Blind Flight? A New Troglobiotic Orthoclad (Diptera, Chironomidae) from the Lukina Jama – Trojama Cave in Croatia

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Blind Flight? A New Troglobiotic Orthoclad (Diptera, Chironomidae) from the Lukina Jama – Trojama Cave in Croatia

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Blind Flight? A New Troglobiotic Orthoclad (Diptera, Chironomidae) from the Lukina Jama – Trojama Cave in Croatia
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PLOS One
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Anderson, Trone
Baranov, Viktor
Hagenlund, Linn Katrine
Ivković, Marija
Kvifte, Gunnar Mikalsen
Pavlek, Martina
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cave animals ( lcsh )
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serial ( sobekcm )

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The genus Troglocladius Andersen, Baranov et Hagenlund, gen. n. is erected based on T. hajdi Andersen, Baranov et Hagenlund, sp. n. collected at 980 m depth in the Lukina jama—Trojama cave system in Croatia. Morphological features such as pale color, strongly reduced eyes and very long legs make it a typical cave animal. Surprisingly, it has also retained large wings and appears to be capable of flight which would make T. hajdi the first flying troglobiont worldwide, disproving previous beliefs that bats are the only animals capable of flying in complete darkness. Morphologically the new species does not readily fit within any described genus, but shares characteristics with genera both in the tribes “Metriocnemini” and “Orthocladiini”. Bayesian molecular phylogenetic analysis using the markers COI, 18S rDNAs, 28S rDNA, CADI, and CADIV groups it with the genera Tvetenia, Cardiocladius and Eukiefferiella in the tribe “Metriocnemini”. Troglocladius hajdi may be parthenogenetic, as only females were collected. The discovery confirms the position of the Dinaric arch as a highly important hotspot of subterranean biodiversity.

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RESEARCHARTICLEBlindFlight?ANewTroglobioticOrthoclad (Diptera,Chironomidae)fromtheLukina Jama – TrojamaCaveinCroatiaTrondAndersen1 ,ViktorBaranov2 ,LinnKatrineHagenlund1 ,MarijaIvkovi 3,Gunnar MikalsenKvifte1,4,MartinaPavlek5,61 DepartmentofNaturalHistory,UniversityMuseumofBergen,UniversityofBergen,Bergen,Norway, 2 LebnizInstituteforFreshwaterEcologyandInlandFisheries,Berlin,Germany, 3 DepartmentofZoology, DivisionofBiology,FacultyofScience,UniversityofZagreb,Zagreb,Croatia, 4 DepartmentofZoology, InstituteofBiology,UniversityofKassel,Kassel,Germany, 5 DepartmentofMolecularBiology,Ru er Bo š kovi Institute,Zagreb,Croatia, 6 CroatianBiospeleologicalSociety,Zagreb,Croatia Theseauthorscontributedequallytothiswork. linn.hagenlund@uib.noAbstractThegenus Troglocladius Andersen,Baranov et Hagenlund,gen.n.iserectedbasedon T hajdi Andersen,Baranov et Hagenlund,sp.n.collectedat980mdepthintheLukinajama — TrojamacavesysteminCroatia.Morphologicalfeaturessuchaspalecolor,strongly reducedeyesandverylonglegsmakeitatypicalcaveanimal.Surprisingly,ithasalso retainedlargewingsandappearstobecapableofflightwhichwouldmake T hajdi thefirst flyingtroglobiontworldwide,disprovingpreviousbeliefsthatbatsaretheonlyanimalscapableofflyingincompletedarkness.Morphologicallythenewspeciesdoesnotreadilyfit withinanydescribedgenus,butsharescharacteristicswithgenerabothinthetribes “ Metriocnemini ” and “ Orthocladiini ” .Bayesianmolecularphylogeneticanalysisusingthe markersCOI,18SrDNAs,28SrDNA,CADI,andCADIVgroupsitwiththegenera Tvetenia Cardiocladius and Eukiefferiella inthetribe “ Metriocnemini ” Troglocladiushajdi maybeparthenogenetic,asonlyfemaleswerecollected.Thediscoveryconfirmsthepositionofthe Dinaricarchasahighlyimportanthotspotofsubterraneanbiodiversity.IntroductionTerrestrialspeciesadaptedtocavelifearegenerallycharacterizedaseithertroglophilesortroglobionts.Whiletroglobiontsareobligatorycave-dwellers,spendingtheirentirelifeinthe caves,troglophilesareessentiallyepigeanspeciesthataresubdividedtoeutroglophiles(ableto maintainpermanentsubterraneanpopulations)andsubtroglophiles(dependentonepigean habitatsforsomebiologicalfunctions).Athirdcategoryofcave-dwellingorganisms,thetrogloxenes,areonlyaccidentallyfoundincaves[ 1 ].Outofmorethan21,000terrestrialcavetaxa worldwide[ 2 ]alltroglobiontsaregrounddwellers.Batsflyinthecompletedarknessofcaves PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 1/15 a11111 OPENACCESS Citation: AndersenT,BaranovV,HagenlundLK, Ivkovi M,KvifteGM,PavlekM(2016)BlindFlight?A NewTroglobioticOrthoclad(Diptera,Chironomidae) fromtheLukinaJama – TrojamaCaveinCroatia. PLoSONE11(4):e0152884.doi:10.1371/journal. pone.0152884 Editor: DonaldJamesColgan,AustralianMuseum, AUSTRALIA Received: August31,2015 Accepted: March20,2016 Published: April27,2016 Copyright: 2016Andersenetal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: Allrelevantdataare withinthepaperanditsSupportingInformationfiles. Funding: Theauthorshavenosupportorfundingto report. CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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usingecholocationfornavigating,theyarehowever,troglophilic,andtherearenoknownflyingtroglobionts. TheDinaridesorDinaricAlpsstretchalongthewesternpartoftheBalkanPeninsulain southernEuropeandareapartoftheAlpinesystemalongtheeastcoastoftheAdriaticSea. Accordingtothemostrecentcensus,theDinarickarstharbors600terrestrialand330aquatic troglobionts[ 3 ].Comparisonofthisnumbertothe1138obligatecavetaxainUnitedStates[ 4 ] revealstheDinaridesastheworld ’ srichestareaofobligatesubterraneanfaunawhencompared tootherregionsofapproximatelythesamesize[ 4 ].Theextremerichnessoftheareacanalso beportrayedviaotherfauna,includingtheworld ’ ssoletroglobioticrepresentativesofPorifera, Cnidaria,BivalviaandPolychaeta[ 5 ]. AlthoughhundredsofdifferentDipteraspecieshavebeenrecordedfromcaves,ithasbeen statedthatDipteracontainsfewtruecavespecies,mostareoccasionalguestsoratmosttroglophiles(seee.g.[ 6 7 ]).However,someDipterahaveevolvedmorphologicalandphysiological adaptationsforatroglobioticlife[ 7 ].Theseadaptationsarepossiblymostpronouncedinthe African Mormotomyiahirsuta Austen,1936(Mormotomyiidae),whichisassociatedwithbats incavesandregardedastroglophilicasitsometimesisfoundoutsideofcaves.Itlackseyes, wingsandpigmentsandhasdevelopedelongate,spindlylegsdenselycoveredwithsensory setae[ 8 ].InthefamilySphaeroceridae,severalspeciesandsubspeciesarefoundincavesand someofthese,suchas Crumomyiaabsoloni (Bezzi,1914),whichisfoundincavesinnortheasternHerzegovina,arepaleandhavereducedeyes,longlegsandshortwingsandhalteres[ 9 – 10 ].Someoftheseadaptationsarealsofoundinothertaxaincluding Allopnyxiapatrizii Freeman,1952(Sciaridae)fromcavesinItaly,theobligatebatparasiticfamiliesNycteribiidaeand Strebliidae,andseveralspeciesinthefamiliesCulicidae,Psychodidae,Mycetophilidae,Keroplatidae,Phoridae,andHeleomyzidae[ 7 11 ]. Chironomidae(Diptera)havebeenrecordedfromcavesinmanypartsoftheworld(e.g. [ 12 – 17 ]).Mostoftheserecordsarefromthefirstfewhundredmetersfromthecaveentrance and,ifidentifiedbeyondfamilylevel,thespeciesrecordedappearmostlytobetrogloxenes. Reeves[ 18 ]categorizes Zavrelimyia nr thryptica (Sublette,1964)fromcavesintheGreat SmokyMountains,U.S.A.,asatroglophile,butuntilnowthereseemstobenorecordsofany truetroglobiontsamongthechironomids. DuringexpeditionstotheLukinajama — TrojamacavesystemintheVelebitMountain RangeinCroatiain2013,severalfemalesofapalechironomidbelongingtothesubfamily Orthocladiinaewerecollectedinachamberat980mbelowthesurface.Thefemaleis describedbelowandplacedinanewgenusbasedonmorphologicalandmoleculardata.Furthermore,itshabitatandnaturalhistoryisoutlinedbasedoncollectiondataand insitu observations.TheLukinaJama — TrojamacavesystemTheLukinajama — TrojamacavesystemissituatedintheHajdu kiandRo anskikukoviStrict Reserve,VelebitNationalPark,a109km2parkinthenorthernpartsoftheVelebitMountain inCroatia.Thereareseveraldeepcavesintheparkwithsomeoftheworld'slargestsubterraneanverticaldrops.TheLukinajama — Trojamacavesystemis1.431mdeepandthusthe deepestcaveinsoutheastEuropeandthe14thdeepestcaveintheworld. Thecavesystemhastwoverticalentrancesat1.438and1.475mabovesea-level( Fig1A ) andinwintersnowfallsintothepitsmakinganicylayerthatcoversthewallsandextends approximatelytoadepthof350minLukinajamaand200minTrojama;someyears,the Lukinajamaentranceiscompletelysealedoffbyanicecap.Airtemperaturegraduallysinks andbecomesmorestableduringthefirst200mofthecavesystem,asinfluencefromthe BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 2/15

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outsidedecreases(at-180mitisaround-0.7C).Fromthere,thetemperatureisverystable yearroundandslowlyrisesagainto2.5Cinthemiddlereaches(aroundSecondCamp, Fig1 ), inthechamberat980mdepthitreaches3.5C,andatthebottomofthepitthetemperatureis around4.9C[ 19 ]. Fig1. A-C. – A.SE-NWprofileoftheLukinajama — TrojamacavesystemintheVelebitMountain,Croatia.ExploredbySpeleologicalCommitteeofthe CroatianMountaineeringAssociation,CroatianSpeleologicalFederationandSlovakSpeleologicalSociety;mappreparedbyDarkoBak š i .Positionsofthe 3campsites(First,secondandthirdcamp)insidethepitaremarkedonthemap. – B.Thechamberat980mdepth,wheremostofthespecimenswere caught. – C.Newlyemergedspecimenof Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.n.,sp.n.,sittingonthecavewall(photoJ.Bedek). doi:10.1371/journal.pone.0152884.g001 BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 3/15

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Thefirst200mofthecavesystemhassubstantialpresenceoficeandsnowyearround. Waterandastrongdraughtofcoldairconstantlyflowdowntheshaft.Theseconditions,along withthecoldtemperaturesmakethispartofthecavesystemunsuitableforsubterranean fauna. Thenewspecieshasonlybeenobservedatdepthsbetween800and980m.Thoughitcannot completelyberuledoutthatthespeciesispresentintheupperpartsoftheLukinajama — Trojamacavesystem,itishighlyunlikelygiventheinhospitableconditions.Thechamberat980m depth( Fig1B — thirdcamp)isabout85x70metersinsizeandcontainsseveralhabitatsincludingdrywalls,hygropetricenvironmentswithwaterseepingonthewalls,smallstreamswith sedimentandasmallpondwithstandingwater — potentialhabitatsfortheimmaturestages. Atotalof54animaltaxahavebeenrecordedfromtheLukinajama — Trojamacavesystem, ofwhich32aretruecavedwellers[ 20 ].Amongthese,thecavesystemhousestheendemicsnail Zospeumtholussum Weigand,2013andoneofthelargestknowncoloniesofthesubterranean leech Croatobranchusmestrovi Kerovec,Ku ini et Jal i ,1999[ 21 – 22 ].MaterialandMethods CollectionofspecimensPermissionforcollectingcavefaunawasissuedbyTheMinistryofEnvironmentalandNature Protection,Croatia.Altogether16femaleshavebeencollectedintheLukinajama — Trojama cavesystem.Duringanexpeditionbetween29thJulyand3rdAugustin2010onefemalewas foundonthewetwallofthecaveatabout800mdepth.Duringasecondexpeditionbetween 1thand3rdAugust2011,twofemaleswerecollectedonthewallinthechamberat980mdepth ( Fig1C ).Duringathirdexpeditionbetween8thand12thAugustin2013altogether13females werecollectedusingstickytrapsplaceddirectlyonthewallsofthecave.Thespecimenswere preservedinalcoholandlatermountedinCanadaBalsamfollowingtheprocedureoutlinedby Sther[ 23 ].MorphologicaldescriptionThegeneralmorphologyfollowsSther[ 24 ].Themeasurementsaregivenasranges,followed bythemeanwhenfourormorespecimensweremeasured,followedbythenumberofspecimensmeasuredinparentheses.DNAextraction,PCRamplificationandsequencingThethoraxandpartoftheabdomenoffivespecimenswerekeptinalcoholandusedforDNA extraction.TheDNAwasextractednon-invasivelyusingtheQIAGENDNeasy1Bloodand TissuekitandamplifiedwithPCRfollowingprotocolsasspecifiedin S1Text and S1Table Themarkers18SrDNA,28SrDNAandCOIwereamplifiedusingprimersasspecifiedin Cranston etal .[ 25 ];GenBankaccessionnumbersandvouchercollectionnumbersforthetwo successfullysequencedspecimensarelistedinthe S2Table .Twofurthermarkers,CADIand CADIV,failedtoamplifyinourstudy,butwereneverthelessincludedinthefinalanalysisfor topologicalstabilitywithrespecttoCranston etal .[ 25 ]. ThegeneregionswerealignedinMegav6.0.6.[ 26 ]usingacombinationofMUSCLE[ 27 ] andmanualalignment.AmbiguousregionswereexcludedinGBlocksv0.91b[ 28 – 29 ],withall theoptionssetforalessstringentselection.Thismethodwassufficientfor28SrDNA;however,for18SrDNAtheamountofhypervariableregionsmadeitnecessarytoalignmanually accordingtosecondarystructure.Thealignmentandreferencesequenceareavailablein S2 and S3 Texts. BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 4/15

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MolecularphylogeneticanalysisThesequenceswerecomparedwithallOrthocladiinaesequencesfromCranston etal .[ 25 ] usingDiamesinaeandProdiamesinaeasoutgroups.Thefinaldatasetconsistsof3.709characters,1.560ofwhichareparsimonyinformative,inamatrixof67taxa.Analyseswerecarried outinPAUP,MEGAandmrBayesv3.2.3,usingBayesianmethods[ 26 30 – 31 ].Modelswere selectedandtestedusingJModeltest[ 32 ]incombinationwithPAUP.Bayesiananalysiswas performedusing10milliongenerations,4MCMCchainreactionsandthemodelGTR+G+Ias selectedbyJModeltestforallgenes.TypematerialTheholotypeand3paratypesaredepositedintheCroatianBiospeleologicalSociety(CBSS) collection,whichisapartofCroatianNaturalHistoryMuseumcollectioninZagreb,Croatia. Threeparatypes,alongwithDNAextractsaredepositedintheUniversityMuseumofBergen, Bergen,Norway(ZMBN)andanadditionalparatypeisdepositedintheZoologischeStaatssammlungMnchen,Munich,Germany(ZSM).NomenclaturalactsTheelectroniceditionofthisarticleconformstotherequirementsoftheamendedInternationalCodeofZoologicalNomenclature,andhencethenewnamescontainedhereinareavailableunderthatCodefromtheelectroniceditionofthisarticle.Thispublishedworkandthe nomenclaturalactsitcontainshavebeenregisteredinZooBank,theonlineregistrationsystem fortheICZN.TheZooBankLSIDs(LifeScienceIdentifiers)canberesolvedandtheassociated informationviewedthroughanystandardwebbrowserbyappendingtheLSIDtotheprefix http://zoobank.org/ ".TheLSIDforthispublicationis:urn:lsid:zoobank.org:pub:5CA9C540440A-4439-8961-4D7BB2FBB781.TheelectroniceditionofthisworkwaspublishedinajournalwithanISSN,andhasbeenarchivedandisavailablefromthefollowingdigitalrepositories: PubMedCentral,LOCKSS,andCristin( www.cristin.no ).Results Troglocladius Andersen,Baranov et Hagenlund,gen.n.urn:lsid:zoobank.org:act:96A93798-6392-482D-A07D-993D2BBF1776 Typespecies Troglocladiushajdi Andersen,Baranov et Hagenlund,sp.nov.urn:lsid:zoobank.org:pub:5CA9C540-440A-4439-8961-4D7BB2FBB781 Etymology .FromtheGreek trogle ,hole,andcladius ,acommonendingofrelatedOrthocladiinaegenera.Thegenderofthenameismasculine. Diagnosis .Thepalecolorincombinationwithhairyeyeswithout,orwithonlyafewommatidia,reducedpalpwithpalpomeres2+3and4+5mostoftenfused,verylonglegs,andlarge wingsandhaltereswillseparatethegenusfromallotherOrthocladiinaegenera. Description. Female .Mediumsizedspecies,winglength1.8 – 2.2mm. Antenna .Femaleantennawith6flagellomeres,antennalratioabout0.41.Flagellomere4 with1seta;flagellomere5with2setae,longestabout5timesaslongasflagellomere;flagellomere6with1apicalseta,abouttwiceaslongasflagellomere.Sensillachaeticapresentonflagellomeres1 – 5,sensillaonflagellomeres2 – 4about1.5timesaslongasflagellomere. Head .Eyereniform,stronglyhairy,withoutorwithfewreducedommatidia.Temporal setaeinsinglerowconsistingofinnerandouterverticals,postorbitalspresentorabsent.Clypeuswithfewsetae.Frontaltubercleabsent.Tentoriumandstipesnormal.Cibarialpumpwith BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 5/15

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anteriormarginstraight.Palpshort,palpomeres2+3and4+5fusedinmostspecimens;third palpomerewithfewsensillaclavataapically. Thorax .Antepronotumwithlobesmeetingmediallyatanteriormarginofscutum,withfew lateralantepronotals.Acrostichalsshort,startingsomedistancefromantepronotum;dorsocentralsabsentorfew;prealarsfew;supraalarabsent.Scutellumwithfewsetaeinsinglerow.Halterelarge. Wing .Broadwithexcavatedanallobe.Membranewithoutsetae,withfinepunctation.Costa moderatelyextended.R3+4endingslightlyclosertoR1thantoR4+5;R4+5endingoppositeto M3+4;FCuaboutoppositetoRM;Cu1weaklysigmoid.Brachiolumwith1seta,R,R1andR4+5withfewsetae,otherveinsbare.Squamawithfewsetae.Sensillacampaniformiaabout3 – 6 basally,5 – 6apically,and2abovesetaonbrachiolumand1basallyonR1. Legs .Alllegsverylong.Alltibiawithonlyonespur,hindtibialcombnormal.Tarsalpseudospursandsensillachaeticaabsent.Pulvillivestigial. Abdomen .Tergitesandsterniteswithfewsetaein3 – 4rows. Genitalia .Withwell-developedgonocoxitewithseveralsetae.TergiteIXdividedinto2setigerousprotrusions.SegmentXnormal.Cercicomparativelylarge.Postgenitalplatewithslightly emarginatedposteriormargin.Coxosternapodemecurved.GonapophysisVIIIdividedinto large,triangularventrolaterallobeandnarrowdorsomesallobe.Apodemeloberelativelyconspicuous.Seminalcapsulespale,large,ovoid,withtriangularneck.Spermathecalductswith loopsandcommonopening;ductsapparentlywithoutmicrotrichia.Labiawithoutmicrotrichia. Systematics. Thespeciesisdifficulttoplacesystematicallybasedonmorphologicalcharactersalone,partlybecauseonlythefemaleisknown,andpartlybecauseitshowsseveralmorphologicaladaptationstocavelife.UsingthepreliminarykeytothefemalesofOrthocladiinae inSther[ 33 ]thespecieswillkeyto Cricotopus vanderWulp,1874ifthedorsocentralsare regardedasshortanddecumbentorto Paratrichocladius SantosAbreu,1918ifthedorsocentralsareregardedaslonganderect.ItclearlyhasadividedgonapophysisVIIIwithalargeventrolaterallobecoveringmostofthedorsomesallobe,ascanbefoundinthe Cricotopus group ofgenera.Further,tergiteIXisdividedintwosetigerousprotrusions,whichalsocanbefound inthe Cricotopus group. ThephylogeneticanalysisbasedonDNAsequencesyieldedatreewhichoverallisverysimilartothatgivenforOrthocladiinaebyCranston etal .[ 25 ].ThetwospecimensfromtheLukina jama — Trojamacavegroupedtogetherandwerewellseparatedfromallothergenerainthe analysis.Theydidnothowever,groupwith Cricotopus intribe “ Orthocladiini ” ,butfellwithin tribe “ Metriocnemini ” togetherwiththegenera Tvetenia Kieffer, Cardiocladius Kiefferand Eukiefferiella Thienemann( Fig2 ).Posteriorprobabilitiesfortheotherhighernodeswithinthis cladewereallsignificant( > 98%),thusexcludingthespecimensfromthesesub-cladeswithhigh confidence.Posteriorprobabilityforthenodewithintribe “ Metriocnemini ” containingthenew species,togetherwith Tvetenia Cardiocladius and Eukiefferiella washowever,only61%possibly duetomissingdataonCADIandCADIV.Themutualarrangementofthegenerawithinthis nodeisthusuncertain.OneshouldalsonotethatmanyOrthocladiinaegeneraremaintobe sequencedandtheinternalrelationshipswithintribe “ Metriocnemini ” maychange. Morphologicallythenewspeciesdoesnotgroupwiththegenera Tvetenia Cardiocladius and Eukiefferiella ,asallthreehaveanundividedgonapophysisVIII[ 33 ]whilethenewspecies clearlyhasadividedgonapophysisVIII.AdividedgonapophysisVIIIwithalargeventrolateral lobecoveringmostofthedorsomesallobeandadividedtergiteIXarehowever,traitsfoundin manyOrthocladiinae,includingboththe Cricotopus groupandseveralgenerawithin “ Metriocnemini ” .Basedonourcurrentknowledge,thenewspeciesapparentlydoesnotfitintoany describedgenus,thussupportingourplacementofthespeciesinanewgenuswithintribe “ Metriocnemini ” BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 6/15

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Fig2.BayesiantreeofOrthocladiinaeshowingthepositionof Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.n.,sp.n.,withinthetribe “ Metriocnemini ” Posteriorprobability(PP)isgivenin percentforthebranches. doi:10.1371/journal.pone.0152884.g002 BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 7/15

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Troglocladiushajdi Andersen,Baranov et Hagenlund,sp.n.(Figs 3 – 6 )Holotype ,f,CROATIA,VelebitMountain,Lukinajama — Trojamacavesystem,N44.766939, E15.026571,980mdepth,3August2010,leg.M.Luki (CBSS). Paratypes .2ffasholotypeexcept1 – 3August2011,leg.J.Bedek;4ffasholotypeexcept9 August2013,leg.M.Pavlek;1fasholotypeexcept10August2013,leg.M.Pavlek(CBSS, ZMBN,ZSM). Etymology .NamedaftertheHajdi,agroupofwinged,dwarf-likecreaturesfromSlavic mythology,wheretheyactedasmessengersoffateandweresaidtodwellincaves.Thenameis toberegardedasanouninapposition. Diagnosis .Seegenericdiagnosis. Description. Female (n=4 – 7,ifnototherwisestated).Totallength1.80(1)mm.Wing length1.79 – 2.17,2.01mm.Totallength/winglength0.98(1).Winglength/lengthofprofemur2.72 – 2.86,2.77. Coloration .Paleyellowish. Antenna ( Fig3C ).Antennalratio(AR)0.39 – 0.45,0.41.Pedicel41 – 55,49 mlong;48 – 61, 55 mwide.Flagellomeres1 – 6length/width(in m):39 – 47,43/23 – 28,26;23 – 28,25/19 – 21,20;29 – 36,33/18 – 19,18;25 – 36,31/14 – 18,16;36 – 43,40/15 – 19,17;63 – 73,70/14 – 18, 15.Apicalseta117 – 144,128 mlong. Head ( Fig3A ).Temporalsetae3 – 7,5including2 – 4,3innerverticals,1 – 2,1outervertical and0 – 2,1postorbital.Eye( Fig3B )with0 – 4,2ommatidia.Clypeuswith4 – 5,5setae.Tentorium,stipesandcibarialpumpasin Fig3D .Tentorium63 – 76(3) mlong,12 – 17(3) mwide. Stipes58(1) mlong.Palpomeres1,2+3,and4+5lengths(in m):11 – 19,15;44 – 97,73;30 – 79,54.Inonespecimenpalpomere4and5distinctlyseparated,fourthpalpomere37 mlong, fifthpalpomere46 mlong.Thirdpalpomerewith2 – 3sensillaclavataapically,longest20 – 22 mlong. Thorax ( Fig3E ).Antepronotumwith3 – 5,3ventrolateralsetae.Acrostichals9 – 12,11;dorsocentrals0 – 2,1;prealars1 – 2,1;supraalarsabsent.Scutellumwith4 – 6,5setae.Haltere344 – 396,368 mlong. Wing ( Fig3G ).Venarumratio(VR)1.01 – 1.02,1.01.Costalextension121 – 133,126 m long.Rwith6 – 9,8setae;R1with1 – 4,2setae;R4+5with6 – 7,6setae,costalextensionwith4 – 7, 6non-marginalsetae;brachiolumwith1seta.Squamawith3 – 4,4setae. Legs .Spurofforetibia21 – 26,23 mlong;spurofmidtibia( Fig3F )15 – 17,16 mlong; spurofhindtibia33 – 43,38 mlong.Widthatapexofforetibia33 – 40,37 m;ofmidtibia36 – 39,37 m;ofhindtibia39 – 48,45 m.Combcomposedof13 – 14,13setae;longest34 – 45, 37 mlong;shortest23 – 28,25 mlong.Lengthandproportionsoflegsasin Table1 Genitalia ( Fig4A – 4E ).SterniteVIIIwith8 – 11,9setaetoeachside.GonocoxiteIXwith 4 – 6,5moderatelystrongsetae.TergiteIXdivided,with14 – 16,15setaeintwodistinctgroups. Cercus98 – 103(2) mlong.Seminalcapsuleovoid;83 – 105,94 mlong;66 – 71,68 mwide; with10 – 14,11 mlong;12 – 16,14 mwideneck.Notum107 – 117,113 mlong.Ventrolateral lobesubtriangular;69 – 76,73 mlong;39 – 48,43 mwideatitswidestpoint;coveredwith microtrichia.Dorsomesallobe54 – 57,56 mlongfromgonocoxapodemetoapex. Maleandimmaturestages .Unknown.DiscussionTruetroglobiontsaregenerallycharacterizedbyseveralmorphologicalfeatureslikelackof integumentarypigment;mostspeciesarewhitishoreventranslucent.Further,sincethereisno lightinthecaves,troglobiontsoftenshowsomedegreeofeyereduction.Theyoftenhaveelongatedlegsandantennae;incaveenvironmentslonglegsandappendagesareclearlyan BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 8/15

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Fig3. A-G. Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.n.,sp.n.,female. – A.Head. – B.Eye. – C.Antenna. – D.Tentorium,stipesandcibarial pump. – E.Thorax. – F.Spurofmidleg. – G.Wing. doi:10.1371/journal.pone.0152884.g003 BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 9/15

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advantageandareoftencoupledwithawell-developedsensoryapparatus.Mostcaveinsects alsolackwingsorthewingsarereduced[ 34 ]. Thenewspeciesdescribedheresharesseveraloftheseadaptations.Thebodyispaleyellowish( Fig5 )andweaklysclerotized.Theeyesarestronglyreducedwithonly0 – 4ommatidia present.Thelegsarecomparativelyverylong( Fig6 ).Theantennaappearsshort,buthavefew, longsetaeonsegments4 – 6andstrongsensillaonsegments1 – 5.Remarkablyhowever,the wingsarewelldeveloped,longandbroadwithanexcavatedanallobe,andthehalteresarealso unusuallylarge. Thecombinationofstronglyreducedeyesandlarge,broadwingsappearstobeunique amongtroglobioticorganismsandmightindicatethatthespeciesisabletoflyslowlyorhover inthetotaldarknessofthecave.Thelongforelegsmightserveas “ feelers ” iftheyarestretched forwardduringflightandthelargehalteresmighthelptheinsectmaintainbalance.Duringthe 2013expedition T hajdi wasneverobservedflying,butsomeofthespecimenscollectedinthe stickytrapsweresittinginthemiddleofthestrips,suggestingthattheyflyatleastoccasionally. Duringfutureexpeditions,effortsshouldthusbemadetoobservethespecies ’ behaviorinthe cave. Fig4. A-E. Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.n.,sp.n.,female. – A.Genitalia,ventralview. – B.TergiteIX. – C.Ventrolaterallobe. – D. Dorsomesallobe. – E.Apodemelobe. doi:10.1371/journal.pone.0152884.g004 BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 10/15

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Ofallfree-livingtroglophilicortroglobioticDipterarecordedsofar, T hajdi hasbyfarthe mosttroglomorphiccharacteristics.Furthermore,whileothertaxaarefoundclosetothe entranceorincomparativelysmallcaves, T hajdi inhabitsadeepandhighlyisolatedpartofa largecavesystemwithverylittlepossibilitytocommunicatewiththeoutsideenvironment. ThediscoveryofthemosttroglomorphicwingedDipteradescribedsofarprovidesfurthersupportfortheDinarickarstasanareaofextremesubterraneanbiodiversity. Thenewspeciesmightwellbeparthenogeneticasonlyfemaleswerecollected.Ininsects, parthenogenesisismostoftenfoundinharshenvironments,wherematingcanbedifficult.Parthenogenesisisnotuncommonamongchironomidsandismostoftenfoundinextremeorisolatedhabitats[ 35 ].ExamplesincludeAntarcticmidgeslike Eretmopteramurphyi Schaeffer, 1914orisolatedislandpopulationslikepopulationsof Limnophyesminimus (Meigen,1818) onGoughandNightingaleIslands[ 36 – 37 ].Parthenogenesiscanalsooccurinhabitatspecialistsrestrictedtofragmentedhabitatssuchas Polypedilumparthenogeneticum Donato et Paggi, 2008livinginwaterheldintheleafaxilsof Eryngiumpandanifolium Chamb et Schlecht(Apiaceae)[ 38 ].Althoughtoofewspecimensof T hajdi havebeencollectedtoconfidentlyruleout theexistenceofmales,weregardparthenogenesisasthemostlikelyreproductivesystemconsideringthespecies ’ isolatedhabitat. Thelarvaeandpupaeof T hajdi arenotknown.Duringtheexpeditionsin2013severallarvaeofthemycetophilid Speoleptaleptogaster Madwar,1937werecollectedonthewallsinthe chamberat980mdepthandwronglythoughttorepresentthelarvaeof T hajdi .However,in the980mchamberthereareseveralshallowstreamswithfinesediment,whichwerenotsampledduringthe2013expeditionandthelarvaeof T hajdi mightinhabitthesestreams.During comingexpeditionstotheLukinajama — Trojamacavesystemortootherdeepcavesinthat area,theimmaturesof T hajdi shouldthereforebesearchedfor.However,ifendemictothe Lukinajama — Trojamacavesystem,greatcareshouldbetakennottocollecttoomanyspecimens,asthepopulationsmightbeverysmallandthusvulnerable. Fig5. Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.n.,sp.n.,female. Photoshowingthepalecolourandthebroadwings. doi:10.1371/journal.pone.0152884.g005 BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 11/15

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Fig6. Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.n.,sp.n.,female. Photoofslide mountedthorax,showingthelonglegsandthecomparativelylargehaltere. doi:10.1371/journal.pone.0152884.g006 Table1.Lengths(in m)andproportionsoflegsof Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.nov.,sp.nov.,female(n=4 – 5). MorphologicalabbreviationsfollowSther[ 24 ]. Fetita1ta2p1711 – 776,745703 – 801,758425 – 531,493253 – 302,278 p2719 – 801,754670 – 703,688310 – 359,343180 – 196,190 p3792 – 850,819735 – 833,810392 – 474,441229 – 253,243 ta3ta4ta5LR p1172 – 196,186106 – 123,11465 – 74,700.61 – 0.67,0.65 p2114 – 131,12774 – 90,8249 – 65,590.45 – 0.52,0.50 p3155 – 188,17890 – 98,9557 – 65,620.53 – 0.57,0.54 BVSVBR p13,01 – 3.23,3.092.95 – 3.33,3.061.46 – 1.50,1,49 p23.66 – 4.12,3.914.09 – 4.53,4.211.07 – 1.47,1.30 p33.54 – 3.64,3.583.53 – 3.90,3.701.12 – 1.71,1.40 doi:10.1371/journal.pone.0152884.t001 BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 12/15

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SupportingInformationS1Table.MastermixforPCRandPCRthermocyclingprotocolsforthegeneticmarkers usedintheanalysis. (DOCX) S2Table.GenBankaccessionnumbersforvouchersLH01andLH02of Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.nov.,sp.nov. (DOCX) S1Text.DNAextractionprotocolforthespecimensof Troglocladiushajdi Andersen,Baranov et Hagenlund,gen.nov.,sp.nov. (DOCX) S2Text.Concatenatedalignmentforallthemolecularmarkersusedintheanalysis. (DOCX) S3Text.Referencesequencewithsecondarystructureannotationforthealignmentof18S rDNA. (DOCX)AcknowledgmentsWeareindebtedtothefollowingspeleologicalorganizations:SpeleologicalSectionoftheUniversityMountaineeringSociety “ Velebit ” ,SpeleologicalSectionoftheCroatianMountaineeringSociety “ ™ eljezni ar ” ,Breganja,CroatianBiospeleologicalSociety,IstrianSpeleological Union,SpeleologicalCommitteeoftheCroatianMountaineeringAssociation,CroatianSpeleologicalFederation,ZagrebSpeleologicalUnion,andtotheNorthernVelebitNationalParkfor organizingandsupportingthecaveexpeditionsandalsotoseveralhundredspeleologistsand otherpeoplewhomadetheexpeditionspossible.ToLouiseLindblom,KennethMelandand SolveigThorkildsenforinvaluablehelpwiththemolecularworkcarriedoutattheBiodiversity Laboratories(BDL,DNASection)attheUniversityMuseumofBergen/DepartmentofBiology,UniversityofBergen,Norway;toMattKrosch,UniversityofQueensland,Australia,for informationonadditionalorthocladsequences;andtoEndreWillassenandBjarteH.Jordal, UniversityMuseumofBergen,forvaluablediscussionsconcerningtheanalysesofthemoleculardata.GladysRamirez,UniversityMuseumofBergen,madetheslidepreparations.Weare alsoindebtedtoDr.JosteinKjrandsen,TromsMuseum,Norwayforidentifyingthelarvae of Speoleptaleptogaster ,andtothetwoanonymousreviewersfortheirconstructivecomments.AuthorContributionsAnalyzedthedata:TALKHGMK.Contributedreagents/materials/analysistools:LKHMIMP VB.Wrotethepaper:TAVBLKHMIGMKMP.Drewtheillustrations:TA.References1. SketB.Canweagreeonanecologicalclassificationofsubterraneananimals?JournalofNaturalHistory.2008;42:21 – 22. 2. JuberthieC,DecouV,editors.EncyclopaediaBiospeologica.Vol1.Moulis&Bucharest:Socitde Biospologie;1994. 3. SketB,ParagamianK,TronteljP.AcensusoftheobligatesubterraneanfaunaintheBalkanPeninsula. InGriffithsHI,KrystufekB,editors.BalkanBiodiversity.PatternandProcessinEurope ’ sBiodiversity Hotspot.Dordrecht,TheNetherlands:KluwerAcademicPublishers;2004.pp.309 – 322. 4. SketB.DiversitypatternsintheDinaricKarst.InWhiteWB,CulverDC,editors.Encyclopediaofcaves. 2nded.Amsterdam:Elsevier/AcademicPress;2012.pp.228 – 238. BlindFlight?ANewTroglobioticOthocladfromCroatia PLOSONE|DOI:10.1371/journal.pone.0152884April27,2016 13/15

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