Evolutionary Applications. 2018;1–13.  | 1wileyonlinelibrary.com/journal/eva

Received:16November2017  |  Accepted:15February2018DOI: 10.1111/eva.12621

O R I G I N A L A R T I C L E

Hurdles in the evolutionary epidemiology of Angiostrongylus cantonensis: Pseudogenes, incongruence between taxonomy and DNA sequence variants, and cryptic lineages

Sirilak Dusitsittipon1,2* | Charles D. Criscione3* | Serge Morand4 |  Chalit Komalamisra5 | Urusa Thaenkham1*

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2018TheAuthors.Evolutionary ApplicationspublishedbyJohnWiley&SonsLtd

*Theseauthorscontributedequallytothiswork.

1DepartmentofHelminthology,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,Thailand2DepartmentsofParasitologyandEntomology,FacultyofPublicHealth,MahidolUniversity,Bangkok,Thailand3DepartmentofBiology,TexasA&MUniversity,CollegeStation,TX,USA4CNRSISEM-CIRADASTRE,FacultyofVeterinaryMedicine,KasetsartUniversity,Bangkok,Thailand5Mahidol-BangkokSchoolofTropicalMedicine,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,Thailand

CorrespondenceCharlesD.Criscione,DepartmentofBiology,TexasA&MUniversity,CollegeStation,TX,USA.Email:ccriscione@bio.tamu.eduandUrusaThaenkham,DepartmentofHelminthology,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,Thailand.Email:urusa.tha@mahidol.ac.th

Funding informationDevelopmentandPromotionofScienceandTechnologyTalentsProject(DPST);AgriculturalResearchDevelopmentAgency,Grant/AwardNumber:CRP5705020410

AbstractAngiostrongylus cantonensis,theratlungworm,isazoonoticpathogenthatisoneoftheleadingcausesofeosinophilicmeningitisworldwide.ThisparasiteisregardedasanemergingpathogenwithaglobalrangeexpansionoutofsoutheasternAsiapost-WWII.Todate,molecularsystematic/phylogeographicstudiesonA. cantonensis have mainlyusedtwomitochondrial(mtDNA)markers,cytochrome c oxidase 1(CO1)andcytochrome b(CYTB),wherethefocushaslargelybeendescriptiveintermsofreport-inglocalpatternsofhaplotypevariants.Inordertolookformoreglobalevolutionarypatterns,wehereinprovideacollectivephylogeneticassessmentusingthesixavail-ablewholemtDNAgenomesamplesthathavebeentaggedasA. cantonensis,A. ma-laysiensis, or A. mackerrasae along with all other GenBank CO1 and CYTB partialsequences that carry these species identifiers. The results reveal three importantcomplicationsthatresearcherswillneedtobeawareof,orwillneedtoresolve,priortoconductingfuturemolecularevolutionarystudiesonA. cantonensis.Thesethreeproblemsare (i) incongruencebetweentaxonomic identificationsandmtDNAvari-ants (haplotypes or whole mtDNA genome samples), (ii) the presence of a CYTB mtDNApseudogene,and(iii)theneedtoverifyA. mackerrasaeasaspeciesalongwithotherpossiblecrypticlineages,ofwhichthereissuggestiveevidence(i.e.,A. canton-ensiscouldbeaspeciescomplex).Weprovidedadiscussionofhowthesecomplica-tionsarehurdlestoourunderstandingoftheglobalepidemiologyofangiostrongyliasis.Wecallforfuturestudiestobemoreexplicitinmorphologicaltraitsusedforidentifi-cations(e.g.,providemeasurements).Moreover, itwillbenecessarytorepeatpriormorphologicalandlife-historystudieswhilesimultaneouslyusingsequencedata inordertoassesspossibleassociationsbetweencriticalepidemiologicaldata(e.g.,bio-geography,virulence/pathology,hostspeciesuse)andspecificlineages.

K E Y W O R D S

Angiostrongylus cantonensis,Angiostrongylus mackerrasae,Angiostrongylus malaysiensis,crypticlineages,cytochrome b(CYTB)gene,cytochrome c oxidase1(CO1)gene,pseudogene,speciescomplex

2  |     DUSITSITTIPON eT al.

1  | INTRODUC TION

Angiostrongylus cantonensis,theratlungworm,isazoonoticpathogenthatisoneoftheleadingcausesofeosinophilicmeningitisworldwide(Barrattetal.,2016).Thisparasiteisregardedasanemergingpatho-genwith a global range expansionout of southeasternAsia post-WWII(Kliks&Palumbo,1992;Wang,Lai,Zhu,Chen,&Lun,2008).DespitecasesofhumaninfectioninmanyoftheareaswhereA. can-tonensishasexpandeditsrange(Wangetal.,2008),therehasbeenlittle attempt to usemolecular evolutionarymethods to elucidatetheglobalepidemiologyofthisimportantpathogen.Indeed,theuseofDNAdataonlycameaboutinthe2000s.Forexample,ribosomalDNAsequenceswereusedtoassessmetastrongylidnematodere-lationships(Carreno&Nadler,2003)ortohelpsurveylarvaefrommolluskintermediatehosts(Fontanilla&Wade,2008;Qvarnstrom,Sullivan,Bishop,Hollingsworth,&daSilva,2007;Qvarnstrometal.,2010),and~360-bpregionofthecytochrome c oxidase 1(CO1)genewasusedtoassessrelationshipstootherspeciesofAngiostrongylus (Eamsobhanaetal.,2010).Todate,molecularsystematic/phylogeo-graphic studiesonA. cantonensishavemainlyused twomitochon-drial (mtDNA) markers,CO1 and cytochrome b (CYTB), where thefocushas largely beendescriptive in termsof reporting local pat-ternsofhaplotypevariants(Aghazadehetal.,2015;Dalton,Fenton,Cleveland,Elsmo,&Yabsley,2017;Dusitsittipon,Criscione,Morand,Komalamisra, & Thaenkham, 2017; Dusitsittipon, Thaenkham,Watthanakulpanich, Adisakwattana, & Komalamisra, 2015;Eamsobhana,Song,etal.,2017;Eamsobhana,Yong,etal.,2017;Lvetal.,2012;Monteetal.,2012;Moreiraetal.,2013;Nakayaetal.,2013;Okano etal., 2014;Rodpai etal., 2016; Simoes etal., 2011;Tokiwa etal., 2012, 2013; Vitta etal., 2016; Yong, Eamsobhana,Song,Prasartvit,&Lim,2015;Yong,Song,Eamsobhana,Goh,&Lim,2015;Yong,Song,Eamsobhana,&Lim,2016).

ComparisonsamongmolecularsystematicstudiesonA. canton-ensiswouldbeuseful to look forevolutionarypatterns that couldaid in epidemiological inference (e.g., biogeographic patterns andsources of introductions, host-associated or disease-associatedlineages).Unfortunately,because studiesdidnot concurrentlyusetheCO1 and CYTBmarkers,suchcomparisonsaredifficulttomake.However, the availability of recent whole mitochondrial genomesequences of A. cantonensis, A. malaysiensis, and A. mackerrasae provides a chance to “anchor”CO1 and CYTB clades because themtDNAdoesnotgenerallyrecombine(Ballard&Whitlock,2004).

Inordertolookformoreglobalevolutionarypatterns,wehereinprovideacollectivephylogeneticassessmentusingthesixavailablewholemtDNAgenomesamplesthathavebeentaggedasA. canton-ensis,A. malaysiensis,orA. mackerrasaealongwithallotherGenBankCO1 and CYTBpartialsequencesthatcarrythesespeciesidentifiers.Ourstudyfocusesonthesethreespeciesduetotheirmorpholog-ical and life-history similarities (Spratt, 2015; details given in theDiscussion).Our results reveal three important complications thatresearcherswillneedtobeawareof,orwillneedtoresolve,priortoconductingfuturemolecularevolutionarystudiesonA. cantonensis.

Thesethreeproblemsare(i)incongruencebetweentaxonomiciden-tifications andmtDNA variants (haplotypes or wholemtDNA ge-nomesamples),(ii)thepresenceofaCYTBmtDNApseudogene,and(iii)theneedtoverifyA. mackerrasaeasaspeciesalongwithotherpossiblecrypticlineages,ofwhichthereissuggestiveevidence(i.e.,A. cantonensis couldbea speciescomplex).Weprovidedadiscus-sionofhowthesecomplicationsarehurdles toourunderstandingoftheglobalepidemiologyofangiostrongyliasis.Wecallforfuturestudiestobemoreexplicitinmorphologicaltraitsusedforidentifi-cations(e.g.,providemeasurements).Moreover,itwillbenecessarytorepeatpriormorphologicalandlife-historystudieswhilesimulta-neouslyusingsequencedatainordertoassesspossibleassociationsbetweencriticalepidemiologicaldata(e.g.,biogeography,virulence/pathology,hostspeciesuse)andspecificlineages.

2  | MATERIAL S AND METHODS

2.1 | Assembly of CYTB and CO1 datasets

Available in GenBank, there are five mitochondrial genomesidentified as either A. cantonensis (NC_013065=GQ398121, Lvetal., 2012; AP017672, BioProject: PRJEB493; KT186242, Yong,Song, etal., 2015; KT947978, Yong etal., 2016) orA. malaysiensis (KT947979=NC_030332;Yongetal., 2016).AwholemtDNAge-nomeidentifiedasA. mackerrasaewasdiscussedinAghazadehetal.(2015),butwheretheactualsequencewasobtainedfromtheap-pendixofthethesisbyAghazadeh(2015).Werefertothesesixsam-plesasthemtDNAgenomereferencesamples.Sampleinformation,forexample,geographiclocation,andhostspecies,ofthesixrefer-encesampleswasobtainedfromtheoriginalpublicationsoravail-ableinformationonGenBankandisincludedinTablesS1andS2.

As prior studies on A. cantonensis do not include both CO1 and CYTB, a primary goal was to determine the correspondencebetweenCO1 and CYTB clades that have been identified in priorwork. Because themtDNAdoes not generally recombine (Ballard&Whitlock, 2004), we used the CYTB and CO1 sequences fromthe above sixmtDNAgenome reference samples to anchorCYTB or CO1haplotypesobtainedfrompreviousstudies.FromGenBank,we assembled all available CYTB or CO1sequencesthathavebeentaggedasA. cantonensis,A. malaysiensis,orA. mackerrasaeandthathave associated publications. TablesS1 and S2 also contain thesample information for all these additional sequences obtainedfromGenBank.Foroutgroups,weusedCYTB and CO1sequencesfrom the mtDNA genome samples of A. costaricensis (NC013067,AP017675,andKR827449)andA. vasorum(NC_018602).

AlignmentswereconductedwithClustalWinBioEdit(Hall,1999)alongwithmanualcorrectionsmadebyeye.Inaligningsequences,ahaplotypefromaparticularstudymayhavebeensubsumed(i.e.,therewasanidenticalmatchintheoverlappingregion)byalongersequencefromanotherstudy.Insuchcases,weonlyusedthelongersequenceinthephylogeneticanalyses.ThematchingGenBankhap-lotypesareindicatedinTablesS1andS2.

     |  3DUSITSITTIPON eT al.

The CYTB alignment consisted of approximately 852 nucle-otides. Primers that amplify this region ofCYTB are described in Dusitsittiponetal.(2017)(also,seebelow).Duetothepresenceofindels in a CYTBpseudogene(seeResults),theCYTBalignmentwasbasedonaminoacidtranslation(translationtable5,theinvertebratemtDNAcode,onGenBank)ofthemtDNAsequences.Subsequentnucleotide alignments (after back translation) were then kept incodonreadingframetofacilitatedownstreammolecularevolutionanalyses. The CYTB dataset contained 38 unique in-group haplo-types (including pseudogene haplotypes; see Results) and threeoutgroupsequences.TableS3containstheCYTBalignmentinfastaformat.

BecausemanyoftheavailableCO1sequenceswereshort(e.g.,~300bases),informationwithregardtotherelationshipsamongthesixmtDNAgenomereferencesampleswouldbelostifwetrimmedthealignment tomatch theshortest sequence.Asourprimary in-terestwasinassessingCO1haplotyperelationshipswithrespecttothesixmtDNAgenomereferencesamples, theCO1alignment re-tained the full-lengthsequencesof referencesamples.The result-inggappedsites(i.e.,sitesthatflanktheoverlappingregion)insuchan alignment are treated asmissing data in amaximum-likelihoodphylogeneticanalysis.WenotethatthereweretwoCO1sequences(MF000735andMF000736)fromDaltonetal. (2017)thatdidnotoverlapwith the regionofCO1thathaspredominatelybeenusedinstudiesofA. cantonensis.Asaresult, thephylogeneticpositionsof theCO1 haplotypes of Dalton etal. (2017) should only be in-terpretedwithrespecttothesixreferencesequencesandnottheremainingCO1haplotypes.The topologyof theCO1tree remainsthesameevenifthesequencesofDaltonetal.(2017)areexcluded(resultsnotshown).TheCO1datasetcontained41uniquein-grouphaplotypesandfouroutgroupsequences.TableS4containstheCO1 alignmentinfastaformat.TablesS1andS2containinformationonthe references fromwhichCO1 or CYTB haplotypesoriginated aswellasmatchinghaplotypes,namingconventions,andsampleiden-tifiersusedintheoriginalpapers.

2.2 | Phylogenetic analyses

PhylogeneticanalyseswereconductedinMEGA6software(Tamura,Stecher,Peterson,Filipski,&Kumar,2013)afterusingthemaximum-likelihoodmodel selection (with the “use all sites’ option”) to de-terminetheevolutionarysubstitutionmodel.ToconfirmthatboththeCO1 and CYTBhaplotypesfromthesixreferencesamplespro-videdthesamephylogeneticsignal,wefirstconductedmaximum-likelihood analyses using the TN93+G and HKY+G models,respectively.Forheuristicpurposesandtodeterminewhetherthetwo mtDNA markers provided similar information, we calculatedpairwisep-distancebetweenthesixreferencesamplesandtestedforacorrelationbetweentheCO1 and CYTBpairwisep-distancesusing a Mantel test. For the complete CO1 and CYTB datasets,maximum-likelihoodanalyseswiththeGTR+GandHKY+Gmod-els, respectively, were used for phylogenetic reconstruction. Onethousandbootstrapswereusedtoassessnodalsupport.Inourprior

study(Dusitsittiponetal.,2017),wefoundstrongassociationsbe-tweenninenuclearmicrosatelliteclustersandCYTBmtDNAclades.Wefollowthenamingconventionsoftheclustersandcladesidenti-fiedinDusitsittiponetal.(2017)tofacilitatediscussioninlinkingtoCO1 clades.

Ingeneral,welinkedCO1 and CYTBcladesbasedonthephylo-geneticpositionsofthereferencesamples.However,bootstrapsup-portwasexpectedtobelowinthecompleteCO1datasetbecauseofthepresenceofmanyshortsequences(e.g.,~300bases).Asaresult,itisimportanttorecognizethatthepositionsoftheshortCO1hap-lotypesaresubjecttochangependingacquisitionofnewsequencedata.Also, for the latter reason,weusedaconservativeapproachwherealinkintheCO1phylogenywasmadeonlybacktothenodethatcontainedthemostrecentcommonancestorofareferencese-quenceunlessacladehad>90%bootstrapsupport.Thereweretwoexceptionstothisrulewhereweusedtherelativephylogeneticpo-sitionsofclades;weconsidertheseexceptionstobehypothesizedclades(seeResults).

2.3 | Pseudogene analyses

Inourpriorstudy(Dusitsittiponetal.,2017),wedescribedhowtheCYTB primer pairs Cytb-F, 5′-TGAATAGACAGAATTTTAAGAG-3′and Cytb-R,5′-ATCAACTTAACATTACAGAAAC-3′ofDusitsittiponetal. (2015) would fail in some samples or produce sequencesthat translated with premature stop codons (henceforth referredto as untranslatable haplotypes). Upon the design of new prim-ers Cytb-F3 5′-ATGTTATCTTGATAAGGTAGAG-3′ and Cytb-R25′-AACCTAAATTCAATATACATACTAT-3′ (see Dusitsittipon etal.,2017),wewere able toobtain translatable sequences from speci-mensthatpreviouslyfailedorproduceduntranslatablehaplotypes.SpecimensamplingandsequencingprotocolsareexplainedindetailinDusitsittiponetal.(2017).Here,wenotethatuntranslatablehap-lotypeswereobtainedfrom41individuals(TableS1givestheindi-vidualidentifiersandlocationsofsamples)usingCytb-FandCytb-R.From30ofthese41individuals,wealsoobtainedtherealCYTB se-quenceusingCytb-F3andCytb-R2(Table1).

As pseudogenes are genes that have lost function, selectionpressures,especiallypurifying,willberelaxedandthepseudogenesmayaccumulateindelsresultinginframeshiftsandpossibleprema-turestopcodons.Also,substitutionrateswillbecomeequalamongcodonpositions;thatis,theratioofnonsynonymoustosynonymoussubstitution rates (dN/dS=ω) will approach 1 (Bensasson, Zhang,Hartl,&Hewitt,2001;Wertheim,Murrell,Smith,KosakovskyPond,& Scheffler, 2015). We used amino acid translated sequences toguide alignment between the CYTB of the six reference mtDNAsamples and the untranslatable haplotypes, that is, candidatepseudogene haplotypes. Manual corrections to nucleotide align-mentsweremadebyeye.Weinspectedthealignmentforindelsthatwouldcauseframeshiftsandprematurestopcodons.

Wethentestedforelevatedωandrelaxedselectioninthehy-pothesized pseudogene lineage. PAML v4.9d (Yang, 2007) via thePAMLX v1.3.1 GUI interface (Xu & Yang, 2013) was used to run

4  |     DUSITSITTIPON eT al.

codeml. The CYTBalignmentfilewasthesameasforthephyloge-neticanalysisabove(TableS3).Gappedsites(i.e.,ambiguouscodons)areignoredintheanalyses.Alikelihoodratiotestofbranchmodelswasusedtodeterminewhethertherewaselevatedωonthehypoth-esizedpseudogenelineage.Thenullmodel(M0)ofoneωratioforallbrancheswascomparedtoanalternativemodeloftwoωratios:onefor the hypothesized pseudogene (foreground branches) and onefortheremainingbranches(backgroundbranches).Likelihoodratiotests1and2describedinZhang,Nielsen,andYang(2005)wereusedtodeterminewhethertheelevatedωwasduetorelaxedselectionasopposedtopositiveselection(e.g.,seePavlovaetal.,2017).Inbothtests,branch-siteModelAisthealternativehypothesis.Branch-siteModelAmodelassumesfoursiteclasses:sitesunderpurifyingse-lection(0<ω0<1);sitesunderneutrality(ω1=1);sitesunderposi-tiveselectionω2>1 intheforeground lineagebutunderpurifyingselection(0<ω0<1) inbackgroundlineages;andsitesunderposi-tiveselectionω2>1intheforegroundlineagebutunderneutrality(ω1=1)inbackgroundlineages.Test1usesasanullthesitesmodelM1a,whichassumestwositeclassesforallbranches:0<ω0<1andω1=1.RejectionofthenullinTest1hasgoodpowertodetectre-laxedselection,buthasahighfalse-positiverateforpositiveselec-tion(Zhangetal.,2005).Test2comparesbranch-siteModelAtoanullwherebranch-siteModelAhasω2=1inforegroundbranches.Test2hasanappropriatetypeIerrorwhentestingforpositivese-lection(Zhangetal.,2005).Thus,asignificantTest1alongwithfail-uretorejectinTest2wouldprovidesupportforrelaxedselection.

3  | RESULTS

3.1 | Phylogenetic analyses

CO1 and CYTBphylogenetictreesbasedonthesixmtDNAgenomereferencesampleswere identical in topology (datanotshown)andmatchedtheirrelationshipsobservedintheresultsbasedonthecom-pleteCO1 and CYTBdatasets(Figures1and2).Thereisasignificantandveryhighcorrelation inCO1 and CYTBpairwisep-distancesofthesixreferencesamples(r = .99;p < .001;Table2).Thus,aswouldbeexpectediftherewasnorecombination,thetwomtDNAgenesshowthesamephylogeneticsignalandareappropriatetouseasan-chorsfortheCYTB and CO1haplotypesobservedinpreviousstudies.

Atthebroadestlevel,twodivergentcladesareobservedintheboth theCO1 and CYTB datasets (Figures1 and 2). Clade I is an-choredby three referencemtDNAsamplesoriginally identifiedasA. cantonensis (NC_013065, AP017672, and KT947978) and thesampleidentifiedasA. mackerrasae,whichisverycloselyrelatedtoAP017672.Clade II isanchoredbyKT947979 (originally identifiedas A. malaysiensis;Yongetal.,2016)andKT186242(originallyiden-tifiedasA. cantonensis,Yong,Song,etal.,2015).Athirdclade,CladeIII, was also identified in theCYTB dataset. Evidence is providedbelowthatshowsCladeIII representsaCYTBpseudogene lineagewithinspecimensthatfallinCladeII.

Comparing the CO1 and CYTB phylogenetic trees, the refer-ence samples enabled the linking of four subclades and relative

phylogenetic positions provided hypothesized links between twoadditional subclades.Within Clade I, Subclade I.1 is anchored bythereferenceKT947978(=CYTBHaplotype6ofDusitsittiponetal.,2017; redclade inFigures1and2).Subclade I.2wasanchoredbybothAP017672andthemtDNAgenomesampleofA. mackerrasae (orangecladeinFigures1and2).ThereferenceNC_013065linkedSubclade I.4 (pink clade in Figures1 and 2). Subclades I.3 and I.5(aquaandblackclades,respectively,inFigures1and2)arethehy-pothesized clades based on relative phylogenetic positions; how-ever, additional reference samples (i.e., samples from which bothCO1 and CYTBhavebeenobtained)areneededtoverifytheselinks.WithinCladeII,theSubcladeII.1(lightgreencladeinFigures1and2)isanchoredbythereferencesamplesKT186242andKT947979(=CYTBHaplotype14ofDusitsittiponetal.,2017).

Generally,thesubcladedesignationshavestrongbootstrapsup-port(>90%)intheCYTBphylogeny(Figure1).Incontrast,bootstrapsupportforsubcladesisoftenlowintheCO1phylogeny(Figure2)becausemanyhaplotypesareshort(e.g.,<500nucleotides).Asare-sultofourconservativeapproach,severalCO1haplotypesthatweresister toSubclades I.1or I.2werenot included in thesesubclades(Figure2).Likewise,noadditional linkingwasmadeinCladeIIduetoalackofadditionalreferencesequencesandthelowphylogeneticresolutionamongtheCO1 or CYTBhaplotypesinthisclade.WealsonotethatinSubcladeII.1,afewhaplotypes(KY439008,KY439011,KY439012)areshort(~300nucleotides),butshowlargedivergence(i.e., long branches; Figure2); hence, their inclusion in this cladeshouldbeconsideredtentative.

3.2 | Pseudogene analyses

From prior studies (Dusitsittipon etal., 2015, 2017), the primersCytb-F andCytb-Rproduced sequenceswithuninterruptedaminoacidtranslations(i.e.,noprematurestopcodons)forspecimensthatonly fallout inClade I (Figure1). In thecurrentstudy, thesesameprimersyieldedfivehaplotypes(H22–H26)withinterruptedaminoacid CYTBtranslationsfrom41specimenscollectedfromvariouslo-cationsacrossThailand(TableS1).UponusingprimersCytb-F3andCytb-R2, we obtainedCYTB sequences with uninterrupted aminoacid translations from30of these41 specimens (Table1).Hence,bothtranslatableanduntranslatablehaplotypeswereobtainedfromthe same individuals. The translatable haplotypes (from primersCytb-F3andCytb-R2)fromall30oftheseindividualsfallinCladeII(Table1,Figure1).

We also highlight that the Cytb-F and Cytb-R primers wereused inastudybyYong,Eamsobhana,Song,etal. (2015)forsam-plesthey identifiedasA. cantonensis and A. malaysiensis.However,upondownloadingfromGenBank,itwasobservedthatallofYong,Eamsobhana,Song,etal.(2015)sequenceslabeledasA. malaysiensis weremarkedas“unverified”onGenBank(TableS1).Indeed,straighttranslation(i.e.,nogapsinthealignmentareinserted)ofthesese-quences revealed premature stop codons.One haplotypewe ob-served,H24,was thesameas theirAMM_1TAKsample (GenBank# KP732098) (Figure1, TableS1). Aligning these “unverified”

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sequenceswithourown samples showedeight different untrans-latablehaplotypes.AfteralignmentoftheseeighttothereferencemtDNA samples, the untranslatable haplotypes had four commonregionswithdeletionsofsix,one,one,andtwonucleotidesinlength.It is after the first twodeletion regions that there is a frameshiftthatleadstoseveralprematurestopcodonsintranslationoftheun-gappedhaplotypes.Theseeighthaplotypesarelargelydistinguishedfromoneanotherbyuniquesingleindelsorafewsinglenucleotidepolymorphisms (TableS3).Thephylogenetic analyses showed thatall eight of the untranslatable haplotypes fall into a single clade,Clade III, which is sister to Clade I (Figure1). Note that becausethemaximum-likelihoodanalysistreatsgapsasmissingdata,someuniqueuntranslatable haplotypes appear identical in thephyloge-netictreeastheironlydifferencesareindels(Figure1).

The branch test with two ω ratios (ωbackground=0.015, ωfore-ground=0.534, lnL=−3,033)was significantly favoredover theM0model of a single ratio (ω=0.019, lnL=−3,037) for all branches(χ2=8, df=1, p = .005). Hence, the hypothesized pseudogenebranchesshowelevatedω.Therewasnodifferenceinthebranch-siteModelA(lnL=−3,025)andModelAwithω2=1(lnL=−3,025),thatis,ω2wasestimatedtobe1inModelA(Test2,χ

2=0,df=1,p = 1).However,Test1wassignificantwereModelAwasfavoredoverM1a(lnL=−3,031;χ2=12,df=2,p = .002).TheresultsofTest1andTest2areconsistentwithrelaxedselectiononthehypothe-sizedpseudogenelineage.

4  | DISCUSSION

4.1 | Incongruence between taxonomic identifications and mtDNA lineages

In Dusitsittipon etal. (2017), we observed two divergent CYTB clades(>11%p-distance)amongwild-caughtlarvalsamplesoriginally

identified asA. cantonensis (based onmorphology of adultwormsfromexperimentallyinfectedrats).Atthetimeofourstudy,wehadusedtwomtDNAgenomesamplesasreferencesinourphylogeneticanalysesbecause theywere identified in their respectivepublica-tionsasA. cantonensis.ThesewereNC_013065(Lvetal.,2012)andKT186242(Yong,Eamsobhana,Lim,etal.,2015;Yong,Song,etal.,2015),bothofwhichoriginatedfromlaboratory-maintainedisolatesinChinaorThailand,respectively.CladeIcontainedNC_013065andCladeIIhadKT186242.Moreover,weobservedthesetwomtDNAcladeswerecompletelycongruentwithtwogeneticallydistantclus-tersthatwerebasedon12nuclearmicrosatelliteloci(Dusitsittiponetal.,2017).AsthemtDNAandmicrosatellitescamefromthesameindividuals,thecompletenon-randomassociationbetweenmtDNAcladesandmicrosatelliteclustersprovidedconclusiveevidencethatthese twoclades representeddistinct species (Dusitsittiponetal.,2017).InthecurrentstudyafteraddingadditionalCYTBhaplotypesalongwith fourmoremtDNA reference samples, these twoCYTB cladesremainedclearlydefined(Figure1).Furthermore,thesetwoclades,anchoredby thesix referencemtDNAsamples,areclearlyobservedintheCO1phylogeny(Figure2).

While the currentmolecular data show clear evidence of twospeciesas reflectedbythetwoclades (Figures1and2), thereareevident incongruencesbetween taxonomic identificationsof sam-plesandmtDNAcladeswhencomparingtheCYTB and CO1phylog-enies.WiththeexceptionoftheA. mackerrasaereference(discussedbelow),allhaplotypes (CYTB or CO1)that fall inClade Ioriginatedfrom samples originally identified asA. cantonensis (TablesS1 andS2).Incontrast,samplesthatfallinCladeIIwereidentifiedaseitherA. cantonensis or A. malaysiensis.

In theCO1 phylogeny, several haplotypes fromwild-collectedlarval samples thatwere identified asA. cantonensis inVitta etal.(2016) fall intoClade II (TableS2,Figure2).Vittaetal. (2016)alsousedKT186242asaCO1referenceintheirphylogeny.Haplotypes

TABLE  1 SamplesfromwhichboththepseudogeneandCYTBgenewereobtained(n=30)

Sample ID Provinces Coordinates Pseudogene haplotype CYTB haplotype

BKK6,7 BKK9

Bangkok 13°45′0″N100°31′1.20″E H22 H24

H14 H14

LB19–21 LopBuri 14°51′21″N100°59′24″E H24 H14

PCK2,5 PCK7,

PrachuapKhiriKhan 12°23′42″N99°55′0″E H22 H22

H13 H16

CTI1,2,16 Chanthaburi 12°36′3″N102°16′14″E H14 H14

CMI3 ChiangMai 18°47′43″N98°59′55″E H22 H18

NWT7 NWT9

Narathiwat 6°25′N101°49′E H22 H25

H16 H19

NKI1–3,6,7 NKI10

NongKhai 17°52′5″N102°44′40″E H26 H26

H19 H20

PNA2,4 PhangNga 8°27′52″N98°31′54″E H22 H14

RNG1–5,7 Ranong 9°57′43″N98°38′20″E H24 H14

KKN6 KhonKaen 16°26′N102°50′E H25 H19

GenBankaccessionnumbersareMG516589-MG516592forH22-H25,respectively.SampleCMI4hadH23(TableS4),butwedidnotobtainthecor-respondingCYTBhaplotypefromthissample.

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from Rodpai etal. (2016), also from wild-caught larval samples,fell intoClade II. Rodpai etal. (2016) referred to their samples asA. malaysiensisinconjunctionwiththefindingoftwodivergedCO1 cladesthatcorrespondedtotwo18SribosomalDNA(rDNA)cladesthathadpriorspecies-designatedreferencesequences(seebelow).Eamsobhana,Yong,etal. (2017) identifiedadult,wild-caught sam-plesasA. malaysiensis,andtheirsamplesalsofallinCladeII(TableS2;Figure2).

Notably, we highlight that the reference sequence KT186242(Yong,Song,etal.,2015)wasbasedonthewhole-genomesequenceof a Thai laboratory isolate identified as A. cantonensis in Yong,Eamsobhana,Lim,etal. (2015).Thesamegroupinthesubsequentyear (Yong etal., 2016) published the mtDNA genome of a wild-caught adult sample fromMalaysia that they identified asA. ma-laysiensis (KT947979). Yong etal. (2016) and Eamsobhana, Yong,etal.(2017)donotcompareKT947979toKT186242.WenotethatthesetwomtDNAgenomes,whichcarrydifferentspeciesnamesinGenBank,fallinCladeII(Figures1and2)andareextremelysimilarattheCO1 and CYTB(<0.2%p-distance,Table2).

Selectiveinclusion/exclusionofhaplotypesamongstudiesalongwith possible misidentifications due to similar morphologies ofA. malaysiensis and A. cantonensis (discussedbelow)have likely ledtotheseincongruencesbetweentaxonomyofsamplesandphyloge-neticrelationshipsoftheirrespectivehaplotypes.Basedonthefact

thatallsamplesforCYTB and CO1 inClade Ihavebeen identifiedas A. cantonensisandthatEamsobhana,Yong,etal.(2017)identifiedwild-caughtadultwormsaprioriasA. malaysiensis,wehenceforthrefertoCladeIasA. cantonensisandCladeIIasA. malaysiensis.

Further support for assigning these species names to theseclades comes from Rodpai etal. (2016) in which they obtainedbothCO1 and 18S rDNA sequences from the same individuals.Rodpaietal.(2016)foundcompletecongruencebetweentwoCO1 clades and two 18S cladeswhere the two 18S clades had priorreferencesequencesdesignatingthemasA. cantonensis(GenBankAY295804;Carreno&Nadler,2003)orA. malaysiensis (GenBankEF514914;Fontanilla&Wade,2008).Tofacilitatefuturestudies,weprovidedTablesS5andS6,whichprovidesampleinformationlinking18ShaplotypesandCO1haplotypesaswell as thealign-mentoftheunique18ShaplotypesofA. malaysiensis and A. can-tonensisinfastaformat.Wealsonotethatthenuclear18Scladesof Rodpai etal. (2016) are completely congruent with the twomain diverged microsatellite clusters observed in Dusitsittiponetal.(2017),therebyreinforcingthespeciesdelimitationofcladesI and II. Of special note, because KT186242 (Yong, Song, etal.,2015)wouldnowbeclassifiedasbeingfromA. malaysiensis,thenthegenomesequenceinYong,Eamsobhana,Lim,etal.(2015)alsoneeds to be reclassified as coming fromA. malaysiensis and notA. cantonensis.

F IGURE  1 Maximum-likelihoodphylogenetictreeofthecompletemtDNACYTBdataset(TablesS1andS3).ThesixreferencemtDNAsamplesareindicatedbycircles.CladeIisanchoredbythreereferencemtDNAsamplesoriginallyidentifiedasAngiostrongylus cantonensis(NC_013065,AP017672,andKT947978)andthesampleidentifiedas A. mackerrasae.CladeIisreferredtoasA. cantonensis(seemaintextforjustification).CladeIIisanchoredbyKT947979(originallyidentifiedasA. malaysiensis;Yongetal.,2016)andKT186242(originallyidentifiedasA. cantonensis,Yong,Song,etal.,2015).CladeIIisreferredtoasA. malaysiensis (seemaintextforjustification).CladeIIIrepresentsaCYTBpseudogenelineagewithinspecimensthatfallinCladeII.OutgroupsincludeA. vasorum (NC_018602),andA. costaricensis (NC_013067,andAP017675).Subcladedesignations(discussedinmaintext)thatlinktheCYTBcladestoCO1 clades are denotedbydifferentcolors.Numbersatnodesindicatedbootstrapsupport(onlynodeswith>50%supportorthatareofparticularinterestareshown)

     |  7DUSITSITTIPON eT al.

4.2 | CYTB pseudogene

WefoundstrongevidenceforaCYTBpseudogenewithinindividu-als that are in the A. malaysiensis clade. First, from 30 individu-als (Table1), we obtained both an untranslatable haplotype thatresides inClade IIIanda translatablehaplotypethat falls inCladeII (Figure1).Second, theuntranslatablehaplotypesshowtheclas-sic signs of a pseudogenized gene in having indels, which causedframeshiftsandthus,downstreamprematurestopcodons.Thefourshareddeletionregions(TableS3)inalleighthaplotypesofCladeIII

suggest thedeletionshappened in theancestralhaplotypeof thisclade.Third,molecularevolutionanalysessupportedrelaxedselec-tionwithelevatedωonthepseudogenelineage.

Incorrect phylogenetic inferences can be drawn if thepseudogene is treated as an ortholog to the real mtDNA genes(Ballard&Whitlock,2004).Yong,Eamsobhana,Song,etal. (2015)usedtheCytb-FandCytb-RprimersonsamplesidentifiedasA. ma-laysiensis and recovered untranslatable haplotypes. As shown inour analyses, these haplotypes of Yong, Eamsobhana, Song, etal.(2015)fallintoCladeIII,thatis,thepseudogeneclade.Fortunately,

F IGURE  2 Maximum-likelihoodphylogenetictreeofthecompletemtDNACO1dataset(TablesS2andS4).ThesixreferencemtDNAsamplesareindicatedbycircles.CladeIisanchoredbythreereferencemtDNAsamplesoriginallyidentifiedasAngiostrongylus cantonensis (NC_013065,AP017672,andKT947978)andthesampleidentifiedasA. mackerrasae.CladeIisreferredtoasA. cantonensis(seemaintextforjustification).CladeIIisanchoredbyKT947979(originallyidentifiedasA. malaysiensis;Yongetal.,2016)andKT186242(originallyidentifiedasA. cantonensis,Yong,Song,etal.,2015).CladeIIisreferredtoasA. malaysiensis(seemaintextforjustification).Outgroupsinclude A. vasorum(NC_018602andNC_013067)andA. costaricensis(AP017675andKR827449).Subcladedesignations(discussedinmaintext)thatlinktheCO1cladestoCYTBcladesaredenotedbydifferentcolors.Numbersatnodesindicatedbootstrapsupport(onlynodeswith>50%supportorthatareofparticularinterestareshown)

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nophylogenetic inferencesweregrosslymisrepresentedasA. ma-laysiensisisstillsistertoA. cantonensisusingtheorthologousCO1 or CYTBgenes(Figures1and2).Nonetheless,appropriategeneticdis-tancecomparisonswouldneedtobemadebetweencladesIandIIandnotcladesIandIII(e.g.,Rodpaietal.,2016;Yong,Eamsobhana,Song,etal.,2015).

4.3 | Need to verify cryptic lineages and A. mackerrasae as species

Another interestingpatternobtainedby collectively analyzing theCO1 and CYTBdataisthatthesamplemtDNAgenomefromA. mack-errasae(Aghazadehetal.,2015)isnestedwithinsamplesthathavebeen identifiedasA. cantonensis (Subclade I.2,Figures1and2). Inparticular, theA. mackerrasaesample isverycloselyrelatedtoandlessthan1%divergent (p-distance)at theCO1 and CYTB (Table2)fromAP017672 (Figures1 and 2), an unpublishedGenBank entrythat was part of the 50 Helminth Genomes Project (BioProject:PPRJEB493).AP017672wasidentifiedasA. cantonensisfromTaiwanwithnootherinformationgiven.

Recently Song, Yong, and Eamsobhana (2017) compared thewholemtDNA ofA. mackerrasae (Aghazadeh, 2015) to KT947978(Thailand isolate of A. cantonensis, Yong etal., 2016) and toNC_013065 (China isolate ofA. cantonensis, Lv etal., 2012). Theyfound across all protein coding genes the following pairwisep-distances: A. mackerrasae-KT947978=1.73%, A. mackerra-sae-NC_013065=3.7%, KT947978-NC_013065=3.52%. TheseoveralldistancesarereflectedintheaverageoftheCO1 and CYTB p-distancesgiveninTable2.Becauseofthegeneticsimilarity,theyconcluded“…thatA. mackerrasaemaybeconspecificwithA. canton-ensis(Songetal.,2017).ItremainstoberesolvedwhetherA. mack-errasae is conspecific with A. cantonensis or undergoing incipientspeciation.”Weagreewith their argument and in general suggestconcordanceinmultiplemarkersbeusedinspeciesdelimitationes-peciallywhenmtDNAdivergenceislow(Janzenetal.,2017).

Our prior study showed concordant patterns between nuclearmicrosatellitemarkersandCYTBlineages(Dusitsittiponetal.,2017).Affiliationsbetweenthesubcladesdesignatedherein,andthesemi-crosatellite clusters are given in Table3. The non-random associa-tions betweenmtDNAhaplotypes andnuclear clusters, alongwithp-distances upwards of 6% between some CYTB haplotypes (e.g.,betweenSubcladesI.1andI.5)withinCladeI,arestrongsuggestive

evidenceofcrypticspecieswithinwhatisregardedasthesinglespe-cies A. cantonensis(Dusitsittiponetal.,2017).Forexample,fromtheNanprovinceofThailand,Dusitsittiponetal.(2017)foundtwoCYTB haplotypes (H7andH9;Subclades I.2andI.3, respectively)eachofwhichcorrespondedtodistinctmicrosatelliteclusters(clusters6and5,respectively).Thus,insympatry,strongmtDNA–nuclearmarkeras-sociations remain intact.Asanotherexample,CYTBhaplotypes13,14,16,17(SubcladeII.1;Figure1)arefoundonlyinsamplesthatbe-longtomicrosatelliteCluster8,butyetthisclusterisdispersedacrosssevenprovinces inThailand(Dusitsittiponetal.,2017).Themainte-nanceofmtDNA–nuclearmarker associationswithin sympatry andacrossgeographyindicatesreproductiveisolation.Hence,wehypoth-esize thatA. cantonensismay in factbea recentlydiverged speciescomplexwhereinthefivesubclades inCladeImayreflectdifferentspecies.

ReturningtodiscussionoftheA. mackerrasaesample,wedrawparticular attention to CYTB Haplotype 7, which was reportedwithin Nakhon Si Thammarat and Nan, Thailand (Dusitsittiponetal.,2017).Haplotype7 is veryclosely related to theA. macker-rasaesample (differsby2of852bp;SubcladeI.2;Figure1).CYTB Haplotype7wasrestrictedtoindividualsthatwereinmicrosatelliteCluster6andnootherhaplotypewasfoundinthiscluster(n = 15;Dusitsittiponetal.,2017).Again,astherewas100%concordancebetween nuclear andmtDNA data, this is suggestive evidence insupport ofA. mackerrasae as a distinct species.Nevertheless, ad-ditionalmultilocusdata areneeded to confirmwhether there areindependentevolutionaryunits(i.e.,distinctspecies)withinCladeI.Forexample,basedonthecollectiveanalysesweconductedherein,wewouldpredictthatifA. mackerrasaeisindeedadistinctspecies,then samples of morphologically identified A. mackerrasae fromAustraliawouldhaveamicrosatelliteprofilethatwouldclusterthemwithCluster6.

4.4 | Evolutionary epidemiological inferences: gaps in linking the phylogenetic data to the life history and morphology

First,we highlight two caveats in linking theCYTB and CO1 sub-clades. (i)We are assuming that there is no effective recombina-tion in themtDNA.Mitochondrial recombination,whichhasbeendocumented ina fewanimalspecies (Ladoukakis&Zouros,2017),wouldprecludeanchoringthetwophylogenies.However,asnoted

TABLE  2 Pairwisep-distancebetweenthesixmtDNAreferencesamples.CO1isabovethediagonalandCYTB is below

AP017672 Aghazadeh KT947978 NC013065 KT947979 KT186242

AP017672 0.009 0.018 0.041 0.090 0.090

Aghazadeh 0.006 0.019 0.041 0.090 0.090

KT947978 0.014 0.014 0.038 0.091 0.091

NC013065 0.036 0.038 0.036 0.086 0.086

KT947979 0.110 0.108 0.109 0.117 0.001

KT186242 0.112 0.110 0.111 0.119 0.002

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inourresults,congruentCYTB and CO1topologiesandpairwisep-distancesamongthesixmtDNAreferencesamplessuggestrecom-binationwasnotpresentinappreciableamounts.(ii)ThemanysmallCO1haplotypesledtolowbootstrapsupportformanyofthesub-clades intheCO1tree.Hence,thesubclade linkswemadeshouldberegardedastentativehypothesesthatneedtobetestedwithad-ditionaldata.Ourgoalwastobeginmakingsenseof theavailablesequencedatainrelationtothebiologyofratlungworms;thus,thesubclade designationswere necessary to facilitate discussion andaddressfutureavenuesofstudy.Belowwerelateourphylogeneticresultstowhatisknownforthelifehistories,morphology,andgeo-graphicdistributionsofA. cantonensis,A. malaysiensis,andA. mack-errasae.Admittedly,weprobablyindicatemoregapsthanlinkstothephylogeneticdata,butbycallingattentiontothesegaps,wehopetohighlightcriticalareasforfuturestudy.Weonlybrieflysummarizesomekey traitsbelow.Foramoreextensivediscussionof the lifehistoriesofthesethreespecies,wereferreaderstoanexcellentre-viewbySpratt(2015).

Angiostrongylus cantonensis, A. malaysiensis, and A. mackerrasae have similar adult morphologies. These three species are largelydistinguished by the adultmale spicule lengths and separation orlengthofbursalrays(Bhaibulaya,1968,1979;Bhaibulaya&Cross,1971;Chen,1935).Bursalrayseparationandlengthareverysubtleand A. malaysiensis and A. cantonensisoverlapintherangeofspiculelengthswithA. cantonensisbeinglargeronaverage.Angiostrongylus mackerrasaehasthesmallestspiculelengthanddoesnotoverlaptheothertwo(Bhaibulaya,1979).Likewise,thelarvaehaveverysubtlemorphologicaldifferences (Bhaibulaya,1975). Inpart, thesesubtle

differencesoroverlap in traits has likely lead to someof the tax-onomic–mtDNA clade incongruences we noted above. Therefore,wecallonfuturestudiestobeexplicitinkeytraitssuchasspiculelength.

Also, A. cantonensis, A. malaysiensis, and A. mackerrasae have beendescribedashaving similar lifehistories.Theyall infect thepulmonaryarteriesandrightventricleofvariousrodents.However,differences in developmental rates and pathological affects inprimates have been reported (Bhaibulaya, 1979; Cross, 1979). Inparticular, Cross (1979) describes howexperimental infections ofmonkeyswith various geographic isolates ofA. cantonensis (mor-phologicallyidentified)resultedingreaterpathologicaleffectsanddeaththancomparedtoinfectionswithA. malaysiensis or A. mack-errasae (monkeys survived infections with the latter species). Inexperimental infections of rats, Cross (1979) recovered a statis-tically lower percentage of adults ofA. malaysiensis compared toA. cantonensis.

Weareawareofonlytwostudiesthathavelookedatmorphol-ogy and/or life history in relation tomtDNA isolates.Monte etal.(2014) examined the morphology of laboratory-maintained wormsthathadtheCO1ac8(SubcladeI.2,Figure2)andCO1ac9(SubcladeI.5,Figure2)haplotypes. Interestingly,CO1ac8 iscloselyrelatedtotheA. mackerrasae sample. They reported significant differences inthespiculelength(mm):1.29forwormswithac8and1.23forthosewithac9.However,bothof thesemeansfall in therangeofA. can-tonensis (1–1.46)andoutof the range forA. mackerrasae (0.4–0.56)(Bhaibulaya,1979).Futurestudiesareneededtodeterminewhetherspicule length (and other traits) is affected by environmental

TABLE  3 SubcladeaffiliationstomicrosatelliteclustersfromDusitsittiponetal.(2017),countrylocations,andhostspecies.Affiliationsweremadefromsampleinformation(TablesS1andS2)fromeithertheCO1 or CYTBhaplotypesthatfallwithintherespectiveclade.(L=laboratory-maintained,W=wild-collected,U=unknownorigin.Ifnosuperscriptisgiven,thenitwaswild-collected.)

Subclade I.1 I.2I.3, hypothesized I.4

I.5, hypothesized II.1

Microsatellitecluster

1–3 6 5 Nodata 4 8

Countries ThailandL,W TaiwanU

ThailandL,W

Australia Japan UnitedStates,mainland Brazil

Thailand ChinaL,W

UnitedStates,HawaiiL

Japan Taiwan Myanmar

Thailand ChinaL,W

Japan Brazil

ThailandL,W

Malaysia Taiwan

Molluschosts Achatina fulica Cryptozona siamensis

Achatina fulica Achatina fulica Achatina fulica Limax marginatus

Achatina fulica Achatina fulica Cryptozona siamensis

Mammalhosts

Rattus rattus Rattus exulans Bandicota indica

Rattus norvegicus Rattus fuscipes Rattus rattus Bandicota indica Diplothrix legata Dasypus novemcinctu Didelphis virginiana

Rattus rattus Rattus norvegicus Rattus rattus

Rattus norvegicus Rattus rattus

Rattus rattus diardii Rattus rattus Rattus norvegicus Bandicota savilei Rattus losea

DuetolowphylogeneticresolutionintheCYTBofCladeIIandduetoalackofadditionalreferencesequences(i.e.,samplesfromwhichbothCO1 and CYTBhavebeenobtained),itisnotpossibletolinktothemicrosatelliteclusters7and9ofDusitsittiponetal.(2017).

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background(e.g.,intermediateordefinitivehostspecies).Monteetal.(2012)alsoreportgreaterlarvaloutputwithinfectionsofCO1ac9.InLeeetal. (2014),morphologyandpathogenicitywereexaminedbe-tweentwoCO1mtDNAisolates,Pingtung(P)andHualien(H),iden-tifiedasA. cantonensisfromTaiwan.Thereisdifficultyininterpretingtheir sequencedata (seeTableS2).Nonetheless, they reported sig-nificantlylowerrecoveryofadultwormsfromratsfortheHisolatecomparedtothePisolateandsmallerfemalebodysizeintheHiso-late.Leeetal.(2014)alsoreportedgreaterpathogenicityandsurvivalin rats infectedwith theP isolate.They indicatednodifferences inmalebodysize,bursalnumber,bursalshape,orspiculemorphology(thoughsizesnotgiven).

In some other interesting life-history studies, cross-breedingexperimentsbyBhaibulaya(1974)andCrossandBhaibulaya(1974)have shown that viable F1 hybrids could be generated betweenA. cantonensis × A. mackerrasae and A. cantonensis × A. malaysien-sis, respectively.However,while the F1 females appeared to pro-duceeggs (whichwe take tomeanoocytes), theF1malesdidnotproduce spermatozoa. In agreement,with the resultsof the lattercross,Dusitsittiponetal.(2017)foundanF1hybridbetweenCladeI (A. cantonensis) and Clade II (A. malaysiensis), usingmicrosatellitemarkers. The mtDNA–nuclear concordance for clades I and II in-dicates there is little tonobackcrossingofF1hybrids,which is inagreementF1sterilityfoundbyCrossandBhaibulaya(1974).

Table3 indicates both host (intermediate and definitive) andgeographiclocationsfromwhichcladeshavebeenrecorded.Somecautionisadvisedininterpretingthesereportsassamplingisnotequalamongstudies.Also,themolecular-basedidentificationsarebasedonthemtDNAresults inFigures1and2.TherearerDNA-basedsurveys.However,wedonotcovertherDNA-basedsurveyshereasthelackofvariationintherDNAprecludesassignmenttothesubcladelevel.Despitetheselimitations,someresultsconfirmpastmorphological-based surveys and some interesting patternshave also emerged. For example, although originally describedfromMalaysia,therearemorphological-basedreportsofA. malay-siensis in Thailand (Spratt, 2015). In particular, amixed infectionofA. cantonensis and A. malaysiensis hasbeen reported from ratsinBangkok,Thailand(Bhaibulaya&Techasoponmani,1972).Thesemorphological reports are now confirmed with the molecular-basedstudiesofRodpaietal.(2016)andDusitsittiponetal.(2017),whereininthelatterstudy,sympatricpopulationsofA. cantonen-sis and A. malaysiensiswere found in Thailand. In addition to thelocations reported for Subclade II.1 (Thailand,Malaysia, Taiwan),othercountrieswherethemtDNAdataindicatetheoccurrenceofA. malaysiensisincludeMyanmarandLaos(TablesS1andS2).Thus,there are nomtDNA-based identifications ofA. malaysiensis out-sideSouth-EastAsia.

In contrast,mtDNA-identified samplesofA. cantonensis,CladeI, havenotonly been found throughout South-EastAsia, but alsoin Japan, Brazil, and the continental United States. Subclades I.1and I.3 have only been reported from Thailand. Subclade I.4 islargelyrestrictedtoSouth-EastAsiaandJapan.TheHawai’ireport(GenBankKP721454inFigure1)inthissubcladeisfromaThailand

laboratory-maintained isolate by way of a Japan laboratory-maintainedisolate(TableS1).TheotherHawai’ireports,CO1haplo-typesac5bandac5(sistertoSubcladeI.2;Figure2),arealsobasedonlaboratory-maintainedisolatesinJapanorThailand(TableS2).AstheHawai’i isolatesarenot in thesamesubclades, itappears thattheremaybemorethanonetypeofHawai’ilaboratoryisolate.Themostgloballydistributed subcladesare I.2 and I.5. Subclade I.2 isinteresting in that it contains theA. mackerrasae sample,which isthesolesamplefromAustralia.Intriguingly,thelarvalsamplesfromDaltonetal.(2017)(GenBankMF000735andMF000736,Figure2)thatwerereportedfromwildlifeinthecontinentalUnitedStatesarecloselyrelatedtoA. mackerrasae(Figure2).Thus,ifSubcladeI.2rep-resentsA. mackerrasae,thencautionisadvisedininterpretingpastglobalreportsofA. cantonensisasitisclearthatSubcladeI.2hasalsobeenintroducedtotheAmericas(Table3).

5  | SUMMARY

OurstudyrepresentsaninitialattempttoconsolidatetheavailablemtDNA data to link the findings of past studies. Assimilating theavailablemtDNAhashighlightedthreecriticalfactorsthatinvestiga-torsneedtobeawareoforresolve infuturestudiesontheevolu-tionaryepidemiologyofA. cantonensis.First,thereareincongruencesbetweenspeciesidentificationsandmtDNAvariants.WeprovidedaresolutiontothisproblemaboveandTablesS1–S4toaidfuturestud-ies.Second,wepresentedstrongevidenceofaCYTBpseudogene.Theprimerswedesigned should enable avoidanceof this pseudo-gene. Third, currentmtDNA–nuclear concordant patterns stronglyhint at the presence of several cryptic species within Clade I andClade II (Dusitsittipon etal., 2017).Moremultilocus-based studiesareneededtoassessspeciesdelimitationsandtotestthevalidityofA. mackerrasae.

EosinophilicmeningitishasonlybeenattributedtoA. cantonen-sis (Barratt etal., 2016), and Spratt (2015) indicates there has notbeenunequivocalevidencetoshowthatA. mackerrasae or A. malay-siensis are zoonotics. However, as discussed above, morphologicalidentificationsofthesespeciesaredifficultduetotheirsimilarities.Thus,experimentsareneededtodeterminetraitdistributionswithinclades(orsubclades)andwherepossible,definitivelylinkmorpholog-icaltraitstothemolecular-basedphylogeneticlineages.Inaddition,currentmoleculardiagnostictoolsrelyonrDNA(Qvarnstrometal.,2010),whichdoesnothaveasmuchvariationasmtDNA.Thegeo-graphicaffiliationsofsubcladesgiveninTable3suggestglobaldistri-butionscouldbelimitedtojustsomesubclades.Finergeneticmarkerresolution is needed because existing studies suggest variation inlifehistoryorpathogenicityamonglineages(Leeetal.,2014;Monteetal., 2012). More studies are necessary to determine whethermtDNAsubclades(possiblyrepresentingaspeciescomplex)areasso-ciatedwithlife-history,infectivity,orpathogenicitytraits.Suchstud-ieswillprovideaclearerpictureoftheevolutionaryepidemiologyofA. cantonensisandcloselyrelatedspeciesaswellasenablemoleculardiagnosticswhenexaminingoutbreaksorvaryingpathologies.

     |  11DUSITSITTIPON eT al.

ACKNOWLEDG EMENTS

This work was supported by Agricultural Research DevelopmentAgency (CRP5705020410), and Development and Promotion ofScienceandTechnologyTalentsProject (DPST;RoyalGovernmentofThailandscholarship).WethankourcolleaguesinCriscioneLab(IsabelCaballero,EmilyKasl,andAndrewSakla)attheDepartmentof Biology, Texas A&MUniversity, TX, USA, for providing helpfulsuggestionsandtechnicalsupport,particularlyforthemicrosatellitework.WealsothanktheDepartmentofHelminthology,FacultyofTropicalMedicine,MahidolUniversity,Thailand, for technicalsup-port,particularlyforpartoftheDNAanalysis.

Dataforthisstudyareavailableat:NewlygeneratedpseudogenehaplotypesaredepositedinGenBank(MG516589–MG516592).Alldata,includingexplicitalignmentfilesandmatchinghaplotypeinfor-mationwithassociatedGenBankaccessionnumbers,areincludedinthesupplementalfiles.

CONFLIC T OF INTERE S T

None declared.

ORCID

Urusa Thaenkham http://orcid.org/0000-0003-0688-2422

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How to cite this article:DusitsittiponS,CriscioneCD,MorandS,KomalamisraC,ThaenkhamU.HurdlesintheevolutionaryepidemiologyofAngiostrongylus cantonensis: Pseudogenes,incongruencebetweentaxonomyandDNAsequencevariants,andcrypticlineages.Evol Appl. 2018;00:1–13. https://doi.org/10.1111/eva.12621