextremelysmall.Insummary,Matticestusn.gen.isanunusuallytiny,spiny,genusofcestodethatseemstoexclusivelyparasitizesaw“shofthegenusPristisThepastalmost2decadeshaveseenaremarkabletransformationinourglobalunderstandingofthetapewormsofsharksandstingrays(i.e.,elasmobranchs).Intotal,202generaarenowrecognizedacrossthe9ordersofcestodesthatparasitizeelasmobranchs(CairaandJensen,2017;HaseliandMalekpourFard,2017).Aphenomenal57(i.e.,28%)ofthesegenerahavebeenerectedinthemere17yrsincetheturnofthe21stcentury.Thispaperaddsanothermembertothisassemblagebyformallyestablishingagenusthathasbeengiventheunof“cialdesignationNewgenus8inthevariousmolecularphylogeneticstudiesinwhichithasbeenincluded(e.g.,Cairaetal.,2014,2017).Thisgenuswas“rstbroughttoourattentionbythelateDr.TomMattis,whohadcollectedmaterialmorethan4decadesagofromanelasmobranchspecimenheidenti“edasthesmalltoothsaw“sh,PristispectinataLatham,offNewOrleans,Louisiana.Unfortu-nately,thatmaterialconsistedofafewmaceratedspecimensthatwereessentiallyimpossibletodescribe,particularlygiventheirtinysize.Manyyearslater,workinginnorthernAustraliaontheparasitesofthedwarfsaw“sh,PristisclavataGarman,incollaborationwithDarwinFisheries,weencounteredspecimensfromP.clavatathatweresimilarto,althoughnotidenticalwith,theMattismaterialfromP.pectinata.Thesespecimenswerenumerousandofsuf“cientlyhighqualitytoallowfortheformalerectionofthegenus.Inhonorofhismanycontributionstothe“eldofcestodology,thisnewgenusbearsthepatronymMatticestusn.gen.OurmaterialfromP.clavataconsistedof2speciesbelongingtothenewgenus,bothofwhicharedescribedbelow.MATERIALSANDMETHODSThecestodesdescribedherecamefrom4individualsof.Theseindividualsco

nsistedof1female(accessionAU-136)98cmintotallength(TL)and3males(accessionsAU-15,AU-36,andAU-138)rangingfrom90to200cminTL.ThehostswerecollectedusinggillnetssetinBuffaloCreeknearDarwin,Australia(12S,130E)between5and15August1997.Asmallsampleoflivertissuefromeachsaw“shwaspreservedin95%ethanoltocon“rmthespeci“cidentityofthehostsusingsequencedata.Theidentitiesofall4saw“shspecimenshavebeencon“rmedbyNayloretal.(2012a).AdditionalinformationofeachhostcanbefoundintheGlobalCestodeDatabase(elasmobranchs.tapewormdb.uconn.edu)bysearchingfortheaccessionnumber,whichisacombinationofcollectioncodeandcollectionnumber(e.g.,AU-15).Thespiralintestinefromeachanimalwasopenedwithamid-ventral,longitudinalincisionand“xedin10%seawater-bufferedformalin(9:1).Before“xation,asubsampleofthewormsfrom1ofthe4hostspecimens(AU-36)wasremovedandpreservedin95%ethanolformolecularwork.MorphologicalmethodsWholemountswerepreparedfollowingconventionaltechniques(seePickeringandCaira,2012).WormswerestainedwithDela“eldshematoxylin,clearedinmethylsalicylate,andmountedinCanadabalsam.MeasurementsweretakenwithanAxioskop2Pluscompoundmicroscope(CarlZeissMicroscopy,Thornwood,NewYork)byusingaSPOTDiagnosticInstrumentDigitalCameraandSPOTversion4.6(SPOTImagingSolutions,SterlingHeights,Michigan).Unlessotherwisestated,measurementsarepresentedinmicrometersasrangevaluesfollowedparentheticallybythemeanSD,wormsamplesize,andtotalnumberofobservationswhenmorethan1measurementperspecimenwasmade,asappropriate.Histologicalsectionswerepreparedforthestrobilaof1specimenofMatticestuskathleenaen.sp.asfollows.Thestrobilawasembeddedinparaf“nandsectionedat8-mintervalsbyusingaCUT4060retractingrotarymicrotome(OlympusCorpo-ration,Melville,NewYork).Sectionsweremountedonglassslides”oodedwith2.5%sodiumsilicateanddriedonaslidewarmerovernight.SectionswerestainedwithGillshematoxylin,counterstainedwitheosin.Theparaf“nwasremovedwithxylene, Received7October2017;revised14January2018;accepted15January*DepartmentofEcologyandEvolutionaryBiologyandtheBiodiversityInstitute,1200SunnysideAvenue,UniversityofKansas,Lawrence,Kansas66045.MarthasVineyardRegionalHighSchool,100EdgartownVineyardHavenRoad,OakBluffs,Massachusetts02577.DOI:10.1645/17-165J.Parasitol.,104(2),2018,pp.133…144AmericanSocietyofParasitologists2018 andthenthesectionsweremountedundercoverslipsonglassslidesinCanadabalsam.Thescolexof3or4specimensand2detachedproglottidsofeachspecies,andawholewormof1species,werepreparedforscanningelectronmicroscopy(SEM)asfollows.Specimenswerehydratedinagradedethanolseries,placedinosmiumtetroxideovernight,dehydratedinagradedethanolseries,placedinhexamethyldisilazane(TedPellaInc.,Redding,California),andallowedtoairdryforatleast1hrinafumehood.Theywerethenmountedonaluminumstubsondouble-sidedPELCOcarbontabs(TedPellaInc.),sputtercoatedwith30nmofgold/palladium,andexaminedwithaLEO/ZeissDSM982Gemini“eldemissionscanningelectronmicroscope(CarlZeissMicros-copy)oraNovaNanoSEM450“eldemissionscanningelectronmicroscope(FEI,Hillsboro,Oregon).StubshavebeenretainedinthepersonalcollectionofC.A.F.Museu

mabbreviationsusedareasfollows:LRP,LawrenceR.PennerParasitologyCollection,UniversityofConnecticut,Storrs,Connecticut,U.S.A.;QM,QueenslandMuseum,Bris-bane,Australia;andUSNM,NationalMuseumofNaturalHistory,SmithsonianInstitution,Washington,D.C.,U.S.A.MicrothrixterminologyfollowsChervy(2009).Hookmeasure-mentletteringsystemfollowsGhoshroyandCaira(2001);proglottidmeasurementsweretakenfromterminalproglottidsunlessotherwiseindicated.MolecularmethodsTotalgenomicDNAwasextractedfromthestrobilaof2or3specimensofeachspeciespreservedin95%ethanolfromhostspecimenAU-36byusingastandardphenol…chloroformprotocol(Hillisetal.,1996).TheD1…D3regionofthe28SrDNAgenewastargetedbecauseofitspreviouslyestablishedutilityinresolvinginterspeci“crelationshipsinothercestodegroups(seeCairaandJensen,2017,andreferencestherein).Forampli“cation,LSU5(Littlewoodetal.,2000)wasusedastheforwardprimerand1500R(Tkachetal.,2003)wasusedasthereverseprimer.Forsequencingreactions,LSU5wasusedastheforwardprimerand1200R(Lockyeretal.,2003)wasusedasthereverseprimer.Inallcases,the28SrDNA(D1…D3)sequencedataweregeneratedfromspecimensofthenewgenusthatwerephotovoucheredbeforeDNAextraction.ThesephotovouchershavebeendepositedintheLRP;accessionnumbersareincludedinthetaxonlabelsinthetreeprovidedhereinandinthedescriptionsofeachspecies,alongwiththecorrespondingGenBankaccessionnumbers.Polymerasechainreaction(PCR)wasperformedin25-lreactionsbyusing2…100ngoftemplateDNA,0.25Mofeachprimer,1MMgCl,0.12mMofeachdNTP,and1.2unitsofTaqDNApolymerase,withannealingtemperaturesbetween50and60C.Cyclesequencingconditionswereasfollows:initialdenaturationfor1minat94C,followedby40cyclesof30secat94C,30secat50…60C,1minat72C,andcompletedby5minat72C.PCRproductswerepuri“edusingNucleospinextractionkit(BDBiosciences,SanJose,California),cyclesequencedwiththeappropriateprimerusingBigDyedideoxyterminatorsversion1.1andsequencedonanABIPRISM3100geneticanalyzer(AppliedBiosystems,FosterCity,California).Toassessthephylogeneticrelationshipsofthenewspecies,andthusalsotoexplorethenovelnatureofthegenus,rDNAsequencedataforadiversityofrelevanttaxawereobtainedfromGenBank.InadditiontothedataforaspecimenMatticestusreferredtoasNewgenus8n.sp.1(TE-92)inCairaetal.(2014),thesespecimensconsistedof13ofthe14otheronchoproteocephalideanspeciesalsoincludedintheanalysesofCairaetal.(2014).Becauseitlacksavoucher,Proteocephalusmacrocephalus(Diesing,1850)LaRue,1914,wasreplacedinouranalysesbyProteocephaluskuyukuyuWoodland,1935fromdeChambrieretal.(2015).Alsoincludedasmembersoftheingroupinouranalysesherewere24adultspecimensrepresenting13speciesofAcanthobothriumBlan-chard,1848beyondthe2includedbyCairaetal.(2014).ThesedatacamefromFyler(2011),Fyleretal.(2009),FylerandCaira(2006,2010),andJensenandBullard(2010).OutgrouptaxaconsistedofCaulobothriumopisthorchisRiser,1955;Paraorygmatobothriumexiguum(Yamaguti,1935)Ruhnke,1994;andRhoptrobothriumcf.gambangiJensenandCaira,2006,allalsofromCairaetal.(2014).Foreachspecimen,themuseumaccessionnumberofthehologenophoreandthecorrespondingGenBankaccessionnumberareincludedinthetax

onlabelinthetreepresentedherein.Specimensforwhichnovelsequencedataweregeneratedhereareindicatedinboldinthat“gure.Sequenceswereassembled,aligned(usingMUSCLEAlignmentwith100iterationsanddefaultsettingsotherwise;Edgar,2004),andtrimmedtoremoveleadingandtrailinggapsbyusingGeneious10.1.3(BiomattersInc.,Newark,NewJersey).Ambig-uouslyalignedregionswereidenti“edandexcludedusingGblocks0.91b(Castresana,2000)underparametersforlessstringentcharacterselectionforexclusion(i.e.,minimumnumberofsequencesforaconservedposition,25;minimumnumberofsequencesfora”ankingposition,25;maximumnumberofcontiguousnonconservedpositions,8;andminimumlengthofablock,5).ThenumberofconstantcharacterswasdeterminedusingPAUP*version4.0a157(Swofford,2003)fromthematrixwiththeambiguouslyalignedregionsexcluded,bothwithandwithoutoutgroups.GTRwasdeterminedtobethemostappropriatemodelofnucleotideevolutionfortheanalysesusingthecorrectedAkaikeInformationCriterion(AICc)implementedinjModeltest2.1.7v20150220(Darribaetal.,2012)followingevaluationofatotalof88models.Maximumlikelihood(ML)analyseswereperformedusingserialimple-mentationofGarli-2.01(Zwickl,2006).TwentyindependentMLrunswereconductedinGarli-2.01byusingdefaultsettingswiththefollowingadjustments:genthreshfortopoterm100000andsigni“canttopochange0.0001.Bootstrapsup-portwasestimatedusingserialimplementationofGarli-2.01.Intotal,200bootstrapreplicateswereconductedusingthesamecon“gurationandmodelaswereusedfortheMLanalyses,withfollowingchangesfromthedefaultsettings:genthreshfortopo-term10000,signi“canttopochange0.01,andtreerejec-tionthreshold20.0.Bootstrapresultsweresummarizedontheoptimaltree(i.e.,thebesttreeresultingfromthe20independentMLruns)byusingSumTrees4.0.0inDendroPy4.0.3(SukumaranandHolder,2010)availableathttps://github.com/jeetsukumaran/DendroPy).134THEJOURNALOFPARASITOLOGY,VOL.104,NO.2,APRIL2018 n.gen.Scolexconsistingofscolexproperandcephalicpeduncle.Scolexproperwith4bothridia;eachbothridiumwithanteriormuscularpadbearing1accessorysuckerand1pairofhooks,dividedinto2loculibytransverseseptum;anteriorloculuslongerthanposteriorloculus;postero-lateralmarginsofanteriorloculuswithlappetsextendingtoposteriormarginofbothridium.Hooksbi-pronged,hollow;accessorypiecebetweenbasesofmedialandlateralhooksabsent;internalchannelofhooksnotextendingintobasesofhooks.Distalandproximalbothridialsurfaceswithacicular“litriches;somesmallgladiatespinitrichesonproximallateralsurfacesofbothridia.Cephalicpeduncleandstrobilawithconspicuousgladiatespinitriches;spinitrichesdenselyarrangedoncephalicpredunclebecomingmoresparsealonglengthofstrobila.Proglottidsacraspedote,non-laciniate,euapolytic.Genitalporeslateral,irregularlyalternating.Testesnumerous,entirelyanteriortoovary,in2to3columns,1layerdeepincrosssection;post-poral“eldoftestespresent.Cirrusarmedwithspinitriches.Ovaryposterior,invertedA-shapedinfrontalview,bilobedincrosssection.Vaginaopeninganteriortocirrussacintogenitalatrium.Vitellariumfollicular;folliclesin2lateralbands;eachbandconsisting1dorsaland1ventralcolumnoffollicles,extendingfromne

aranteriorofproglottid,stoppingshortofposteriormarginofovary,interruptedbyterminalgenitalia.Uterussaccate,medioventral,beginningatovarianisthmus,notreachinganteriormarginofproglottid.Excretoryducts4,arrangedin1dorsaland1ventralpair.Eggsspherical.Parasitesofsaw“shofthegenusLinck(Rhinopristiformes:PristidaeBonaparte);circumglobalindistribution.Typespecies:Matticestusanneaen.gen.,n.sp.Additionalspecies:Matticestuskathleenaen.sp.ThisgenushonorsthelateDr.TomMattisnotonlyforhisdiscoveryofthe“rstofitsmembersbutalsoforhisremarkablecontributionstoourknowledgeofthelifecyclesofelasmobranch-hostedcestodes.Inthemostrecenttreatmentoftheelasmobranch-hostedonchoproteocephalideans,Cairaetal.(2017)recognized11validgenera.Matticestusn.gen.isreadilydistinguishedfrommorethanhalfofthesebasedonthepresenceandcon“gurationofitshooks.UnlikeProsobothriumCohn,1902,itbears,ratherthanlacks,hooks.EachofthehooksinapairinMatticestusn.gen.isbi-pronged,ratherthantri-pronged,asinPhoreiobothriumLinton,1889andTriloculatumCairaandJensen,2009,bothofwhich,unlikethenewgenus,alsobearaposterior-mostloculusthatisdividedbysubloculi.Thebi-prongednatureofthehooksMatticestusn.gen.alsodistinguishesitfromOnchobothriumdeBlainville,1828andPotamotrygonocestusBrooksandThorson,1976,bothofwhichbearuni-prongedhooks.Itfurtherdiffersfromthesegenerainitspossessionof2,ratherthan3or1,loculiperbothridium,respectively.UnlikeAcanthobothriumAcanthobothroidesBrooks,1977,PinguicollumRiser,1955,andPlatybothriumLinton,1890,thebothridiaofMatticestus,eachbear2,ratherthan3,loculi.ItfurtherdiffersfromAcanthobothriuminthattheinternalchannelofeachhookdoesnotextendintothehookbase;itpossesses,ratherthanlacks,postero-lateralbothridiallappetsthatextendtotheposteriormarginofthebothridia;anditpossesses,ratherthanlacks,gladiatespinitrichesthatextendthroughoutmuchofthelengthofthestrobila.ItfurtherdiffersfromPinguicolluminthatitlacksalayeroftissuecoveringitsbothridiaandfromPlatybothriuminthatthetalonsofitshooksareintheformoftuberclesontheproximalsurfacesoftheaxialprongs,ratherthanextendingfreelybetweentheaxialandabaxialhookprongs.ThenewgenusmostcloselyresemblesMegalonchosBaerandEuzet,1962andspeciescurrentlyassignedtoUncibilocularisSouthwell,1925(seeJensenandCaira,2008)initspossessionofbothbi-prongedhooksand2loculiperbothridium.However,unliketheformergenus,itpossesses,ratherthanlacks,post-poraltestesanditsuterusextendswellbeyond,ratherthanstopsshortof,thecirrussac.UnlikeUncibilocularis,speciesofMatticestusn.gen.bearlarge,gladiatespinitrichesthroughoutthelengthoftheirstrobila.Further-more,thehooksofthenewgenuslackthematrixofdarktissuethatsurroundsthebasesofthehooksofspeciesofUncibilocu-laris.Insummary,Matticestusn.gen.isatiny,spiny,genusofcestodethatseemstoexclusivelyparasitizesaw“shofthegenusPristisMatticestusanneaen.gen.,n.sp.(Figs.1,2)Description(basedon11completeworms;1matureand2graviddetachedproglottids;and1worm,3scoleces,and2detachedproglottidsexaminedwithSEM):Worms860…1,100(95211)long,greatestwidthatlevelofterminalproglottid,7…9(7.80.6;11)proglottidsperwo

rm;eupolytic.Scolexconsistingofscolexproperandcephalicpeduncle.Scolexproperwith4bothridia,135…173(14611;11)longby93…120(1049;11)wide.Bothridiafreeposteriorly,45…56(503;11)wide,eachwithspecializedanteriorregioninformofmuscularpadand2loculi;muscularpad36…50(444;9;18)longby35…55(449;17)wide,triangularinshape(Fig.2B),bearingaccessorysuckerand1pairofhooksatposteriormargin;accessorysucker14…254;6;9)longby21…30(273;6;9)wide;anteriorloculus68…87(755;11;20)longatcenter,withconcaveposteriormarginandpostero-laterallappets;lappetsextendingtoposteriormarginofbothridium;posteriorloculus22…26(241;11;20)long;posterior-to-anteriorloculuslengthratio1:2.8…3.6(3.20.3;11;20).Velumbetweenmedialmarginsofdorsalandventralpairsofbothridiainconspicuous.Hooksbi-pronged,hollow,withtubercleonproximalsurfaceofeachaxialprong;internalchannelsofaxialandabaxialprongscontinuous,smooth,notextendingintohookbases;axialprongoflateralhookslightlyshorterthanabaxialprong;axialandabaxialprongsofmedialhookapproximatelyequalinlength;lateralandmedialhooksapproximatelyequalinsize.Lateralhookmeasurements:A23…271;11;21),B37…51(434;11;21),C40…58(505;11;21),D52…72(635;11;21).Medialhookmeasurements:A27(252;11;22),B42…61(505;11;22),C40…59(5011;22),D60…79(705;11;22).Basesoflateralandmedialhooksapproximatelyequalinlength.Cephalicpeduncle33…12025;11)longby22…44(345;11)wideatposteriormargin.Muscularpad(Fig.2F)anddistal(Fig.2G)andproximal(Fig.2I,H)surfacesofbothbothridialloculicoveredwithacicular“litriches;proximalsurfaceoflateralmarginofanteriorloculusofCAIRAETAL.—ANEWCESTODEGENUSFROMSAWFISH135 1.„LinedrawingsofMatticestusanneaen.gen.,n.sp.()Wholeworm(holotype,QMG236758).()Scolex(holotype,QMG236758).(Hooks(paratype,USNM1470750).()Terminalproglottid(paratype,USNM1470750).()Terminalgenitaliafromdetachedmatureproglottid(paratype,QMG236763).136THEJOURNALOFPARASITOLOGY,VOL.104,NO.2,APRIL2018 2.„ScanningelectronmicrographsofMatticestusanneaen.gen.,n.sp.()Wholeworm;smallletterindicateslocationofdetailinD.(Scolex;smalllettersindicatelocationsofdetailsinE…I.()Detailofapicalregionofbothridiumandhooks.().Microtrichesoncephalicpeduncle.(Narrowbandofgladiatespinitrichesonproximallateralsurfaceofanteriorloculus.()Acicular“litrichesonmuscularpad.()Acicular“litrichesondistalbothridialsurface.()Acicular“litrichesonmedialproximalbothridialsurfacerim.()Acicular“litrichesonproximalbothridialsurface.(Partiallyevertedcirrus;smalllettersindicatelocationsofdetailsinKandL.()Rostratespinitrichesandacicular“llitrichesonbaseofcirrus.(Uncinatespinitrichesandcapilliform“litrichesonmoredistalportionsofcirrus.CAIRAETAL.—ANEWCESTODEGENUSFROMSAWFISH137 bothridiaadjacenttorimwithnarrowbandofsmallgladiatespinitriches(Fig.2E).Cephalicpeduncle(Fig.2A,B)andstrobilacoveredwithgladiatespinitrichesandcapilliform“litriches;gladiatespinitrichesbecominglessdensealonglengthofstrobila(Figs.1A,2A).Proglottidsacraspedote,protandrous.Immatureproglottids6…8(70.7;11)innumber;matureproglottids1innumber.Terminalproglottid386…525(43458;11)longby116…160(13615;11)wide,length-to-widthratio2.5

…4(3.20.5;11):1;detachedmatureproglottid(n1)689longby162wide,length-to-widthratio4.25:1;detachedgravidproglottids(n2)608…721longby267…284wide,length-to-widthratio2.1…2.7:1.Genitalporesmarginal,irregularlyalternating,51…62%(563;11)ofproglottidlengthfromposteriorend;57%indetachedmatureproglottid(n1);60…62%indetachedgravidproglottids(nTestesirregularlyovalinfrontalview,18…50(368;11;42)longby20…50(358;11;41)wide,arrangedin2regularcolumnsanteriortoovary,1layerdeep,9…13(111;11;21)intotalnumber,1…2(1.20.4;11;21)inpost-poral“eld.Vasdeferenscoiledatanterio-medialmarginofcirrussac.Cirrussacspherical,55…83(7110;11)longby40…55(476;1)wide;78longby72wideindetachedmatureproglottid(n1);104…110longby90…96wideindetachedgravidproglottids(n2),containingcoiledcirrus;baseofcirruscoveredwithsparselyarrangedrostratespinitrichesandacicular“litriches(Fig.2K),followeddistallybyrobustlarge,uncinatespinitrichesandcapilliform“litriches(Fig.2L).Vaginarelativelythick-walled,straighttoweaklysinuous,extendingalongmediallineofproglottidfromootypetoanteriormarginofcirrussac,thenlaterallyalonganteriormarginofcirrussactoopenintocommongenitalatriumanteriortocirrus;vaginalsphincterobservedonlyingravidproglottids;seminalreceptaclenotseen.Ovaryoccupyingposteriorthirdofproglottid,invertedA-shapedinfrontalview,bilobedincrosssection,withlobulatemargins,38…90(6315;11)wideatisthmus,asymmetrical,withaporallobeslightlylongerthanporallobe;porallobe108…18022;11)long;aporallobe110…208(13927;11);ovarianisthmusinanteriorhalfofovary.Mehlisglandposteriortoovarianisthmus.Vitellariumfollicular,in2lateralbands;eachbandconsistingof1dorsaland1ventralcolumnoffollicles,extendingfromnearanteriorlimitof“eldoftestestonearovarianisthmus,interruptedbyterminalgenitalia;vitellinefolliclesirregularinshape.Uterusmedio-ventral,saccate,thin-walled,extendingalongmediallinefromovarianisthmustonearanteriorlimitof“eldoftestes.Excretoryducts4,arrangedin1dorsaland1ventralpair.Eggsindetachedgravidproglottidsspherical,12…13(12.50.2;2;8)longby12…13(12.50.4;2;8)wide.TaxonomicsummaryTypeandonlyknownhost:PristisclavataGarman,dwarfsaw“sh(Rhinopristiformes:Pristidae).Siteofinfection:Spiralintestine.Typelocality:BuffaloCreeknearDarwin(12E),NorthernTerritory,Australia,TimorSea,IndianFourof4hostsexamined(100%).ThisspecieshonorsAnneSt.OngeinrecognitionofherdecadesofserviceastheremarkablegraduatestudentcoordinatorofBiologicalSciencesattheUniversityofConnect-Specimensdeposited:Holotype(QMG236758)and5paratypes(4wholewormsand1detachedmatureproglottid;QMG236759-G236763),4paratypes(3wholewormsand1detachedgravidproglottid;USNM1470750-1470753),and4paratypes(3wholewormsand1detachedgravidproglottid;LRP9315…9318);specimenspreparedforSEMretainedwithJ.N.C.attheUniversityofConnecticut.Photographicvouchersofspecimensforwhich28SrDNA(D1…D3)sequencedataweregeneratedaredepositedintheLRP(9313…9314).Matticestuskathleenaen.sp.(Figs.3,4)Description(basedon8completematureworms,3maturedetachedproglottids,crosssectionseriesof1matureproglottid,and4scolecesand2detachedproglottidsexaminedwithSEM)

:Worms1,465…2,925(1,985458;8)long,greatestwidthatlevelofterminalproglottid,10…18(163;8)proglottidsperworm;eupolytic.Scolexconsistingofscolexproperandcephalicpeduncle.Scolexproperwith4bothridia,180…208(19111;8)longby160…186(17211;7)wide.Bothridiafreeposteriorly,70…88(816;8)wide,eachwithspecializedanteriorregioninformofmuscularpadand2loculi;muscularpad35…55(456;8;12)longby48…73(647;8;12)wide,triangularinshape(Figs.3B,4A,4B),bearingaccessorysuckerand1pairofhooksatposteriormargin;accessorysucker23…28(262;6;10)longby26…40(334;10)wide;anteriorloculus98…118(1066;8;15)longatcenter,withconcaveposteriormarginandpostero-laterallappets;lappetsextendingtoposteriormarginofbothridium;posteriorloculus33…47(404;8;15)long;posterior-to-anteriorloculuslengthratio1:2.23…3.38(2.690.3;8;15).Velumbetweenmedialmarginsofdorsalandventralpairsofbothridiainconspicuous.Hooksbi-pronged,hollow,withtubercleonproximalsurfaceofeachaxialprong;internalchannelsofaxialandabaxialprongscontinuous,smooth,notextendingintohookbases;abaxialprongoflateralhookslightlyshorterthanaxialprong;axialandabaxialprongsofmedialhookapproximatelyequalinlength;lateralandmedialhooksapproximatelyequalinsize.Lateralhookmeasurements:A29…34(311;8;16),B50…63(573;8;16),C44…62(515;8;16),D73…87(804;8;15).Medialhookmeasurements:A27…34(302;8;17),B55…68(604;8;15),C45…62(525;8;17),D72…90(815;8;14).Basesoflateralandmedialhooksapproximatelyequalinlength.Cephalicpeduncle108…238(19841;8)longby42…61(516;8)wideatposteriormargin.Muscularpad,distal(Fig.4E),andproximal(Fig.4F,G)surfacesofbothbothridialloculicoveredwithacicular“litriches;proximalsurfaceoflateralmarginofposteriorregionofanteriorloculusalsowithrestrictedpatchofsmallgladiatespinitriches(Fig.4D).Cephalicpeduncle(Fig.4C)andstobilacoveredwithgladiatespinitrichesandcapilliform“litriches;gladiatespini-trichesdenseoncephalicpedunclebecomingverysparsetowardsposteriorofstrobila(Fig.1D).Proglottidsacraspedote,protandrous.Immatureproglottids10…17(152;8)innumber;matureproglottids1innumber.Terminalproglottid327…732(516140;8)longby140…285(21344;8)wide,length-to-widthratio1.7…3.8(2.50.8;8):1;detachedmatureproglottids(n3)729…810longby259…308wide,length-to-widthratio2.5…2.9:1.Genitalporesmarginal,138THEJOURNALOFPARASITOLOGY,VOL.104,NO.2,APRIL2018 3.„LinedrawingsofMatticestuskathleenaen.sp.()Wholeworm(holotype,QMG236764).()Scolex(holotype,QMG236764).(Hooks(paratype,QMG236765).()Terminalproglottid(paratype,USNM1470754).()Terminalgenitalia(paratype,USNM1470754).CAIRAETAL.—ANEWCESTODEGENUSFROMSAWFISH139 irregularlyalternating,47…57%(524;8)ofproglottidlengthfromposteriorend;54…61%indetachedmatureproglottids3).Testesirregularlyovalinfrontalview,15…35(256;8;32)longby36…76(5912;8;32)wide,arrangedin2…3irregularcolumnsanteriortoovary,1layerdeepincrosssection,27…34(302;8;21)intotalnumber,2…3(2.60.5;8;21)inpost-poral“eld.Vasdeferenscoiledatanterio-medialmarginofcirrussac.Cirrussacspherical,80…125(11115;8)longby43…85(5613;8)wide,containingcoiledcirrus;cirrusdenselycoveredwithnarrow,uncinatespinitrichesandcapilliform“litriche

sthroughoutitslength(Fig.4H,I).Vaginarelativelythick-walled,straighttoweaklysinuous,extendingalongmediallineofproglottidfromootypetoanteriormarginofcirrussac,thenlaterallyfollowinganteriormarginofcirrussactoopenintocommongenitalatriumanteriortocirrus;vaginalsphincterabsent;seminalreceptaclenotseen.Ovaryoccupyingposteriorthirdofproglottid,invertedA-shapedinfrontalview,bilobedincrosssection,withweaklylobulatemargins,73…125(1008)wideatisthmus,essentiallysymmetrical;porallobe85…250(16056;8)long;aporallobe95…258(16151;8)long;ovarianisthmusinanteriorhalfofovary.Mehlisglandposteriortoovarianisthmus.Vitellariumfollicular,consistingof2lateralbands;eachbandconsistingof1dorsaland1ventralcolumnoffollicles,extendingfromnearanteriorlimitof“eldoftestestonearposteriorofovary,interruptedbyterminalgenitalia;vitellinefolliclessomewhatirregularinshape.Uterusmedio-ventral,saccate,thin-walled,extendingalongmediallinefromovarianisthmustonearanteriorlimitof“eldoftestes.Excretoryducts4,arrangedin1dorsaland1ventralpair.Eggsnotseen. 4.„ScanningelectronmicrographsofMatticestuskathleenaen.sp.()Scolex;smalllettersindicatelocationsofdetailsinC…G.()Detailofapicalregionofbothridiumandhooks.()Microtrichesoncephalicpeduncle.()Narrowbandofgladiatespinitrichesonproximallateralbothridialsurface.()Acicular“litrichesondistalbothridialsurface.()Acicular“litrichesonproximallateralbothridialsurface.()Acicular“litrichesonproximalmedialbothridialsurface.()Partiallyevertedcirrus;smallletterindicateslocationofdetailsinI.()Narrowuncinatespinitrichesandcapilliform“litrichesoncirrus.140THEJOURNALOFPARASITOLOGY,VOL.104,NO.2,APRIL2018 TaxonomicsummaryTypeandonlyknownhost:PristisclavataGarman,dwarfsaw“sh(Rhinopristiformes:Pristidae).Siteofinfection:Spiralintestine.Typelocality:BuffaloCreeknearDarwin(12E),NorthernTerritory,Australia,TimorSea,IndianFourof4hostsexamined(100%).ThisspecieshonorsKathleenTeboforhermorethan2decadesofphenomenalserviceasAdministrativeAssistantfortheDepartmentofEcologyandEvolutionaryBiologyattheUniversityofConnecticut.Specimensdeposited:Holotype(QMG236764)and3paratypes(2wholewormsand1detachedmatureproglottid;QMG236765-G236767);3paratypes(2wholeworms,1detachedmatureproglottid;USNM1470754-1470756);and7paratypes(3wholewormsand1detachedmatureproglottid[LRP9322…9325];2SEMvouchers[LRP9326…9327];andcrosssectionseriesofmatureproglottidanditsscolexvoucher[LRP9328…9330]);specimenspreparedforSEMretainedwithJ.N.C.attheUniversityofConnecticut.Photographicvouchersofspecimensforwhich28SrDNA(D1…D3)sequencedataweregeneratedaredepositedintheLRP(9319…9321).Matticestuskathleenaen.sp.iseasilydistinguishedfromitsonlydescribedcongener,M.anneae,initslargersize(1,465…2,925vs.860…1,100intotallength);greaternumberofproglottids(10…18vs.7…9);and,mostconspicuously,initsgreaternumberoftestes(27…34vs.9…13).AssumingthebaseofthecirruswasvisibleinbothspeciesexaminedwithSEM(Figs.2J,4H),thecirrusarmatureofthe2speciesalsoappearstodiffersubstantiallyinboththetypeandarrangementofspinitriches(Fig.2J…Lvs.Fig.4H,I).Finally,thehooksofthe2speciesdiffe

rsomewhatinshape;whereasinM.anneaetheabaxialprongsarestraight,intheabaxialprongsareslightlycurved.MolecularphylogenyThematrixofsequencesfromall49specimenswas1,406bpinlength,ofwhich124bpwereexcludedusingGblocks.Thereduceddatamatrixcomprised765constantand517variablecharacters,377ofwhichwereparsimonyinformative.Intheingroupalone,therewere790constantcharactersand492variablecharacters,349ofwhichwereparsimonyinformative.The4specimensofkathleenaewereidenticalinsequence.The2specimensofM.anneaedifferedfrom1anotherby2bp.Thetreeresultingfromphylogeneticanalysisofthepartial28SrDNAsequencedataisshowninFigure5. 5.„Topologyofoptimaltree(i.e.,withalikelihoodscoreof8945.7066)resultingfromMLanalysis;non-elasmobranchhostedonchoproteocephalideansindicatedwith“shicons.NodalsupportgivenasMLbootstrapvalues;onlybootstrapvalues50%arepresented.Taxonlabelsinclude(inparentheses)specimennumbers,accessionnumbersofvouchers,andGenBanknumbers;*indicatesspecimensforwhichonlyphotovouchershavebeendeposited;specimensforwhichnovelsequencedataweregeneratedareindicatedinbold.ScalebarlengthindicatessubstitutionsperCAIRAETAL.—ANEWCESTODEGENUSFROMSAWFISH141 Theanalysisyielded2highlysupportedcladesofMatticestusspecimens,eachofwhichconsistedofthereplicatespecimensofthe2newspecies.Incombination,these2groupscomposedawell-supportedcladethatgroupedamong,butindependentlyfrom,the8othergeneraofelasmobranch-hostedonchoproteocephalideansincludedintheanalysis.OurassignmentofMatticestustotheorderOnchoproteoce-phalideaiswellsupportedfromamorphologicalstandpoint.Itsspeciesresemblemostoftheotherelasmobranch-hostedspeciesintheorderintheirpossessionofascolexwith4bothridia,eachofwhichisarmedwithapairofhooks,andproximalbothridialsurfacesandcephalicpedunclewithlarge,gladiatespinitrichesthatextendthroughoutthelengthofthestroblia(Figs.1A,D,2A,3A,D).Fromamolecularstandpoint,consistentwiththeresultsofCairaetal.(2014),thetreeresultingfromouranalysis28SrDNAdataplacethegenusamongtheelasmobranch-hostedonchoproteocephalideans.Thatsaid,herewefoundittogroupassistertoacladeconsistingoftheProteocephalidaethesinglerepresentativesofPotamotrygonocestusspeciesofAcanthobothriumandthesinglespeciesofUncibilocularis,ratherthanassistertoacladeconsistingofonlythelatter2generaaswasfoundbyCairaetal.(2014).Clearly,theaf“nitiesofMatticestuswithintheonchoproteocephalideansremainuncer-taingiventhehypothesizedsister-groupofthegenuswasnotwellsupportedinthetreesresultingfromeithertheCairaetal.(2014)orouranalyses.Ourworkdoesnothingtohelpinformtheaf“nitiesof,whichwasincludedhereasaningroupforthesakeofcompleterepresentationofputativegeneraofelasmo-branch-hostedonchobothriideansforwhichcomparablesequencedataareavailable.Basedonitsconspicuousmorphologicalresemblancetotheelasmobranch-hostedonchoproteocephali-deans,Cairaetal.(2014,2017)persistinconsideringitamemberofthatorder.Thatordinalplacementis,however,notsupportedbytheresultsofexistingmolecularanalyses,inwhichitispositionedatvariouspointsacrossthecestodetree,oftenoutsideoftheOnchoproteocephalidea.Resolutionofthisconundrumawa

itstheapplicationofsequencedataforadditionalmolecularTheunusuallysmallsizeofspeciesofMatticestus,giventhesizeoftheirde“nitivehosts,whichmayattainatotallengthofmorethan3m(Lastetal.,2016),raisesinterestingquestionsaboutthepossible“nalintermediatehostsofmembersofthisgenus.ThereisnoreasontobelievethebiologyofMatticestusdiffersfromthatofessentiallyallothermarinecestodes(CairaandReyda,2005).Thus,wehypothesizethatthelifecycleofspeciesofMatticestusinvolvesasequenceofseveraldifferenthosts,butalsothatthemetacestodesaretrophicallytransmittedamongthesehosts.Inthiscontext,thedietdataavailableforclavataareinterestingtoconsider.IntheKimberleyregionofWesternAustralia,Thorburnetal.(2008)reportedthepopeyemullet(Rhinomugilnasutas[DeVis])asthemajorpreyitemtheywereabletoidentifyinthestomachcontentsofP.clavataalthoughevidenceofprawnswasalsofound.InhisunpublishedMastersthesis,Peverell(2009)reportedthestomachcontentsofP.clavatainnorthernAustraliatoincludesciaenidssuchasNibeasquamosaSasaki,leptobramidssuchasLeptobramamuelleriSteindachner,clupeids,andmugilids.Beyondtheseteleosts,healsoreported“ndingcrustaceanssuchasthebananashrimp,PenaeusmerguiensisdeMan,inthestomachsofsomespecimens.Asaconsequence,alloftheaboveshouldbeconsideredpossibleintermediatehostsforthemetacestodesofspeciesofMatticestusTheglobalsurveyworkwehaveconductedoverthepastseveraldecades(seeCairaandJensen,2017)leadsustobelievethatspeciesofMatticestusareexclusivelyparasitesofsaw“shes.Thishighlysimpli“esestimationoftheglobaldiversityofthiscestodegenusbecausethenumberofsaw“shspeciesinhabitingtheplanetisextremelylimited.Inthemostrecenttaxonomictreatmentoftheraysoftheworld,Lastetal.(2016)followingtheworkofFariaetal.(2013),recognizedthefollowingvalidspeciesofsaw“shes:Anoxypristiscuspidata(Latham),P.clavataP.pectinataPristispristis(L.),andPristiszijsronBleeker.RecognitionofPristismicrodonLathamasasixthvalidspecieshasyettoberesolved.NADH2sequencedatageneratedbyNayloretal.(2012a)provideevidencethattheAustralianandAtlanticspecimensattributedtoP.pristisaredistinct;thus,microdonshouldperhapsbeconsideredvalidratherthanasynonymofP.pristis.Regardless,theglobaltotalofpristidswouldnotexceed6species.Beyondthe2speciesofMatticestusdescribedherefromP.clavata,thespecimensfromDr.TomMattiscon“rmtheexistenceofathirdmemberofthegenusinpectinata,andourcollectionsoverthepastseveraldecadesfromP.zijsronandP.pristis(orP.microdon)fromAustralianwatershaveyieldedyetadditionalspeciesofthenewgenus,allofwhichawaitformaldescription.Thus,wepredictthatspeciesofPristiswillultimatelybefoundtohost,atamaximum,10speciesofMatticestusintotal.Incontrast,wepredictthatthemonotypicAnoxypristisWhiteandMoy-ThomasisnotanappropriatehostofspeciesofMatticestus.Since1991,wehavehadtheopportunitytoexamine7specimensofA.cuspidatafromseveraldifferentlocalitiesinAustralia.UnlikespeciesofPristis,noneofthesespecimenswasfoundtohostMatticestus,suggestingthat,amongsaw“shes,MatticestusislikelyrestrictedtothegenusPristis.ThisisconsistentwiththeresultsofthephylogeneticanalysesofNayloretal.(2012b),providingeviden

cethatAnoxypristisisthesistertaxonoftheguitar“shgenusGlaucostegusBonaparte,ratherthanofPristis„andthusthesaw“shesdonotrepresentamonophyleticgroup.FurthersupportingtherestrictedhostassociationsofMatticestusisthefactthatwehavealsoexamined3…19specimensofall6speciesofGlaucostegusrecognizedbyLastetal.(2016)andnonewerefoundtohostthenewgenus.Wearegratefultoseveralreviewerswhoprovidedvaluablesuggestionsforimprovingthismanuscript.WethankJulieLloydandRichardMounseyoftheFisheriesDivisionoftheDepart-mentofPrimaryIndustryandFisheriesDarwinforarrangingforpermissionforustoexaminesaw“shinBuffaloCreekandforassistingwiththeactualcollectionofthesehosts.ElizabethJockuschoftheUniversityofConnecticutprovidedthefacilitiesforthemolecularelementsofthisstudy.SEMwasconductedintheBioscienceElectronMicroscopyLaboratoryattheUniversityofConnecticut.Thisworkwassupportedinpartbyfundsfrom142THEJOURNALOFPARASITOLOGY,VOL.104,NO.2,APRIL2018 NationalScienceFoundationawards0818696,0818823,1457762,and1457776.LITERATURECITED,J.N.,K.J(eds.).2017.Planetarybiodiversityinventory(2008…2017):Tapewormsfromvertebratebowelsoftheearth.UniversityofKansas,NaturalHistoryMuseum,SpecialPublicationNo.25,Lawrence,Kansas,464p.,J.N.,K.JV.A.I.2017.Onchoproteo-cephalideaIICaira,Jensen,Waeschenbach,Olson&Little-wood,2014.Planetarybiodiversityinventory(2008…2017):Tapewormsfromvertebratebowelsoftheearth,J.N.Caira,andK.Jensen(eds.).UniversityofKansas,NaturalHistoryMuseum,SpecialPublicationNo.25,Lawrence,Kansas,p.,J.N.,K.J,A.W,P.D.OT.J.L.2014.Ordersoutofchaos„molecularphylogeneticsrevealsthecomplexityofsharkandstingraytapewormrelationships.InternationalJournalforParasitol-AIRA,J.N.,ANDF.B.REYDA.2005.Eucestoda(trueMarineparasitology,K.Rohde(ed.).CSIROPublishing,Collingwood,NewSouthWales,Australia,p.92…,J.2000.Selectionofconservedblocksfrommultiplealignmentsfortheiruseinphylogeneticanalysis.MolecularBiologyandEvolution,L.2009.Uni“edterminologyforcestodemicrotriches:AproposalfromtheInternationalWorkshopsonCestodeSystematicsin2002…2008.FoliaParasitologica,D.,G.L.T,R.DD.P.2012.jModelTest2:Moremodels,newheuristicsandparallelcomputing.NatureMethods,A.,A.W,M.F,T.SJ.M.2015.Alarge28SrDNA-basedphylogenycon“rmsthelimitationsofestablishedmorphologicalchar-actersforclassi“cationofproteocephalideantapeworms(Platyhelminthes,Cestoda).ZooKeys,R.C.2004.MUSCLE:Multiplesequencealignmentwithhighaccuracyandhighthroughput.NucleicAcidsResearch,V.V.,M.T.M,P.C,T.R.W,C.A.G.J.P.N.2013.Speciesdelinea-tionandglobalpopulationstructureofcriticallyendangeredsaw“shes(Pristidae).ZoologicalJournaloftheLinnean,C.A.2011.Anextremelyhyperapolyticspecies(Cestoda:Tetraphyllidea)fromtheJapanesewobbe-Orectolobusjaponicus(Elasmobranchii:Orectolobi-formes)inTaiwan.ComparativeParasitology,C.A.,J.N.C.2006.Fivenewspeciesof(Tetraphyllidea:Onchobothriidae)fromthefreshwaterstingrayHimanturachaophraya(Batoidea:Dasya-tidae)inMalaysianBorneo.JournalofParasitology,C.A.,J.N.C.2010.Phylogeneticstatusoffournewspeciesof(Cestoda:Tetraphyllidea)parasiticonthewedge“shRhynchobatuslaevischii:Rhynchobatidae):i

mplicationsforinterpretinghostassociations.InvertebrateSystematics,C.A.,J.N.CK.J.2009.FivenewspeciesAcanthobothrium(Cestoda:Tetraphyllidea)fromanunusualspeciesof(Rajiformes:Dasyatidae)fromnorthernAustralia.FoliaParasitologica,S.,J.N.C.2001.Fournewspeciesof(Cestoda:Tetraphyllidea)fromthewhiptailDasyatisbrevisintheGulfofCalifornia,Mexico.JournalofParasitology,M.,Z.M.2017.AnewgenusandspeciesoftheTrypanorhynchfamilyOtobothriidaeDollfus,1942fromtheslenderweaselsharkParagaleusrandalliCompagno,Krupp&Carpenter(Hemigaleidae)inthePersianGulf.SystematicParasitology,D.M.,C.MB.K.M(eds.).1996.Molecularsystematics.SecondEdition.SinauerAssociates,Sunderland,Massachusetts,655p.,K.,S.A.B.2010.Characterizationofadiversityoftetraphyllideanandrhinebothriideancestodelarvaltypes,withcommentsonhostassociationsandlife-cycles.InternationalJournalforParasitology,K.,J.N.C.2008.ArevisionofSouthwell,1925(Tetraphyllidea:Onchobothriidae)withthedescriptionoffournewspecies.ComparativeParasitologyAST,P.R.,W.T.WHITE,M.R.DARVALHO,B.SRET,M.F.W.STEHMANNANDG.J.P.NAYLOR(eds.).2016.Raysoftheworld.CornellUniversityPress,Ithaca,NewYork,690p.,D.T.J.,M.CE.A.H2000.TheinterrelationshipsofProseriata(Platyhelminthes:Seriata)testedwithmoleculesandmorphology.MolecularPhylogeneticsandEvolution,A.E.,P.D.OD.T.J.L.2003.UtilityofcompletelargeandsmallsubunitrRNAgenesinresolvingthephylogenyoftheNeodermata(Platyhelmin-thes):Implicationsandareviewofthecercomertheory.BiologicalJournaloftheLinneanSociety,G.J.P.,J.N.C,K.J,K.A.M.R,N.C.L.2012b.Elasmobranchphylogeny:Amitochondrialestimatebasedon595species.biologyofsharksandtheirrelatives,J.C.Carrier,J.A.Musick,andM.R.Heithaus(eds.).CRCPress,Taylor&FrancisGroup,BocaRaton,Florida,p.31…56.,G.J.P.,J.N.C,K.J,K.A.M.R,W.T.WP.R.L.2012a.ADNAsequence…basedapproachtotheidenti“cationofsharkandrayspeciesanditsimplicationsforglobalelasmobranchdiversityandparasitol-ogy.BulletinoftheAmericanMuseumofNaturalHistoryEVERELL,S.C.2009.Saw“sh(Pristidae)oftheGulfofCarpentaria,Queensland,Australia.M.S.ThesisJamesCookUniversity,Townsville,Queensland,Australia.,M.,J.N.C.2012.AnewhyperapolyticTriloculariaebertisp.n.(Cestoda:Tetraphyllidea),(Squaliformes:Squalidae)offSouthAfricawithcommentsonitsdevelopmentandfecundity.FoliaParasitologicaCAIRAETAL.—ANEWCESTODEGENUSFROMSAWFISH143 ,J.,M.T.H.2010.DendroPy:APythonlibraryforphylogeneticcomputing.Bioinformatics,D.L.2003.PAUP*.PhylogeneticAnalysisUsingParsimony(*andOtherMethods).Version4.SinauerAssociates,Sunderland,Massachusetts.,D.C.,D.L.M,A.J.R,H.S.GE.P.2008.Lifehistorynotesofthecriticallyendangereddwarfsaw“sh,Pristisclavata,Garman1906fromtheKimberleyregionofWesternAustralia.EnvironmentalBiologyofFishes,V.,D.T.J.L,P.D.O,J.M.KZ.S.2003.MolecularphylogeneticanalysisoftheMicrophalloideaWard,1901(Trematoda:Digenea).SystematicParasitologyWICKL,D.J.2006.Geneticalgorithmapproachesforthephylogeneticanalysisoflargebiologicalsequencedatasetsunderthemaximumlikelihoodcriterion.Doctoraldisserta-tion.UniversityofTexasatAustin,Austin,Texas,125p.144THEJOURNALOFPARASITOLOGY,VOL.104,NO.2,APRIL201