Ecology and Evolution. 2019;1–14.  | 1www.ecolevol.org

Received:18August2018  |  Revised:19November2018  |  Accepted:27November2018DOI: 10.1002/ece3.4846

O R I G I N A L R E S E A R C H

The Klingon batbugs: Morphological adaptations in the primitive bat bugs, Bucimex chilensis and Primicimex cavernis, including updated phylogeny of Cimicidae

Gonzalo Ossa1 | Joseph S. Johnson2 | Anna I. E. Puisto3 | Veikko Rinne3 |  Ilari E. Sääksjärvi3 | Austin Waag2 | Eero J. Vesterinen3,4  | Thomas M. Lilley5,6

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.© 2019 The Authors. Ecology and EvolutionpublishedbyJohnWiley&SonsLtd.

1ConserBatEIRL,SanFabian,Chile2DepartmentofBiologicalSciences,OhioUniversity,Athens,Ohio3BiodiversityUnit,UniversityofTurku,Turku,Finland4DepartmentofAgriculturalSciences,UniversityofHelsinki,Helsinki,Finland5InstituteofIntegrativeBiology,UniversityofLiverpool,Liverpool,UK6FinnishMuseumofNaturalHistory,UniversityofHelsinki,Helsinki,Finland

CorrespondenceThomasMLilley,FinnishMuseumofNaturalHistory,UniversityofHelsinki,Helsinki,Finland.Email:thomas.lilley@helsinki.fiandEeroJVesterinen,BiodiversityUnit,FI-20014UniversityofTurku,Turku,Finland.Email:ejvest@utu.fi

Funding informationH2020MarieSkłodowska-CurieActions,Grant/AwardNumber:706196;JanejaAatosErkonSäätiö;BettyVäänäsenSäätiö

AbstractTheCimicidaeisafamilyofblood-dependentectoparasitesinwhichdispersionca-pacityisgreatlyassociatedwithhostmovements.Batsaretheancestralandmostprevalenthostsforcimicids.Cimicidshaveaworldwidedistributionmatchingthatoftheirhosts,buttheglobalclassificationisincomplete,especiallyforspeciesoutsidethemostcommonCimicidaetaxa.Inthisstudy,weplacealittle-studiedcimicidspe-cies,Bucimex chilensis,withinacomprehensivemolecularphylogenyofCimicidaebysequencing thegenomic regionsof thisandotherclosely relatedspecies.For thisstudy,we collectedB. chilensis females fromMyotis chiloensis in Tierra del Fuego,1,300kmfurthersouththanpreviouslyknownsouthernmostdistributionboundary.WealsosequencedCOIregionsfromPrimicimex cavernis,aspecieswhichtogetherwith B. chilensiscomprisetheentiresubfamilyPrimiciminae.UsingBayesianposteriorprobability and maximum-likelihood approaches, we found that B. chilensis and P. cavernisclusteredclosetoeachotherinthemolecularanalyses,receivingsupportfromsimilarmorphologicalfeatures,agreeingwiththemorphology-basedtaxonomicplacementofthetwospecieswithinthesubfamilyPrimiciminae.Wealsodescribeapreviouslyunrecognizedmorphologicaladaptationofthetarsalstructure,whichal-lowstheaustralbatectoparasite,B. chilensis,toclingontothepelageofitsknownhost,theChileanmyotis(Myotis chiloensis).Throughamorphologicalstudyandbe-havioral observation,weelucidatehow this tarsal structureoperates, andwehy-pothesizethatbyclinginginthehostpelage,B. chilensisisabletodisperseeffectivelytonewareasdespitelowhostdensity.ThisisauniquefeaturesharedbyP. cavernis,theonlyotherspeciesinPrimiciminae.

K E Y W O R D S

Chiroptera,Cimicinae,dispersal,ectoparasite,tarsalstructure

2  |     OSSA et Al.

1  | INTRODUC TION

Parasitismisawidespreadlifestyle,withparasiticorganismsfoundinmanytaxaandconstitutingasmuchas50%ofanimalbiodiversity(Poulin&Morand,2000;Weinstein&Kuris,2016).Thus,parasitesareimportantnotonlyfortheirnotableinteractionswiththeirhosts,butalsoevolutionarily,astheyprovideopportunitiestotestnumer-oushypothesesonspeciation(Morand&Poulin,2003).Constructingparasitephylogeniesusingmolecularmethodshasopenedthedoorforresearchinthisarea,aswellasprovidingabroaderunderstand-ingofrelationshipsamongparasitesandtheirhosts.Therefore,phy-logeniesofdiverseorwidespreadgroupsofparasitesareuseful instudiesofparasitespeciation,orcoevolutionofparasitesandtheirhosts(Hafner&Nadler,1988).

TheCimicidae(Heteroptera)areanecologicallyimportantfam-ily of parasites with a phylogeny which could benefit from moreattention despite recent advances made by Balvín (2015), Balvín,Munclinger,Munclinger, Kratochvíl, and Vilímová (2012). Cimicidsareobligatehematophagousectoparasitesthataredistributedacrosstheglobeandcontain110describedspecieswithin24generainsixsubfamilies(Henry,2009).Bothsexesfeedexclusivelyonblood,anddevelopmentintoasubsequentinstar,aswellaseggproductioninadultfemalesandspermproductioninmalesrequiresabloodmeal(Reinhardt&Siva-Jothy,2007;Waage,1979).Cimicidsareproposedtohaveevolvedfrompredatoryheteropteranancestors,butroughly60%ofextantcimicidspeciesspecializeonparasitizingbats(Poulin& Morand, 2000) (Chiroptera). Bats are considered the ancestralhosts of cimicids, althoughhumans andother vertebratesmaybeusedassecondaryhosts(Hornoketal.,2017;Usinger,1966).

Bats, the second largest mammalian order, are highly socialanimals (Kerth, 2008). During pregnancy and lactation, many batspeciesestablishmaternitycoloniesinroostswithrelativelystableclimaticconditions togivebirth to theiryoung.Both thebatsandtheirroostsprovideasuitableenvironmentforarthropodectopara-sites(Lucan,2006),butthesocialbehaviorsofbatsalsorepresentsrisks to these parasites. Social grooming has been observed in anumberofbatspecies,whichexposestheseparasitestoothermem-bersofthesocialgroupofhosts(Kerth,Almasi,Ribi,Thiel,&Lüpold,2003). However, it is difficult for bats to protect themselves bygroomingagainstcimicids.Cimicidsareableproduceadefensesub-stanceandbatsrefusetobitethem(Usinger,1966),althoughitisnotcompletelyunlikelyforthistooccur (Bartonicka,2008).Generally,adultcimicidsfeedinperiodsoffewdays,andonlywhilebatsarenormothermic (Bartonicka, 2008), after whichmost species leavetheimmediatevicinityofthehosttodigestthemealwithinthecon-finesoftheroostandcansurviveuptoa1.5yearswithoutfeedingagain(Johnson,1941).Thelatterallowsthemtooverwinteratsum-merroostsevenafterbatshavemigratedtohibernationsites.Theability tosurvive longperiodswithoutmealsmaybeanespeciallyimportant adaptive trait inCimicidae,whichappear tohavea lowinherentcapacityfordispersaloverlongdistances,andevenshortdistancemovements seem to be limited (Talbot, Vonhof, Broders,Fenton, & Keyghobadi, 2016; Usinger, 1966). In fact, without the

ability to fly, it is unlikely that adult cimicids are able to dispersewithoutthehost(Balvín,Sevcik,etal.,2012;Brown&Bomberger-Brown, 1996;Usinger, 1966).Although the phylogenetic topologybetweencimicidsandtheirspecificbathostshavenotbeenstudiedingreatdetail,thebiologyandecologyofmanycimicidsappearstobestronglyinfluencedbythehostspeciesandtheirecology(Balvin,Bartonicka,Simov,Paunovic,&Vilimova,2014;Balvín,Munclinger,etal.,2012;Hafner&Nadler,1988).

Inseminatedcimicidfemalesareobservedattachedtoforearmsof bats outside roostsmoreoften thanmales or non-inseminatedfemales, suggesting cimicids primarily travel on bats to disperse(Balvín,Sevcik,etal.,2012;Heise,1988).However,transmissionap-pears tobeuncommon (Talbotet al., 2016), possiblybecause thismodeofdispersalposes inherentriskstocimicids,ascimicids lackmorphological adaptations toproperly attach to thehost for pro-longed periods of time and are easily discarded during grooming.Thus,althoughcimicidshostsarehighlymobile,cimicidpopulationsmaybemoregeneticallyisolatedthanthoseoftheirhosts(Talbotetal.,2016).Unfortunately,cimicidsspendmostoftheirlivesincrypticbat roosts and are therefore seldomavailable for study.AlthoughtheEuropeanfaunaofcimicidsarewelldescribed,numerousgapsremaininglobalcimicidtaxonomy,hostspecificityandecologyfromothercontinents.Fillingthesegapswillprovideopportunitiestotestnovelhypothesesontheecologyandevolutionoftheseuniqueec-toparasitesofbats,whichbeingtheonlyflyingmammals,arehighlymobileanddistributedacrosstheglobe.

Untilrecently,theonlyphylogenyofHeteropterawasbuiltexclu-sivelyonmorphology(Usinger,1966).Thisphylogeny,andtheposi-tioningofCimicomorphawithinHeteroptera,exhibitedanumberofinconsistenciescomparedtomoleculardatapresentedbyBalvínetal.(2015),whichhowever,concentratedonthegenusCimex,ratherthanthefamilyasawhole.Morerecentmolecularphylogeniesaddtothis(Hornoketal.,2017),butbesidesahypotheticalphylogenybyReinhardtandSiva-Jothy(Reinhardt&Siva-Jothy,2007),theydonotprovideacomprehensivedescriptionofCimicomorpha,orCimicidae(Li, Tian, Zhao, & Bu, 2012; Schuh,Weirauch, &Wheeler, 2009).ThiswasrectifiedbyBalvínetal. (2015),buteventheydidnotin-cludesomeofthesistergroupsoutsidethefourcommonCimicidaespeciesgroupswithin thegenusCimex (Cimex lectulariusL., Cimex pilosellus (Horvath, 1910), Cimex hemipterus (Fabricius, 1803) andCimex pipistrelli (Jenyns,1839).

Hereinwedescribenovelmorphologicaladaptationsinthetarsalstructureofthebatectoparastite,Bucimex chilensisUsinger,1963,whichmayallowfor itsmoreeffectivedispersal.WecomparethemorphologyofB. chilensis to itsclosestknownrelative,Primicimex cavernisBarber,1941,whichsharesmanyofthesamedistinguish-ablefeatures(Usinger,1966).ThesetwospeciesaretheonlyknowndescribedtaxaofthesubfamilyPrimicimicinae,andareclassifiedinmonotypicgenera(Usinger,1966).Bothspeciesareassociatedsolelywithbatsinthewesternhemisphere.UsingDNAsamplesfrombothspecies,weaddtothephylogenyofthefamilyCimicidaeusingbothnuclearandmitochondrialsequencedataaswellasdescribeanewgeographicrecordforB. chilensis.

     |  3OSSA et Al.

2  | MATERIAL AND METHODS

2.1 | Bat capture methods and location

We captured two adult female Myotis chiloensis (Waterhouse,1840, Capture permit #1253-2016 by the Servicio Agrícola yGanadero,Chile [SAG]) atKarukinkaReserve in southernTierradel Fuego (54°S, 69°W; elevation 159 meters above sea level)with B. chilensisfemalesattachedtothepelagesonthedorsalsur-faces(Figure1).Thecaptureswerefromtwoconsecutiveyears,November2016andDecember2017.Thiscapturesiteislocated1,300km south of the previously described southernmost dis-tribution ofB. chilensis (Usinger, 1966, Figure 2). TheKarukinkareserve is situated in the sub-antarctic phytogeographic prov-ince,withprecipitationbetween450and1,100mm/year, andameanannualtemperatureof7°C(Arroyoetal.,1995).Thehabitatsurroundingthecapturesite isdominatedbyNothofagus pumilio (Poepp. & Endl.) Krasser, a deciduous tree species, mixed withNothofagus betuloides (Mirb.) Oerst., an evergreen tree species(Arroyo et al., 1995) and a high diversity ofmosses and lichens(Armestó, Villagrán, & Kalin Arroyo, 1996). In addition to theB. chilensis from Tierra del Fuego, two individuals of P. cavernis wereobtainedfromtheSmithsonianInstitutionNationalMuseumofNaturalHistoryUSA,forphotographyandsequencing.ThesespecimenswerecollectedinNeyCaveinMedinaCounty,Texas,USA.We collected samples fromCimex pilosellus and C. adjunc‐tusfromManitoba,Canada.Additionally,wereceivedsamplesforC. lectularius fromvarious locations inFinland, including regionsofTurku,Tampere,Oravainen,andKemiö.SeeTable1fordetailsallsamplesinthisstudy.

2.2 | Digital layer imaging

PicturesweretakenwithCanonEOS7DMark IIcameraattachedtoanOlympusSZX16microscope.Focusingandcamerawerecon-trolled by Deep Focus module for QuickPHOTO 3.1 (Promicra).Focus stackingof thepictureswasdonebyCombineZP (availableathttp://combinezp.software.informer.com/download/).Specimenswerekeptinethanolwhilephotographed.

2.3 | DNA extraction

WegatheredsamplesfrommultipleCimicidaespeciesforourphy-logeneticanalysisasdetailed inTable1.DNAwasextracted fromthewholespecimeninthecaseoftheCimexsp.samples,orlegsinthecaseofthefreshB. chiloensisandmuseumP. cavernisspecimen.DNAwasextractedusing theNucleoSpin®TissueKit (productnr740,952,Macherey-Nagel),accordingtotheinstructionsforstand-ard protocol (User manual, version June 2014/Rev. 14) providedwiththekit.TheP. cavernis museumsampleswerecleanedbeforetheextractiontoremoveallthenon-targetmaterialfromthesam-ple surfaceas follows: (a) sampleswerevortexedbriefly ina tubecontaining2%bleachand incubatedfor10min, (b)bleachwasre-movedandsampleswerewashedbyadding99%ethanol,andthen(c)rinsedwithdd-H2Oandfinallydried,andthenextractedasabove.The laboratoryandtheequipmentweresterilizedbeforeeachex-tractionbatch.

2.4 | PCR and sequencing

Foreachextract,weamplifiedfivegenes,bothnuclearandmitochon-drial, usingprimers andprotocols afterBalvinet al. (2015). Shortly,thecytochromeoxidasesubunitI(COI)wasamplifiedusingLep1Fdeg/Lep3R (Hajibabaei, Janzen, Burns, Hallwachs, & Hebert, 2006),16S ribosomal gene (16S) using 16S_LR-J (Kambhampati & Smith,1995)/16S_LR-N(Simonetal.,1994),18Sribosomalgene(18S)intwooverlappingfragments:18S-1/18S-3and18S-2/18S-4(Tian,Zhu,Li,Xie,&Bu,2008),Internaltranscribedspacer(ITS2)usingCAS5p8sFc/CAS28sB1d(Kim&Lee,2008),andfinallyElongationfactor1subunitα(EF1a)withrcM52.6(alsoknownasShirley;Choetal.,1995)/M2412(alsoknownasProwler;Damgaard,Andersen,&Sperling,2000).Forold museum sample P. cavernis, we first tried LCO1490/HCO2198(Folmer,Black,Hoeh,Lutz,&Vrijenhoek,1994)whichfailedtoyieldresults,andsubsequentlyreceivedproductwithLCO1490withC_R(Shokrallaetal.,2015).ThefollowingPCRsetupwasusedforallsam-ples:2µlofthetemplateDNAwasmixedwith300nMofeachprimer,5µlof2×MyTaqRedMix (Bioline)andthereactionwasfilledupto10µlwithdouble-distilledwater.ThePCRcyclingconditionswereasfollows:initialdenaturationfor5minin95°C,then35cyclesofdena-turationfor30sin95°C,annealingfor30sin42–57°C(theanneal-ingtemperaturewasgene-specificasdetailedinBalvínetal.(2015),andelongationfor30sin72°C,endingwithfinalelongationstepfor5minin72°C.AblankcontrolwasincludedineachPCRbatch.FortheP. cavernissample,wetriedtoincreasePCRsuccessbyaddingmore

F I G U R E 1   Bucimex chilensis(whitearrow)atthebaseofthetail,onthedorsalsurfaceofMyotis chiloensis

4  |     OSSA et Al.

DNA(upto6µl),byincreasingthetotalvolume(upto20µl),andbyincreasingthenumberofPCRcyclesto50.Forallgenes,successfulPCRproductswerecleanedbyadding1µlofExonucleaseIand1.0µlofFastAP (both included in theA'SAPcleankit; productnr80350,ArcticZymes,Trømssa,Norway)toeachproduct,andbyheatingthemixto37°Cfor10minand85°Cfor5min.Afterthat,sequenceswereshippedtoMacrogenEurope(Macrogen,Seoul,Rep.ofKorea)forse-quencing.Resultingsequencesweretrimmedforsequencingprimersandnon-reliablepoor-qualityregionsandthenalignedpergeneusingGeneiousR6(Kearseetal.,2012).

2.5 | Phylogenetic analysis

ToconstructCimicidaephylogeny,wedownloadedall sequencesusedbyBalvínetal.(2015).TheseincludedsequencesfromCimex pipistrelle Jenyns, 1839, C. adjunctus Barber, 1939, C. japonicus

Usinger,1966,C. hemipterus Fabricius,1803,C. lectularius L.,Cimexsp.,C. latipennis Usinger&Ueshima,1965,C. pilosellus,C.cf.anten‐natus,Cacodmus vicinus Horvath,1934,Cacodminae sp.,Oeciacus vicarius Horvath, 1912, O. hirundinis (Lamarck, 1816), Paracimex setosus Ferris & Usinger, 1957, Aphrania elongata Usinger, 1966.Additionally,wedownloadedsequencesofLeptocimex inordinatus Ueshima, 1968 from GenBank. Similarly, for phylogenetic out-group,weretrievedCimicomorphasequencesfromRhodnius pro‐lixusStål,1859(Reduviidae),Lygus elisus VanDuzee,1914(Miridae),and Orius niger(Wolff,1811)(Anthocoridae)followingBalvínetal.(2015). The accession codes are listed inTable1.Unfortunately,despite rather comprehensive data set, we could not retrievefreshsamplesorsequencesforalltheCimicidaespeciesfoundinSouth America, for example those collected from Argentina (DiBenedetto, Autino, González, & Argoitia, 2017). All the sampleswithaccessioncodesandothermetadataarecollectedinTable1.

F I G U R E 2  MapofaustralSouthAmerica.PreviouscollectionsitesofBucimex chilensisareindicatedwithreddots.Thepresentsamplecollection site is indicated with a red triangle

     |  5OSSA et Al.

TAB

LE 1

 Samplesusedinstudyandaccessionnumbersforsequences(Ossaetal.)

Sam

ple

IDSp

ecie

sCo

llect

ion

coun

try

Refe

renc

esIT

S216

SEF

1CO

I18

S

KT380159*

Lept

ocim

ex in

ordi

natu

sTh

aila

ndPotiwat,Sungvornyothin,

Samung,Payakkapol,and

Apiwathnasorn(2016)

––

–KT380159

–

KT380160*

Lept

ocim

ex in

ordi

natu

sTh

aila

ndPotiwatetal.(2016)

––

–KT380160

–

KT380161*

Lept

ocim

ex in

ordi

natu

sTh

aila

ndPotiwatetal.(2016)

––

–KT380161

–

Pcav-1

*Pr

imic

imex

cav

erni

sUSA

Thisstudy

––

–MK141690

–

Buci-1

Buci

mex

chi

lens

isChile

Thisstudy

MK205317

MK190909

–MK141702

MK201662

Buci-2

Buci

mex

chi

lens

isChile

Thisstudy

MK205318

MK190910

–MK141694

MK201663

BB-2

Cim

ex p

ilose

llus

Canada

Thisstudy

MK205330

MK190908

MK213763

MK141693

MK201659

BB-9

Cim

ex a

djun

ctus

Canada

Thisstudy

MK205332

--

MK141701

MK201660

BB-4

Cim

ex a

djun

ctus

Canada

Thisstudy

MK205331

--

MK141691

MK201661

ORA-2

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205323

--

MK141703

MK201664

TKU-8

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205325

--

MK141706

MK201667

TKU-5

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205322

--

MK141704

MK201665

TKU-6

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205327

--

MK141705

MK201666

TRE-1

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205326

-MK213764

MK141695

MK201668

WE-1

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205319

-MK213765

MK141696

MK201669

WE-2

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205328

-MK213766

MK141692

MK201670

WE-3

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205324

-MK213767

MK141697

MK201671

WE-4

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205329

-MK213768

MK141698

MK201672

WE-5

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205320

-MK213769

MK141699

MK201673

WE-6

Cim

ex le

ctul

ariu

sFinland

Thisstudy

MK205321

-MK213770

MK141700

MK201674

48Ci

mex

pip

istre

lli g

roup

2CzechRep.

Balvínetal.(2015)

KC503542

GU

9855

53KC503545

GU

9855

31KC503546

52Ci

mex

pip

istre

lli g

roup

1CzechRep.

Balvínetal.(2015)

KC503543

GU

9855

49KC503545

GU985527

KC503547

57Ci

mex

pip

istre

lli g

roup

2CzechRep.

Balvínetal.(2015)

KC503542

GU

9855

55KC503545

GU

9855

33KC503548

61Ci

mex

pip

istre

lli g

roup

2CzechRep.

Balvínetal.(2015)

KC503543

GU

9855

51KC503545

GU

9855

29KC503549

62Ci

mex

pip

istre

lli g

roup

2CzechRep.

Balvínetal.(2015)

KC503542

GU

9855

54KC503545

GU

9855

31KC503550

73Ci

mex

pip

istre

lli g

roup

1CzechRep.

Balvínetal.(2015)

KC503542

GU

9855

50KC503545

GU

9855

28KC503551

83Ci

mex

pip

istre

lli g

roup

2U.K.

Balvínetal.(2015)

KC503543

GU

9855

56KC503545

GU

9855

34KC503552

103

Cim

ex p

ipist

relli

gro

up 2

Bulgaria

Balvínetal.(2015)

KC503542

GU

9855

52KC503545

GU

9855

30KC503553

350

Cim

ex p

ipist

relli

gro

up: C

imex

ja

poni

cus

Japan

Balvínetal.(2015)

KF018700

KF018727

KF018744

KC503541

KF018713

140

Cim

ex a

djun

ctus

(C.

pilo

sellu

s gro

up)

USA

Balvínetal.(2015)

KF018699

GU

9855

58KF018742

GU

9855

36KF018712

(Con

tinue

s)

6  |     OSSA et Al.

Sam

ple

IDSp

ecie

sCo

llect

ion

coun

try

Refe

renc

esIT

S216

SEF

1CO

I18

S

141

Cim

ex a

djun

ctus

(C.

pilo

sellu

s gro

up)

USA

Balvínetal.(2015)

KF018698

GU985557

KF018741

GU

9855

35KF018712

142

Cim

ex a

djun

ctus

(C.

pilo

sellu

s gro

up)

USA

Balvínetal.(2015)

KF018699

GU

9855

59KF018743

GU985537

KF018712

TM_C10

Cim

ex c

f. an

tenn

atus

(C.

pilo

sellu

s gro

up)

USA

Balvínetal.(2015)

KF018705

KF018732

KF018749

KF018760

KF018718

KR_C18

Cim

ex la

tipen

nis (

C.

pilo

sellu

s gro

up)

Canada

Balvínetal.(2015)

KF018707

KF018734

KF018750

KF018758

KF018720

KR_C19

Cim

ex la

tipen

nis (

C.

pilo

sellu

s gro

up)

Canada

Balvínetal.(2015)

KF018706

KF018733

KF018750

KF018757

KF018719

KR_C20

Cim

ex p

ilose

llus

USA/Canada

Balvínetal.(2015)

KF018704

KF018731

KF018748

KF018759

KF018717

145

Cim

ex h

emip

teru

sMalaysia

Balvínetal.(2015)

KF018695

KF018724

KF018710

KF018754

KF018739

801

Cim

ex h

emip

teru

sIn

dia

Balvínetal.(2015)

KF018696

KF018725

KF018710

KF018755

KF018739

26Ci

mex

lect

ular

ius

CzechRep.

Balvínetal.(2015)

KF018697

GU

9855

46KF018740

GU

9855

24KF018711

39Ci

mex

lect

ular

ius

CzechRep.

Balvínetal.(2015)

KF018697

GU

9855

48KF018740

GU

9855

26KF018711

46Ci

mex

lect

ular

ius

CzechRep.

Balvínetal.(2015)

KF018697

GU985547

KF018740

GU

9855

25KF018711

110

Cim

ex le

ctul

ariu

sFrance

Balvínetal.(2015)

KF018697

GU

9855

45KF018740

GU

9855

23KF018711

133

Cim

ex le

ctul

ariu

sSerbia

Balvínetal.(2015)

KF018697

KF018726

KF018740

KF018756

KF018711

120

Oec

iacu

s hiru

ndin

isCzechRep.

Balvínetal.(2015)

KF018691

GU

9855

65KF018736

GU

9855

43KF148594

130

Oec

iacu

s hiru

ndin

isGermany

Balvínetal.(2015)

KF018692

GU985567

KF018736

GU

9855

44KF148594

149

Oec

iacu

s vic

ariu

sUSA

Balvínetal.(2015)

KF018694

GU

9855

63KF018738

GU

9855

41KF018709

KR_88-10n1

Oec

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s vic

ariu

sUSA

Balvínetal.(2015)

KF018694

KF018723

KF018738

KF018753

KF018709

KR_88-10n3

Oec

iacu

s vic

ariu

sUSA

Balvínetal.(2015)

KF018694

KF018722

KF018738

KF018752

KF018709

897

Cim

ex sp

.Japan

Balvínetal.(2015)

KF018693

GU

9855

64KF018737

GU

9855

42KF018708

C9

Para

cim

ex se

tosu

s–

Balvínetal.(2015)

–KF018735

KF018751

KF018761

KF018721

240

Caco

dmus

vic

inus

Jordan

Balvínetal.(2015)

KF018701

KF018728

KF018745

KF018762

KF018714

244

Caco

dmin

ae sp

.Mauretania

Balvínetal.(2015)

KF018702

KF018730

KF018747

KF018764

KF018716

243

Aphr

ania

elo

ngat

aMauretania

Balvínetal.(2015)

KF018703

KF018729

KF018746

KF018763

KF018715

ONI

Anth

ocor

idae

: Oriu

s nig

er–

Huaetal.(2008);Jungetal.

(2010)

–NC012429

–NC012429

GQ258418.1

LEL

Miri

dae:

Lyg

us e

lisus

–Wheeler&Shuh(unpubl.)

–AY252785.1

–HM215068.1

AY25

2310

.1

RPR

Redu

viid

ae: R

hodn

ius

prol

ixus

–Gaunt&Miles(2002);

García,Manfredi,Fichera,

andSegura(2003)

AF324519.1,

EU822954.1

ACPB02032738.1

AF449138.1

AY34

5868

.1

* Onlyincludedinthedataset2.

TAB

LE 1

 (Continued)

     |  7OSSA et Al.

Forthesequencesproducedinthisstudy,theprimersandlowquality regions were trimmed of the sequences, and all the se-quences including references from GenBank were aligned withMUSCLEplugin (Edgar,2004)using softwareGeneious (Kearseetal.,2012).First,weusedGenBankBLASTanalysistocheckwhetherourtrimmedsequenceswerefreefromcontamination.Forsomeofthesamples,onlyCOIsequenceswereavailable,sowepreparedtwodifferentdatasets:1(multilocus:52-taxonset)and2(COI:56-taxonset).SeeTable1fordetailsofsamples ineachdataset.Forthesetwodata sets, twomodel-basedmethods (Bayesian inferenceandmaximumlikelihood)wereusedtoanalyzethedata.

Bayesianphylogeneticanalyseswerecarriedoutusingthepro-gramMrBayesv3.2.3×64(Huelsenbeck&Ronquist,2001) inCSCservers (www.csc.fi). The GTR+G (with four rate categories forGamma)modelofsubstitutionwasfittedtoeachdataset.Thedatasetswere subjected to two runs of onemillion generations each,withevery1000thgenerationsampledandthefirst2,500sampledgenerationsdiscardedasburn-in.Similarly,weconstructedamaxi-mum-likelihoodtreewith100bootstrapreplicates(othersettingsasdefault)usingcommandlinePhyML(version20120412)(Guindon&Gascuel,2003)atCSCservers.TheposteriorprobabilitytreefromBayesiananalysisandconsensustreefromMLwasretrievedandim-portedtoGeneioustodrawthefinaltree.

3  | RESULTS

3.1 | Morphological characters in the Primicinae

The Primicimicinae individuals obtained were morphologicallyidentified as B. chilensis, P. cavernis (Figure 3). The subfamilyPrimicimicinae, towhichboth species examinedbelong to, differsfromotherCimicidaebyhavingmottledtibiae(Figures3and4a,b,c),labrumovertwiceaslongaswideandtarsiwithseveralerectcte-nidea(spines)atinnerapexinappositiontoclaws(Figure4a,b,c),forwhichtheecologicalfunctionhasnotbeensuggestedpriortothisstudy.ThetwoPrimicimicinaespeciesaresimilarinappearance,butmaybeseparatedby the relative lengthof femoraand the lengthof firstantennalsegment,which isas longas thesecondsegmentin P. cavernis and much shorter in B. chilensis. Primicimex cavernis lacksthemycetomesandspermalegefoundinothercimicidgroups.Bucimex chilensis,ontheotherhand,exhibitsmycetomesandawell-developedspermalege(Figure3).ThePrimicimicinaetarsidiffersig-nificantlyfromthetarsiofC. lectularius (Figure4d),whichlacktheerectctenideaattheinnerapexinappositiontoclaws.Inadditiontothis,theC. lectulariusfeatureadditionalspinesandaspikeatthejointbetweenthetibiaandtarsus.Theotherspecies in thisstudyweremorphologically identified toC. lectularius (Finnishspecimen)or Cimexsp.(Canadianspecimen).

3.2 | Molecular identification of the specimens

In the BLAST analysis, the closest match for B. chilensis COI se-quenceinGenBankwasarecordofOrius minutus(Linnaeus,1758)

(Hemiptera, Cimicoidae, Anthocoridae) with very low similarity(82%, E-value 7e-151; BLAST was performed online 22nd March2017).ForP. cavernis,wewereonlyabletoretrieve309bpsequenceofCOIfromthetypespecimensinthisanalysis.Forthissequence,the closestmatch (83%, E-value 1e-68)was to Liorhyssus hyalinus (Fabricius,1794) (Hemiptera,Coreoidea,Rhopalidae).Thepercent-age identity between query and subject sequence was naturallytoo low tomakeanyconclusionsaboutphylogenetic relationshipsbasedontheBLASTanalysis.Finnishsamplesweremolecularlycon-firmedasC. lectularius, and theCanadiansampleswereconfirmedtoincludebothC. pilosellus and C. adjunctus.Allthesequencespro-ducedinthisstudywereuploadedtoGenBankwithaccessioncodesMK141690–MK141706.

3.3 | Phylogenetic analysis of the Cimicidae

The multilocus analysis of five genes using 52 taxa (in data set1; Table 1) placed the B. chilensis samples into the base of theCimicidae,nexttotheoutgroupfamiliesAnthocoriidae(Cimicoidea),Reduviidae,andMiridae (Figure5).Thesamepatternsoccurred inbothBayesian andML trees (Figure5).Moreover, theCOI phylo-geneticanalysisusing56taxa (indataset2;Table1)producedal-mostidenticalpatternscomparedtothemultilocustree(Figure6).InCOItree,theB. chilensis and P. cavernis cluster close to each other forminganowncladeatthebaseofCimicidae(Figure6).SubfamilyCacodminae appears to be a monophyletic group, and genusLeptocimexisconfirmedasamemberofCacodminae(Figure6).Ontheotherhand,thegenusOeciacusappearstobeparaphyletic,thetwospeciesnotclusteringtogether,andfurthermore,theCimex pip‐istrellisplitsintotwodistinctgroupstogetherwithC. japonicus and C. sp. All C. lectulariusspecimens,the“bedbug,”clusterinthesamecladeregardlessofgeographicalorigin(Figures5and6).TheotherCimex species (hemipterus, pilosellus, cf. antennatus, and adjunctus) formseparateclustersinallanalysis(Figures5and6).Inbothmulti-locusandCOIphylogenies,thegenusCimexseemstobepolyphyl-etic,despitehighsupportforallspeciesgroupings(Figures5and6).

4  | DISCUSSION

For the first time, we place the two known taxa in subfamilyPrimicimicinaeintoamolecularphylogenyofCimicidae.Inourphy-logeny,PrimicimicinaeisasistergrouptoallotherCimicidae,asde-scribedmorphologicallybyUsinger (1966).WealsofoundthatthetwomembersofthePrimiciminaesubfamilyhaveuniquetarsalmor-phology(ctenidium)forattachingtotheirbathosts,andthatthesetraitsmaybeancestral among thecimicids.Cimicids in theC. pip‐istrellus groupsandC. lectularius areoften loosely attached to thewings, forearmsanduropatagiumof thebat (Balvín,Sevcik, et al.,2012;Heise,1988).However,themorphologyofB. chilensis tarsi is verydifferenttoothercimicids.ThestructureoftheB. chilensis tar-susappearstobeanadaptationtoclingingontopelageofthehost,whichisalsowherebothindividualswerefound:anatypicallocation

8  |     OSSA et Al.

forothercimicids.This“cling-on”behavior,andtheresemblanceoftheabdomentotheheadofthefierceextra-terrestrialwarriorspe-ciesinthepopularTV-show,StarTrek,suggestthedescriptivenick-name,“TheKlingonbatbug.”.

The B. chilensis individualwas locatedondorsal surfaceof thebat,graspingontothepelageof thehost (Figure1).After there-movalofthecimicid,weobservedbathairclampedinbetweenthetarsalclawsandtheerectctenidiumbetweenthem,displayingthemechanisminaction(Figure4a).Thissamemorphologicalcharacter-isticisalsofeaturedontheP. cavernis,suggestingitmayalsospendextendedperiodson thehost.This feature,absent inothercimic-ids,appearstobeonlysharedbythetwomembersofthesubfamilyPrimicimicinae. A similar functional adaptation has evolved in thefamily Polyctenidae (Heteroptera), which have several functionalmorphological adaptations that facilitate the obligate associationwithbatsandcontinuouslivingonthehostspecimen:forexample,lackofwings,shortantennaeandmostimportantly,incomparisonwith Bucimex chilensis, form of tibiae, claws and associated erectctenidea (Maa,1964). Inaddition, the tarsal clawshavebeenpro-posedtobethemostimportantstructureforhostattachmentalsoinbatflies(Dick&Patterson,2006).Thisbehaviorisverydifferenttomoremanyothercimicids,which,fortheirlimiteddispersal,useaverydifferentmechanismtoattachtotheplagiopatagiumortibiaofthebatasdepictedinBalvín,Sevcik,etal.,2012).Thismodeofattachmentmaybefacilitatedbythespecializedsetaelocatedapi-coventrallyonthetibia,aswellasthestiffspines,whichflankthetibiaandtarsusjoint(Figure3d).

Beforethediscoveryofoursample,individualsofBucimex chilen‐sis described inUsinger (1966) had been obtained fromChile fromAraucaria araucana (Molina)K.KochtreesatTolhuaca(38°S,71°W);in a Nothofagus sp.hollowinLonquimay(38°S,71°W),Araucaníare-gion and in Nothofagus sp.atDalcahue(42°S,73°W),LosLagosregion,associated with either M. chiloensis or Histiotus magellanicusPhilippi,

1866batcolonies(Usinger,1966)(Figure2).OurnewrecordinTierradelFuegois1,300kmtothesouth(58°S,69°W)ofthispreviousre-cord.BecauseM. chiloensisisnotamigratingspecies(Rodriguez-SanPedro,Allendes,&Ossa,2016),thenewgeographicalrecordismostlikelynotduetoarangeexpansion,butratherreflectsthelackofre-searchonbatsor the associated invertebrates in the southern lati-tudes.Asforthenorthernrange,theB. chilensishasnotbeenreportedfromnorthernArgentina(Autino,Claps,Sanchez,&Barquez,2009),butthedistributionmostlikelyextendsfurthernorthof38°SinChile.

Theresultsofthemolecularphylogeneticanalysisusingamul-tilocusapproachareinaccordwiththepreviousphylogeniesbasedon morphology and molecular data (Balvín et al., 2015; Hornoketal.,2017;Lietal.,2012;Reinhardt&Siva-Jothy,2007;Usinger,1966), with the addition of Primicimicinae and associated taxaadded. The single locus results, using theCOI gene and includingthe P. cavernis (and Leptocimex inordinatus) samples, ofwhich onlythis single genewas retrieved, also reflected the aforementionedstudies.BothphylogeniesconstructedinthisstudyplaceP. cavernis next to theB. chilensis, indicating a strong phylogenetic signal. Tofurtherstrengthenourfindings,thesetwospeciessharemanyprim-itivefeatures,suchasthestructureofthemaleandfemalegenitals,aswellas featuresthatarespecific tothesubfamilyPrimiciminae,suchasthetarsalstructureandmottledtibiae,whichareabsentinother cimicids. Therefore, the subfamily status of Primiciminae issupported. However, themissing spermalege of P. cavernis, whichUsinger (1966)regardsasaprimitivetraitmayratherbeaderivedone (Reinhardt & Siva-Jothy, 2007), because the spermalege ispresent inAnthocoridae, the sistergroupofCimicidae (Hangayetal., 2008). Although the close relatedness between Bucimex and Primicimexseemstoberobust.Clearly,moreworkisneededtofullyresolve thephylogenetic relationshipswithin familyCimicidae (es-peciallytheparaphylyofOeciacus),superfamilyCimicoidea,andthewholeinfraorderCimicomorpha.

F I G U R E 3   Dorsal and ventral views ofBucimex chilensis(left)andPrimicimex cavernis(right).BlackarrowindicatesspermalageonB. chilensis,whichismissingfromP. cavernis

     |  9OSSA et Al.

The dispersal of individuals between roosts is crucial inmain-taininglocalandrangewidegeneticdiversityofbatbugs,butalsoallowsinvasionofnewortemporarilyabandonedroosts.However,this appears to happen rather infrequently, with only 3% of sur-veyedNyctalus noctula(Schreber,1774)carryingC. pipistrellusbugsinalargestudybyHeise,(1988).ThepredominanceofadultfemaleCimexfoundonbathostsintheoutsideroostenvironmentsupportstheideathatremainingattachedtothehostisdeliberateandservesthepurposeofdispersal (Balvín,Sevcik,etal.,2012;Heise,1988).Becauseasingle-matedfemalehastheabilitytoinitiateanewinfes-tation,theyarethemosteffectiveagentsofdispersal(Bartonicka&Gaisler,2007;Usinger,1966).ThismayalsobetrueforP. cavernis,forwhichhabitat,hostchoice,andfeedingbehaviorhavebeende-scribedindetailbyUeshima(1968).Thetarsalstructuresmostlikelyfacilitate dispersal in this cave-dwelling species,which has accesstothousandsofhosts.However,TierradelFuego iscave-freeandpopulationdensityofhostsislow.Oneoftheradiotrackedhostin-dividuals(M. chiloensis) inthisstudyappearedtoroostsolitaryinahollow tree, which is a relatively unsheltered roostwith fluctuat-ingclimaticconditions.Anindividualtreecanonlybeconsidereda

semi-permanentroost,oftenonlyusedbybatsforsomeyears,whentreesareatacertaindegreeofdecay(Lacki&Baker,2003;Lacki,Baker,&Johnson,2012).Tree-roostingbatsalsouseseveralroostswithin their home range and showahighdegreeof roost switch-ingwithinaseason(Kerth,Ebert,&Schmidtke,2006;Lewis,1995).Therefore, a low host population density and temporary use ofroostsbythehostsmaynecessitateamorepermanent,ectoparasiticlife-historyforB. chilensis attached to its host.

Most cimicids are generalist when it comes to host specieschoice,althoughhostassociationcaninfluencevariationinsalivarygene proteins in populations specializing in specific host species(Talbot,Vonhof,Broders,Fenton,&Keyghobadi,2018).BothC. lect‐ularius and C. pipistrellii have been described frommany bat hostspecies(Balvinetal.,2014).Primicimex cavernishasbeendescribedasexpressinghostspecificitytowardTadarida brasiliensis(Ueshima,1968).Neycavein,MedinaCo.Texas,wherethespecieshasbeendescribed from (Usinger, 1966), is a seasonal roost forT. brasilien‐sis as well as Mormoops megalophylla,whichmay act as a second-aryhost (M.Meierhofer,pers.comm.).Sofar,B. chilensishasbeendescribedonM. chiloensis and Histiotus magellanicus (Usinger,1966).

F I G U R E 4   (a)TarsalclawsclingingontoMyotis chiloensisfur.(b)Tarsalclawsanderectctenidea(blackarrow),whichfacilitategraspinghosthair.(c)AsimilartarsalstructureonPrimicimex caverniswithctenidea(blackarrow).(d)ThetibiaandtarsiofCimex lectulariarus with specializedsetaeonthejoint(blackarrow),whichmaybeusedtofastenthebugtotheplagiopatagiumofthebat

(a)

(b) (c) (d)

10  |     OSSA et Al.

Convergentphenotypesinectoparasitescanoftenbeseenamongdifferentlineagesofahighertaxonorevenwithinasinglespecies(McCoy et al., 2005). For instance,C. lectularius and C. pipistrellus,havebeenfoundtobeaninterestingmodelforthestudyofwithin-species morphological diversification (Balvín, Munclinger, et al.,2012).Thedevelopmentofconvergentphenotypes,orinanextremesituation, alloxenic speciation, could bemediated by reproductivebarriers,whicharelikelyassociatedwithlocaladaptationofthepar-asiteandshiftinitshostspecificity(Poulin,2007).BothbatspeciesknowntohostB. chilensisusetreesandbuildingstoformtheircolo-nies(Mann,1978),andmaybesharedbetweenthespecies,butthe

coloniesofH. magellanicusaresmallerthancoloniesofM. chiloensis insouthernChile.Altamiranoetal(2017)describedtheuseoftreeholesofH. magellanicus at theAraucanía region, showing that thecolonieswereformedbynomorethan10adultindividualsandtheychangeroostfrequentlyduringtheyear(Altamiranoetal.,2017).Onthe other hand,when buildings are used, colonies ofM. chiloensis canconsistofhundredsofindividuals(Ossaetal.,2010).However,wehaveyettoobserveB. chilensis on H. magellanicusoranyotherbatspecies.FurtherelucidatingthehostspecificityandecologyofB. chilensiswouldrequireabetterunderstandingofroostingbehav-ior of the host species and acquiring specimens froma variety of

F I G U R E 5  AmultilocusDNA-basedphylogenyofCimicideausingfivegeneswithsupportvaluesforallthemaincladesbasedonbothBayesianposteriorprobability(leftnumber)andmaximum-likelihoodanalysiswith100bootstrapreplicates(right).ThreeCimicomorphafamiliesoutsideCimicidaeareusedasanoutgrouptorootthetree.ThehighertaxawithinCimicoideaaremarkedintheclades.Themorphologicaldifferencesinthetarsiareillustratedforcomparison:Orius niger(representingAnthocoridaeandotheroutgroups),Bucimex chilensis (Primiciminae),andCimex lectularius (Cimicinae+Cacodminae)

Bucimex chilensis

Rhodnius prolixus (Reduviidae)Lygus elisus (Miridae)Orius niger (Anthocoriidae)

Cacodminae sp.

Cacodmus vicinusAphrania elongata

Cimex lectularius

Cimex latipennis

Cimex pilosellus

Cimex adjunctus

Paracimex setosus

Cimex cf. antennatus

Cimex hemipterus

Oeciacus hirundinis

Cimex pipistrelli group 1

Cimex japonicus

Cimex sp.

Cimex pipistrelli group 2

Oeciacus vicarius

100/100

100/100

-/100

100/100

100/100

100/97

99/98

100/100100/89

100/100

100/78 100/100

100/100

100/79

99/79

100/100

100/100

100/100

100/100

100/100

100/100

100/100

100/100C

imic

oide

a

Cim

icid

aePrimicimicinae

Cimicinae

Cac

odm

inae

     |  11OSSA et Al.

hostspeciesandgeographicareas.Forinstance,becauseofitshabitofattaching to thepelageof thehost, convergentphenotypesondifferent host species and geographic areasmay requiremorpho-logicalchangesintheclawstructuretofacilitatethedifferencesinhair structure.

Here,wedescribetheKlingonbatbuganditsabilitytoadheretotheirhostatthesouthernmostdistributionofthespeciesrange,1,300kmtothesouthofthepreviouslyknownsouthernmostdis-tributionboundary inChile.Our findings show that basal cimic-idspossessadaptationsforgraspingontothepelageofhosts.In

contrast,morederivedspeciesusesetaeandspinesonthetibiaforbrieflyadheringtothewingofthehost.ThegreaterdiversityofmorederivedspecieswithinCimididae,adaptationsforattach-ing to thewing, instead of clinging to the pelage, suggests thismethod could have yet undiscovered advantages and warrantsfurtherinvestigation.Ourresultsaremostlycoincidentwithpre-viousphylogeniesbasedonmorphology.Becauseofthedifficul-ties in obtaining cimicid specimens from austral SouthAmerica,thisstudyfillsagapintheknowledgeofthiscrypticparasite-hostrelationship.

F I G U R E 6  AphylogenyofCimicideausingCOIgenewithsupportvaluesforallthemaincladesbasedonbothBayesianposteriorprobability(leftnumber)andmaximum-likelihoodanalysiswith100bootstrapreplicates(right).BothPrimiciminaespecies(B. chilensis and P. cavernis) cluster close to each other at thebaseofCimicidaewithhighsupport.ThesubfamilyCacodminaeisalsostronglysupported,butsubfamilyCimicinaeispoorlyresolved,suchaspolyphyleticgenus Cimex

Primicimex cavernis

Bucimex chilensis

Rhodnius prolixus (Reduviidae)

Lygus elisus (Miridae)Orius niger (Anthocoriidae)

Cacodminae sp.

Cacodmus vicinusAphrania elongata

Leptocimex inordinatus

Cimex lectularius

Cimex latipennis

Cimex pilosellus

Cimex adjunctus

Paracimex setosus

Cimex cf. antennatus

Cimex hemipterus

Oeciacus hirundinis

Cimex pipistrelli group 1

Cimex japonicus

Cimex sp.

Cimex pipistrelli group 2

Oeciacus vicarius

99/-99/80

100/96

98/88

99/77

99/100

100/67

100/-

100/64

92/63 100/100

100/100

99/98

100/100

99/73

100/85

100/100

100/99

100/100

100/100

100/100

100/81

Prim

icim

icin

ae

Cac

odm

inae

Cimicinae

Cimicidae

12  |     OSSA et Al.

ACKNOWLEDG MENTS

We thank The Rufford Foundation (RSG 19502-1), MarieSkłodowska-Curie Actions, Betty Väänänen Foundation and JaneandAatos Erkko Foundation for financial support.We also thankthe Wildlife Conservation Society for permission to work at theKarukinkaReserveinTierradelFuego.WearegratefultoT.HenryattheSmithsonianInstitutionNationalMuseumofNaturalHistoryforassistanceacquiringsamplesofP. cavernis,andtheWillisLabattheUniversityofWinnipegforsupplyingC. pilosellussamples..WethankDr.C.NielsonofOhioUniversityfortheuseofthemicroscopeandcamera hardware and software.We acknowledgeCSC—ITCenterforScienceLtd.,Espoo,Finland,fortheallocationofcomputationalresources.WethankDr.P.SihvonenforvaluablecommentstothemanuscriptandMaijaK.Laaksonenforartisticbugdrawings.

AUTHOR CONTRIBUTIONS

TML,GO,andJSJdesignedthestudy,conductedthefieldwork,andproducedthefirstdraftofthemanuscript.APandEJVconductedthelaboratorywork,sequencedthesamples,andanalyzedthedata.VR and IES contributed to systematics and taxonomy.All authorscontributedtothefinalversionofthemanuscript.

DATA ACCE SSIBILIT Y

Alldataandsequencesare inGenBank.PleaseseeTable1forac-cessionnumbers.

ORCID

Eero J. Vesterinen https://orcid.org/0000-0003-3665-5802

Thomas M. Lilley https://orcid.org/0000-0001-5864-4958

R E FE R E N C E S

Altamirano,T.A., Ibarra, J. T.,Novoa, F.,Vermehren,A.,Martin,K.,&Bonacic, C. (2017). Roosting records in tree cavities by a forest-dwelling bat species (Histiotus magellanicus) in Andean temperateecosystemsofsouthernChile.Bosque Valdivia,38,421–425.https://doi.org/10.4067/S0717-92002017000200020.

Armestó, J., Villagrán, C., & Kalin Arroyo, M. (1996). Ecología de losbosquesnativosdeChile.VicerrectoríaAcadémica,UniversidaddeChile,SantiagodeChile:ComitédePublicacionesCientíficas.

Arroyo, M., Donoso, C., Murúa, R., Pisano, E., Schlatter, R., & Serey,I.(1995).Haciaunproyectoforestalecológicamentesustentable:con-ceptos,análisisyrecomendaciones.InformehechoporlaComisiónCientífica Independiente del Proyecto Río Cóndor a Bayside Ltd,EEUU.Inf.NoPubl.SantiagoChile.

Autino,A.G.,Claps,G.L.,Sanchez,M.S.,&Barquez,R.M.(2009).Newrecordsofbatectoparasites(Diptera,HemipteraandSiphonaptera)fromnorthernArgentina.Neotrop. Entomol.,38,165–177.https://doi.org/10.1590/S1519-566X2009000200002

Balvin,O.,Bartonicka,T.,Simov,N.,Paunovic,M.,&Vilimova,J.(2014).DistributionandhostrelationsofspeciesofthegenusCimexonbatsinEurope.Folia Zoologica,63,281–289.

Balvín, O., Munclinger, P., Kratochvíl, L., & Vilímová, J. (2012).MitochondrialDNAandmorphologyshow independentevolution-aryhistoriesofbedbugCimex lectularius(Heteroptera:Cimicidae)onbats and humans.Parasitology Research,111, 457–469. https://doi.org/10.1007/s00436-012-2862-5

Balvín,O.,Roth,S.,&Vilímová,J.(2015).MolecularevidenceplacestheswallowbuggenusOeciacusStålwithinthebatandbedbuggenusCimexLinnaeus(Heteroptera:Cimicidae):ThegenusOeciacuswithinthe genusCimex. Systematic Entomology,40, 652–665. https://doi.org/10.1111/syen.12127

Balvín, O., Sevcik, M., Jahelkova, H., Bartonicka, T., Orlova, M. V.,& Vilimova, J. (2012). Transport of bugs of the genus Cimex(Heteroptera: Cimicidae) by bats inwestern Palaearctic.Vesperilio,16,43–54.

Bartonicka, T. (2008). Cimex pipistrelli (Heteroptera, Cimicidae)and the dispersal propensity of bats: An experimental study.Parasitology Research, 104, 163–168. https://doi.org/10.1007/s00436-008-1175-1

Bartonicka, T., & Gaisler, J. (2007). Seasonal dynamics in the num-bers of parasitic bugs (Heteroptera, Cimicidae): A possiblecause of roost switching in bats (Chiroptera, Vespertilionidae).Parasitology Research, 100, 1323–1330. https://doi.org/10.1007/s00436-006-0414-6

Brown,C.,&Bomberger-Brown,M.(1996).Coloniality in the Cliff Swallow. Chicago,Illinois:TheUniversityofChicagoPress.

Cho,S.,Mitchell,A.,Regier,J.C.,Mitter,C.,Poole,R.W.,Friedlander,T.P.,&Zhao,S.(1995).Ahighlyconservednucleargeneforlow-levelphylogenetics: Elongation factor-1 alpha recovers morphology-basedtreeforheliothinemoths.Molecular Biology and Evolution,12,650–656.

Damgaard, J.,Andersen,N.M.,&Sperling,F.A.H. (2000).Phylogenyof the water strider genus Aquarius Schellenberg (Heteroptera:Gerridae) based on nuclear and mitochondrial DNA sequencesand morphology. Insect Syst. Evol., 31, 71–90. https://doi.org/10.1163/187631200X00327

Di Benedetto, I.M. D., Autino, A. G., González, C. A., & Argoitia,M.A. (2017). Propicimex tucmatiani (Wygodzinsky, 1951) (Hemiptera,Cimicidae, Cimicinae): A new bat ectoparasite for the Corrientesprovince, Argentina. Check List, 13, 475–478. https://doi.org/10.15560/13.5.475

Dick,C.W.,&Patterson,B.D.(2006).Batflies:Obligateectoparasitesofbats.In:S.Morand,B.R.Krasnov,&R.Poulin(eds),Micromammals and Macroparasites(pp.179–194).Tokyo:Springer.

Edgar,R.C.(2004).MUSCLE:Multiplesequencealignmentwithhighac-curacyandhighthroughput.Nucleic Acids Research,32,1792–1797.https://doi.org/10.1093/nar/gkh340

Folmer,O.,Black,M.,Hoeh,W.,Lutz,R.,&Vrijenhoek,R.(1994).DNAprimers for amplification of mitochondrial cytochrome c oxidasesubunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol.,3,294–299

García, B. A., Manfredi, M., Fichera, L., & Segura, E. L. (2003). Shortreport:Variation inmitochondrial12Sand16SribosomalDNAse-quences in natural populations of Triatoma infestans (Hemiptera:Reduviidae). The American Journal of Tropical Medicine and Hygiene,68,692–694.https://doi.org/10.4269/ajtmh.2003.68.692.

Gaunt,M.W.,&Miles,M.A. (2002).An insectmolecular clock datesthe origin of the insects and accords with Palaeontological andBiogeographic landmarks.Molecular Biology and Evolution,19,748–761.https://doi.org/10.1093/oxfordjournals.molbev.a004133

Guindon,S.,&Gascuel,O.(2003).Asimple,fast,andaccuratealgorithmto estimate large phylogenies by maximum likelihood. Systematic Biology,52,696–704.https://doi.org/10.1080/10635150390235520

Hafner,M.S.,&Nadler,S.A.(1988).Phylogenetictreessupporttheco-evolutionofparasitesandtheirhosts.Nature,332,258–259.https://doi.org/10.1038/332258a0

     |  13OSSA et Al.

Hajibabaei,M.,Janzen,D.H.,Burns,J.M.,Hallwachs,W.,&Hebert,P.D.N.(2006).DNAbarcodesdistinguishspeciesoftropicalLepidoptera.Proceedings of the National Academy of Sciences, 103, 968–971.https://doi.org/10.1073/pnas.0510466103

Hangay,G.,Gruner,S.V.,Howard,F.W.,Capinera,J.L.,Gerberg,E.J.,Halbert,S.E.,…Cherry,R. (2008).MinutePirateBugs(Hemiptera:Anthocoridae), In J. L. Capinera (Ed.), Encyclopedia of Entomology (pp. 2402–2412). Dordrecht, Netherlands: Springer. https://doi.org/10.1007/978-1-4020-6359-6_4633

Heise, G. (1988). Zum Transport von Fledermauswanzen (Cimicidae)durchihreWirte.Nyctalus,2,469–473.

Henry,T. (2009).BiodiversityofHeteroptera. InR.G.Foottit,&P.H.Adler (Eds.), Insect Biodiversity: Science and Society. Oxford, UK:BlackwellPublishing.

Hornok,S.,Szőke,K.,Boldogh,S.A.,Sándor,A.D.,Kontschán,J.,Tu,V.T.,…Estók,P.(2017).Phylogeneticanalysesofbat-associatedbugs(Hemiptera:Cimicidae:CimicinaeandCacodminae)indicatetwonewspecies close to Cimex lectularius. Parasit. Vectors, 10, https://doi.org/10.1186/s13071-017-2376-1

Hua,J.,Li,M.,Dong,P.,Cui,Y.,Xie,Q.,&Bu,W. (2008).Comparativeand phylogenomic studies on the mitochondrial genomes ofPentatomomorpha (Insecta: Hemiptera: Heteroptera). BMCGenomics,9,610.https://doi.org/10.1186/1471-2164-9-610

Huelsenbeck,J.P.,&Ronquist,F.(2001).MRBAYES:Bayesianinferenceofphylogenetictrees.Bioinforma. Oxf. Engl.,17,754–755.https://doi.org/10.1093/bioinformatics/17.8.754

Johnson, C. G. (1941). The ecology of the bed-bug, Cimex lectulariusL., in Britain. J. Hyg. (lond.), 41, 345–461. https://doi.org/10.1017/S0022172400012560

Kambhampati,S.,&Smith,P.T.(1995).PCRprimersfortheamplificationoffourinsectmitochondrialgenefragments.Insect Molecular Biology,4,233–236.https://doi.org/10.1111/j.1365-2583.1995.tb00028.x

Kearse,M.,Moir,R.,Wilson,A.,Stones-Havas,S.,Cheung,M.,Sturrock,S.,…Drummond,A. (2012).GeneiousBasic:An integratedandex-tendabledesktopsoftwareplatformfor theorganizationandanal-ysisofsequencedata.Bioinforma. Oxf. Engl.,28,1647–1649.https://doi.org/10.1093/bioinformatics/bts199

Kerth,G.(2008).Causesandconsequencesofsocialityinbats.BioScience,58,737–746.https://doi.org/10.1641/B580810

Kerth,G.,Almasi, B., Ribi,N., Thiel,D.,& Lüpold, S. (2003). Social in-teractions among wild female Bechstein’s bats (Myotis bechsteinii) livinginamaternitycolony.Acta Ethologica,5,107–114.https://doi.org/10.1007/s10211-003-0075-8

Kerth,G.,Ebert,C.,&Schmidtke,C. (2006).Groupdecisionmaking infission–fusion societies: Evidence from two-field experiments inBechstein’sbats.Proceedings of the Royal Society B‐Biological Sciences,273,2785–2790.https://doi.org/10.1098/rspb.2006.3647

Kim,H.,&Lee,S.(2008).MolecularsystematicsofthegenusMegoura(Hemiptera: Aphididae) using mitochondrial and nuclear DNA se-quences. Molecules and Cells,25,510–522.

Lacki,M.J.,&Baker,M.D.(2003).Aprospectivepoweranalysisandreviewofhabitatcharacteristicsusedinstudiesoftree-roostingbats.Acta Chiropterologica,5,199–208.https://doi.org/10.3161/001.005.0211

Lacki,M. J., Baker,M.D.,& Johnson, J. S. (2012). Temporal dynamicsofroostsnagsoflong-leggedmyotisinthePacificNorthwest.USA. J. Wildl. Manag.,76,1310–1316.https://doi.org/10.1002/jwmg.376

Lewis,S.E.(1995).Roostfidelityofbats:Areview.Journal of Mammalogy,76,481–496.https://doi.org/10.2307/1382357

Li,M., Tian,Y., Zhao,Y.,&Bu,W. (2012).Higher level phylogeny andthe first divergence time estimation of Heteroptera (Insecta:Hemiptera)basedonmultiplegenes.PLoS ONE,7,e32152.https://doi.org/10.1371/journal.pone.0032152

Lucan,R.K.(2006).RelationshipsbetweentheparasiticmiteSpinturnixandegavinus(Acari:Spinturnicidae)anditsbathost,Myotis dauben‐tonii (Chiroptera: Vespertilionidae): Seasonal, sex- and age-related

variation in infestation and possible impact of the parasite on thehostconditionandroostingbehaviour.Folia Parasitologica(praha),53,147–152.https://doi.org/10.14411/fp.2006.019

Maa,T.C. (1964).Areviewoftheoldworldpolyctenidae.Pac. Insects,6,495–516.

Mann,G.(1978).LospequeñosmamiferosdeChile.Gayana Concepc.,40,1–342.

McCoy,K.D.,Chapuis,E.,Tirard,C.,Boulinier,T.,Michalakis,Y.,Bohec,C.L.,…Gauthier-Clerc,M.(2005).Recurrentevolutionofhost-spe-cialized races in a globally distributed parasite. Proceedings of the Royal Society of London. Series B: Biological Sciences,272,2389–2395.https://doi.org/10.1098/rspb.2005.3230

Morand,S.,&Poulin,R. (2003).Phylogenies, thecomparativemethodand parasite evolutionary ecology. Advances in Parasitology, 54,281–302.

Ossa,G.,Ibarra,J.T.,Barboza,K.,Hernández,F.,Gálvez,N.,Laker,J.,&Bonacic,C.(2010).Analysisoftheecholocationcallsandmorphom-etry of a population ofMyotis chiloensis (Waterhouse, 1838) fromthesouthernChilean temperate forest.Cienc. E Investig. Agrar.,37,131–139.https://doi.org/10.7764/rcia.v37i2.177

Potiwat, R., Sungvornyothin, S., Samung, Y., Payakkapol, A., &Apiwathnasorn, C. (2016). Identification of Bat EctoparasiteLeptocimex Inordinatus from Bat-dwelling Cave, KanchanaburiProvince,Thailand.The Southeast Asian Journal of Tropical Medicine and Public Health,47,16–22.

Poulin,R.(2007).Evolutionary Ecology of Parasites,2ndedition.Princeton,NJ:PrincetonUniversityPress.

Poulin,R.,&Morand,S.(2000).Thediversityofparasites.The Quarterly Review of Biology,75,277–293.https://doi.org/10.1086/393500

Reinhardt, K., & Siva-Jothy, M. T. (2007). Biology of the bed bugs(Cimicidae).Annual Review of Entomology,52, 351–374. https://doi.org/10.1146/annurev.ento.52.040306.133913

Rodriguez-San Pedro, A., Allendes, J. L., & Ossa, G. (2016). ListaActualizada de los murciélagos de Chile con comentariossobretaxonomía,ecología,ydistribución.Biodivers. Nat. Hist.,2,18–41

Schuh,R. T.,Weirauch,C.,&Wheeler,W.C. (2009). Phylogenetic re-lationships within the Cimicomorpha (Hemiptera: Heteroptera): Atotal-evidence analysis. Systematic Entomology,34, 15–48. https://doi.org/10.1111/j.1365-3113.2008.00436.x

Shokralla, S., Porter, T. M., Gibson, J. F., Dobosz, R., Janzen, D. H.,Hallwachs, W., … Hajibabaei, M. (2015). Massively parallel multi-plexDNAsequencingforspecimen identificationusingan IlluminaMiSeqplatform.Scientific Reports,5,9687.https://doi.org/10.1038/srep09687

Simon,C.,Frati,F.,Beckenbach,A.,Crespi,B.,Liu,H.,&Flook,P.(1994).Evolution,weighting,andphylogeneticutilityofmitochondrialgenesequencesandacompilationof conservedpolymerasechain reac-tionprimers.Annals of the Entomological Society of America,87,651–701.https://doi.org/10.1093/aesa/87.6.651

Talbot,B.,Vonhof,M. J.,Broders,H.G.,Fenton,B.,&Keyghobadi,N.(2016).Range-widegeneticstructureanddemographichistoryinthebatectoparasiteCimex adjunctus. BMC Evolutionary Biology,16,268.https://doi.org/10.1186/s12862-016-0839-1

Talbot,B.,Vonhof,M. J.,Broders,H.G.,Fenton,B.,&Keyghobadi,N.(2018). Host association influences variation at salivary proteingenesinthebatectoparasiteCimexadjunctus.Journal of Evolutionary Biology,31,753–763.https://doi.org/10.1111/jeb.13265

Tian,Y.,Zhu,W.,Li,M.,Xie,Q.,&Bu,W.(2008).Influenceofdatacon-flict and molecular phylogeny of major clades in Cimicomorphantrue bugs (Insecta: Hemiptera: Heteroptera). Molecular Phylogenetics and Evolution,47, 581–597. https://doi.org/10.1016/j.ympev.2008.01.034

Ueshima, N. (1968). Cytology and bionomics of Primicimex cavernis Barber.(Cimicidae:Hemiptera).Pan‐Pac. Entomol.,44,145–152.

14  |     OSSA et Al.

Usinger, R. L. (1966).Monograph of Cimicidae (Hemiptera, Heteroptera). CollegePark,MD:EntomologicalSocietyofAmerica.

Waage, J. K. (1979). The evolution of insect/vertebrate associations.Biological Journal of the Linnean Society, 12, 187–224. https://doi.org/10.1111/j.1095-8312.1979.tb00055.x

Weinstein,S.B.,&Kuris,A.M.(2016).IndependentoriginsofparasitisminAnimalia.Biology Letters,12,20160324.https://doi.org/10.1098/rsbl.2016.0324

How to cite this article:OssaG,JohnsonJS,PuistoAIE,etal.TheKlingonbatbugs:Morphologicaladaptationsintheprimitivebatbugs,Bucimex chilensis and Primicimex cavernis,includingupdatedphylogenyofCimicidae.Ecol Evol. 2019;00:1–14. https://doi.org/10.1002/ece3.4846