Asymmetrical hybridization and gene flow between Eisenia andrei and E. fetida lumbricid earthworms

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PLOSONE|https://doi.o r g/10.13 7 1/journal.p o ne.0204 4 6 9 Septembe r21,2018 1^/16

RESEARCHARTICLE

Asymmetricalhybridizationandgeneflowbetwee nEiseniaandreiandE.fetidalumbricidearthworm s

BarbaraPlytycz¹*,JanuszBigaj¹,TomaszPanz²,PawełGrzmil³

1DepartmentofEvolutionaryImmunology,InstituteofZoologyandBiomedicalResearch,JagiellonianUniver sity,Krakow,Poland,2FacultyofBiochemistry,BiophysicsandBiotechnology,JagiellonianUniversity,Krako w,Poland,3DepartmentofGeneticsandEvolution,InstituteofZoologyandBiomedicalResearch,Jagiellonian University,Krakow,Poland

*Barbara. p lytycz@uj.e d u.pl

Abstract

UniformlypigmentedEiseniaandrei(Ea)andstripedE.fetida(Ef)lumbricidearthwormsare hermaphroditescapableofself-fertilization,cross-fertilization,andasymmetricalhybridiza- OPENACCESS

Citation:PlytyczB,BigajJ,PanzT,GrzmilP(2018)A symmetricalhybridizationandgeneflowbetweenEis eniaandreiandE.fetidalumbricidearthworms.PLoS ONE13(9):e0204469.https://doi.org/10.1371 / journ al.pone . 0204469

Editor:P.Pardha-

Saradhi,UniversityofDelhi,INDIA

Received:August1,2018 Accepted:September8,2018 Published:September21,2018

Copyright:©2018Plytyczetal.Thisisanopenacce ssarticledistributedunderthetermsoftheCreativeC ommonsAttributionLicense,whichpermitsunrestri cteduse,distribution,andreproductioninanymediu m,providedtheoriginalauthorandsourcearecredite d.

DataAvailabilityStatement:Allrelevantdataare withinthepaperanditsSupportingInformationfile.

AllthesequencesweredeclaredintheGenBank(M G030809-MG030998for28SandMG030999- MG031156forCOI.

Funding:Investigationsarefinanciallysupportedb ytheNationalScienceCentre,POLAND(2016/23/B/

NZ8/00748)andMinistryofScienceandHigherEduc ationPOLAND(K/ZDS/005405).Thefundershadno roleinstudydesign,datacollectionandanalysis,deci siontopublish,orpreparationofthemanuscript”.

tion.ThelatterwasdetectedbygenotypingofF1andF2progenyofthecontrolledEa+Efpairsbysp ecies-

specificsequencesofmaternalmitochondrialCOIgenesandmaternal/paternalnuclearS28rRN Agenes.AmongF1offspringtherewereself-fertilizedEa(aAA),Ef(fFF),andcross-

fertilizedfertileEa-

derivedhybrids(aAF);thelattermatedwithEaandgavenewgenerationofEaandhybrids,whilem atedwithEfgaveEa,Ef,Ea-derivedhybridsandsterileEf-

derivedhybrids(fFA).CoelomicfluidofEaexhibitsuniquefluorescencespec- tracalledheretheM-

fluorescenceconsideredasamolecularbiomarkerofthisspecies.Sincesimilarfluorescencewas detectedalsoinsomeEf(hypotheticalhybrids?),theaimofpresentinvestigationswastoidentifyth eM-

positiveearthwormsamongfamiliesgenotypedpreviously.Itwasassumedthatfactor/sresponsi bleformetabolicpathwaysleadingtopro-ductionofundefinedyetM-

fluorophoremightbeencoded/controlledbyallelesofhypotheti- calnucleargeneofEiseniasp.segregatingindependentlyfromspecies- specificS28rRNAnucleargenes,where‘MM’or‘Mm’allelesdetermineM- positivitywhile‘mm’allelesdeter-mineM-negativephenotypes.SpectraofM- fluorescenceweredetectedinall10Ea(aAAMM)and19Ea-

derivedhybrids(aAFMm),threeoffourEf-

derivedhybrids(fFAMm)andone‘atypical’Ef(fFFMm)among13Efearthworms.Amongprogeny of‘atypical’M-pos-itiveEf(fFFMm)reappeared‘typical’M-

negativeEf(fFFmm),confirmingsuchhypothesis.Alternatively,theM-

fluorescencemightbedependentonunknowngeneproductsofverti-cally-transmittedEa- specificsymbioticbacteriasexuallytransferredtotheEfpartner.Hypothesesofintrinsicandexter naloriginofM-fluorescencemightcomplementeachother.Thepresence/absenceofM- fluorophoredoesnotcorrespondwithbodypigmentationpat-terns;Ef-

characteristicbandingappearedinposteriorpartsofhybridsbody.Inconclusion,Ea/Efhybridizati

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PLOSONE|https://doi.o r g/10.13 7 1/journal.p o ne.0204 4 6 9 Septembe r21,2018 2^/16 onmayserveforfurtherstudiesonbi-

directionalgeneflow.

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GeneflowbetweenEiseniaandreiandE.fetidaearthworms

Competinginterests:Theauthorshavedeclaredth

atnocompetinginterestsexist.

Introduction

LumbricidearthwormsfromEiseniasp.arevaluablemodelsinvariousscientificdisciplineslikebioche mistry,ecotoxicology,andbiomedicine[1–

5]whereproperspeciesdelimitationiscrucial.ThisconcernmainlyuniformlypigmentedEiseniaandr ei(Ea)andstripedE.fetida(Ef),originallydescribedaspigmentationmorphsoftheonespeciesspelled asEiseniafoetida,thenasitstwosubspecies,andlateronastwoindependentspecieswithreproductiv ebarrier[6]formingtwodistinctcladesonphylogenetictreebasedonspecies-

specificDNAsequences[7–9].

Bodypigmentationisoftennotconclusive,thusduringourearlierstudiesofEa/Efdeliv- eredfromFrancewehaveusedvariousmethodsforproperdistinctionofspecimensofthesetwosp ecies[10];amongothers,coelomicfluidwasanalyzedinrespectofpresenceoffluores-

cencespectraconsideredtobeafingerprintofE.andrei,hypotheticallyderivedfrom4-methy- lumbelliferylβ-D-glucoronide[11],calledtheMUGfluorophore[10;12;13],andhereshortlytheM- fluorophore.Contrarytoourexpectation,wehavedetectedsuchfluorescentbiomarkernotexclus ivelyinEabutalsoinsomeEfspecimens–

thusweconsideredthemashypotheticalhybrids[10;12].Justthisobservation,togetherwithawid espectrumofpigmentationpatternsofearthwormsfromourEa/Efculturespromptedustotestahy pothesisabouttheexistenceofinter-

specifichybridsbetweenEaandEf,bothofthembeingsimultaneoushermaphrodites[14]capable toself-fertilization[15].

HybridizationwasdetectedbygenotypingofF1andF2progenyofthecontrolledEa+Efpairsbys pecies-specificsequencesofbothhaploidmitochondrialCOIgenesofmaternalorigin[16;17]

(‘a’or‘f’forEaorEf,respectively)anddiploidnuclear28SrRNAgenesofmaternal/paternalorigin(e ither‘A’forEaor‘F’forEf).AmongF1offspringtherewereself-

fertilizedEa(aAA),Ef(fFF),andcross-

fertilizedfertilehybrids(aAF)derivedfromEaova;theaAFhybridsmatedwithEagavenewgen erationofEaandhybrids,andwhilematingwithEfgaveEa,Ef,aAFandsterilefFAhybridsderivedfro mEfova.Usingthemethodsofthecombinedmitochondrialandnuclearmarkerswedetectedonthe EabranchoftheCOI-

basedphylogramboththe‘pure’Easpecimens(aAA)andrelativelycommoninter-

specifichybrids(aAF),whileontheEfbranchtherewereboth‘pure’fFFspecimensandafewsterilef FAh y b r i d s[18].

SinceearthwormgenotypingwasperformedonDNAextractedfromamputated(andthenregener ating[19])tailtips,thesameparental,F1,andF2earthwormsservedasdonorsofcoe-

lomicfluid(thatwasgraduallyrestored[12;13])foranalysisinrespectofpresence/absenceofM- fluorophore.TheaimofsuchanalyseswasansweringthequestionhowmolecularmarkerspecificforE .andreicouldbetransferredtosomeE.fetidaearthworms?

Hypothetically,theM-

fluorescencemightbedependenteitheronthemetabolicpathway/sofEiseniasp.itself,ormightbe derivedfromverticallytransmittedE.andrei-specificsym-

bioticbacteriathatcan‘infect’partnersofcopulation.TheresultsoftrackingtheM-

positiveearthwormswithintheirfamiliesfrompreviousinvestigationswereconsistentwithhypothe -sisoftheintrinsicoriginoffluorophore;thedominantM-allelemightbetransmittedfromM- positiveEa(aAAMM)tofertileEa-derivedM-positivehybrids(aAFMm)andthento‘atypical’M- positiveEf(fFFmM)earthwormandsterileEf-

derivedhybrids(fFAmM).SuchintrinsicpathwaywasalsoconsistentwithreappearanceofM- negativeEf(fFFmm)earth-wormsinlong-lastingculturesofatypicalM-

positiveEf(fFFMm).However,hypotheticalparticipationofmicrobiome-

derivedfactorsinproductionofM-fluorophorecannotbeneglected.Thepresence/absenceofM- fluorophoredoesnotcorrespondwithbodypigmen-tationpattern.

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Materialsandmethods Experimentalanimals

AdultcompostingearthwormsEiseniaandrei(Ea)andEiseniafetida(Ef)fromlaboratorycolo-

niesattheLilleUniversity(France)wererearedforgenerationsintheInstituteofZoologyandBiomedical ResearchoftheJagiellonianUniversity,Krakow,Poland.

Themainanalysesofcoelomicfluidwereperformedon46outof158descendantsoflabo- ratory-pairedM-positiveEaandM-

negativeEfspecimensgenotypedpreviously[18].Inshort,duringpreviousinvestigationsthepair soffreshlyhatchedearthwormswerecultureduntilcocoonproduction/reproduction.Supravitall yamputatedtailtipsoftheseparentalspecimensandtheiroffspringservedasasourceofindividua llynumberedDNAsamplesgeneticallyana-lyzedintwoways:1)byspecies-

specific(maternallyderived)haploidmitochondrialDNAsequencesoftheCOIgenebeingeither‘

a’forwormsfromEaovaor‘f’forwormsfromEfova;2)bythediploidmaternal/paternalspecies- specific(AforEaandFforEf)nuclearDNAsequencesof28Sribosomalgene.Thedescriptionofg enotypeswereasfollow:‘aAA’forEa,‘fFF’forEf,andaAForfFAfortheirhybridsderivedeitherfrom the‘aA’or‘fF’ova,respec-

tively.AmongoffspringofEa+EfpairsthereweremainlyaAAandfFFearthwormsresultedfromth efacilitatedself-

fertilizationandsomeaAFhybridsfromaAovabutnonefFAhybridsfromfFova.TheaAFhybrids matedwithEagaveanewgenerationofEaandaAFhybrids,whilematedwithfFFgavefFF,aAF,a ndsterilefFAhybrids.Pairsofhybrids,bothaAFandfFA,producedplentycocoonsbutnohatchlin gs[18].

Proof-of-conceptinvestigationswereperformedoncoelomicfluidofspecimensfromlong- lastingculturesofM-positiveEa(EaMp),‘typical’M-negativeEf(EfMn),and‘atypical’M-pos- itiveEf(EfMp).TheEfMpindividualswereidentifiedin2013duringourpreviousstudies[10].

PigmentationpatternswerephotographicallydocumentedwiththeDSLcamera(SonySLT- A58).

AnalysisofM-fluorescenceincoelomocyte-containingcoelomicfluid

Formainexperiments,46geneticallyidentifiedaAA,fFF,aAF,orfFAspecimensfromprevi- ousstudy[18]ofsimilarbodyweights(X=0.77+0.18g),wereusedforanalysesofcoelomicfluid.

SpectrofluorimetricanalysisoftheM-fluorophoreinnon-

invasivelyretrievedcoelomicfluidwasperformedbyslightlymodifiedmethoddescribedpreviousl y[10;12;13].Afterover-

nightdepurationonmoistfilterpapers,earthwormwereimmersedin3mL0.9%Natriumchloratum (Kutno,Poland)andelectrostimulatedfor30secwithamildelectriccurrent(4.5V)forcoelomicfluid extrusionthroughdorsalporesduringanimalbodymovements.Afterfluidextrusiontheearthwor mswerereturnedtotheiroriginalboxes.OnemLoftheextrudedcoelo-mocyte-

containingcoelomicfluidwassupplementedwith20uLofTriton(Sigma-

Aldrich)andshakedfor20minonElponLaboratoryShakertype358Stodissolvecellularcompone nts.ThensampleswereadjustedwithPBSto2mLandfinal1%TritonlysateswereanalyzedusingP erkin-ElmerSpectrofluorimeterLS50B.Aspreviously[10;12;13],emissionspectraofM-

fluorophorewererecordedbetween340and480nm(lambdaat320nm,peakat380nm)whileexcit ationspectrabetween260and360nm(lambdaat380nm,peakat320nm).Fluoro-

phoresaregraduallyrestoredincoelomicfluidofelectrostimulatedarthworms[12;13]thus–

whennecessary–

theprocedureofcoelomicfluidextrusion/analysiswasrepeatedforthesamespecimensafterearthwo rms’4-weekrecoveryinsoil.

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Results

M-positiveandM-negativespecimensamonggenotypedEiseniasp.earthworms

TheM-

positive(Mp)earthwormsexhibiteddistinctspectraoffluorescencewithapeakofabsorbanceat314–

320nm(λ=380)andapeakofemissionat370–380nm(λ=320),whiletheM-

negative(Mn)earthwormsweredevoidofsuchfluorescenceinTriton-lysatesofcoelo- micfluid(InsetinFig1).Asvisibleonphylogenetictreeof46descendantsofEa+Efearth-

wormsarrangedonthebasisofmitochondrialCOIgeneofmaternalorigin,all29specimensderivedfro mE.andreiova,i.e.10aAAand19aAFhybrids,wereM-positive.Thirteenspeci-

mensfromE.fetidaovawereM-

negative,amongthem12fFFearthwormandonefFAhybrid;onlyonefFFspecimenandthreefFAhybri dswereM-positive(Fig1).

GenealogyofM-positiveandM-negativeearthworms

GenealogyofM-positiveandM-

negativedescendantsofEa+EfpairshasbeenshownonFig2.AmongF1offspringofpairsofparentals pecimensEa+EfthereareM-positiveEa,M-negativeEf,andM-

positiveaAFhybridsfromEaova,butnonefFAhybridfromtheEfova.TheaAFMphybridspairedwithaA AMpspecimensgaveF2generationofaAAMppureEaspeci-

mensandaAFMphybrids.TheaAFMphybridspairedwithM-

negativefFFearthwormsgavefourkindsofF2specimens,i.e.‘typical’fFFMnearthworms,oneM- positiveEfearthworm(fFFMp)andalsofourhybridsfromEfova,ofwhichthreewereM-

positive(fFAMp)andonewasM-negative(fFAMn)(Fig2).

SpeculationsongenotypesofM-positiveandM-negativeEiseniasp.earthworms

Hypotheticallythefactor/sresponsibleforM-

fluorescencemightbeencoded/controlledbythenucleardominant‘M’alleleofsomeunknownge ne/sofEiseniasp.whiletworecessive‘mm’allelesdetermineM-

negativephenotype.ThusgenotypesofphenotypicallyM-positiveearth-

wormsareeitherofMMorMm,whilegenotypesofM-negativespecimensarealways‘mm’.

Hypothetically,M/mallelessegregateindependentlyfromthenuclearA/Fsequencesof28Sr RNAgene.ThereforethegenotypeofM-

positiveEaspecimensmaybeeitheraAAMMoraAAMm,whilethegenotypeofM- negativeEfspecimensmaybeonlyfFFmm.Inter-

specifichybridsmightbeeitheraAFMm/aAFmMorfFAMm/fFAmM,withthefirstwrittenalleleofea chgenebeingofmaternalorigin,whileMm/mMhavethesamephenotypiceffects.

AsillustratedonFig3,duringhybridizationexperimentsstartingwithEa+Efpairs,theEaMpspecime nofaAAMMgenotypeshallproduceonlyonetypeofova,i.e.aAM,andonekindofspermatozoa,AM.Th eEfspecimens,fFFmm,shallproduceonlyfFmovaandFmspermatozoa.TheaAMovamaybeeitherse lf-fertilizedbyAMspermatozoagivingaAAMMspecimensorcross-

fertilizedbyFmspermatozoaofEfpartnergivingtheM-

positiveaAFMmhybrid.ThefFmovaoffFFmmpartnermaybeself- fertilizedbyFmspermatozoagivingfFFmmM-

negativeEfearthwormsorbytheAMspermatozoaoftheEapartnergivingM-pos-

itivefFAmMhybrids(Fig 3a and3b ).However,fFAmMhybridsfromEfovawereabsentamonginvestiga tedspecimens(framedinFig3b),thatpointedoutonasymmetricalhybridiza-

tionofEaandEf,withhybridsderivedpreferentially(orexclusively)fromtheEaova.

Theoretically,theM-

positiveaAFMmhybridsmightproducefourtypesofoocytes,aAM,aAm,aFM,andaFm,thetwolatt

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ergenotypeslessprobableduetomitochondrial-nuclear(aF)incompatibility[20–

22],andfourtypesofspermatozoa,AM,Am,FM,andFm.Onecouldexpectanypossiblecombinatio nresultingfromself-fertilizationofhybrids(seeS1Fig).

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Fig1.FluorescencespectraofM-fluorophoreanditspresence/absenceonphylogramofEa,Ef,andtheirhybrids.Themaximum- likelihoodphylogramconstructedaccordingtosequencesofthematernalmitochondrialCOIgene(‘a’or‘f’)ofEa/Efindividuallycodede arthwormscharacterizedalsobysequencesoftheirnucler28SrRNAgenes(‘AA’,‘fFF’,‘aAF’or‘fFA’)andphenotypesoftheirM- fluorophoreasM-positive(Mp)orM-negative(Mn).&:atypicalMug-negativehybridfFA149/194Mn;#:atypicalMUG-

positivespecimenfFF158/190.Genebankaccessionnumbersaregivenin[18].Inset:Examplesoffluorescencespectraofexcitation(left) andemission(right)incoelomicfluidofMp(orangesolidlines)andMn(bluedottedlines)specimens.

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Fig2.RelationshipswithinfamiliesofEa,EfandtheirhybridswithandwithouttheM-

fluorophore.ProgenyofEaandEfparentalspecies(aAA+fFF),andcrossesbetweenaAFhybridsandEa(aAF+aAA)orEf(aAF+fFF)earth worms.CodedspecimensareeitherM-positive(Mp)orM-negative(Mn).SymbolsarethesameasonFig1.

However,theM-negativeaAAmmandaAFmm,whichtheoreticalmightresultfromself-fertil- izedhybridovabyanyofhybridsperm,wereabsentamong46investigatedearthworms.Moreover ,accordingtoourpreviouswork,pairsofhybridsgavenoviableoffspring[18].Nev-

erthelesswecannotexcludeofparticipationofhybridself- fertilizationduringmatingofaAFhybridswithparentalspecies.

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Fig3.HypotheticalgenotypesofEa(aAAMM)andEf(fFFmm)pairsandtheiroffspring.a)Schemeofparentalcells,theirga metes(ova,spermatozoa)andzygotes,andb)thePunnettsquare.AssumptionisthatM-

fluorescencemightbeencoded/controlledbythenucleargenewiththedominant‘M’alleleandtherecessive‘m’allelesegregating independentlyfromthenuclearA/Fsequencesof28srRNAgene.The‘MM’and‘Mm/mM’determinestheM-

positive(Mp)phenotype(inorange)while‘mm’genotypedeterminestheM-

negative(Mn)phenotype(inblue).Punnettsquaresareadaptedtopairsofhermaphroditicearthwormsabletoself- fertilization;ovainyellow,spermatozoain

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green.Ineachpairthefirstalleleisthatofmaternalorigin.Framedgenotypeswereapparentlyabsentamonginvestigat edearthworms.Ovaandresultedoffspringwithmito-nuclearincompatibilityarecrossedout.

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TheaAFMmhybridsgaveaprogenyinpairswithEaorEfspecimensillustratedonFig4,whereprogeny frommito-nuclearincompatibleovaareexcluded,andaAFself-

fertilization(shownonS1 Fig )isomittedforaclarity.

OnlyM-

positiveoffspring(aAAMM;aAAMm/aAAmM)appearedintheaAFMm+aAAMMpairs,thatwascon sistentwithdataonFig4a.

TheoffspringofaAFMm+fFFmmpairsfromthehybrid’sova(aAMandaAm),excludingthosew ithmito-nuclear-incompatibility(aFMandaFm),mightgivebothM-positive(aAFMm)andM- negative(aAFmm)hybrids(Fig4b),butthelatterwereabsentamonginves-

tigatedearthworms.TheoffspringfromtheEfova(fFm)consistedofbothM-positiveandM- negativehybrids(fFAmMandfFAmm),the‘atypical’M-

positiveEfspecimenfFFmM,andmostcommonM-negativeEf(fFFmm)earthworm(Fig4b).

Fig5showsthatevenoneuniqueM-positivefFFMmspecimenmightinitiatepropagationofM- positivephenotypein‘traditional’Ef(fFFmm)culture;fFFMn/fFFMMgenotypesmightappearbys elf-fertilizationandcross-fertilizationwitha‘typical’M-

negativefFFmmpartner(Fig5a),andthenbymatingwithnewly-

createdotherfFFMnearthworms(Fig5b).Ontheotherhand,Fig5 b illustrate showintheprogenyof phenotypically‘atypical’M-positiveEfearthwormsmightreappearthe‘typical’M-

negativeEfspecimens,thathashappenedinearth-wormsusedforourproof- ofconceptinvestigations(seebelow).

Proof-ofconceptinvestigations:ReappearanceofM-

negativeEfspecimensamongdescendantsof‘atypical’M-positiveEfearthworms

EarthwormsfromFranceweretestedforpresence/absenceofMUF-

fluorophorein2013[10]andthengroupsofthemwereculturedfurtherseparatelyasEaMp,EfMn,an d‘atypical’EfMnspecimens.Fouryearslater,amongprogenyofEaMpandEfMntherewereexclusi velytheM-positiveEaandM-

negativeEfspecimens,respectively.Amongrandomlysampled7specimensfromthedescendantsof‘

atypical’EfMpearthwormstherewerefiveM-positive(fFFMm)andtwoM- negativespecimens(fFFmm).Theresultsofourproof-of-

conceptinvestigationswereconsistentwithhypothesisaboutinheritanceofundefinedgenewithth edominantM-alleleresponsibleforM-fluorophoreinEiseniasp.(Fig5b).

PigmentationpatternsofgenotypedM-positiveandM-negativeearthworms

AsshownonphotosinFig6,thepresence/absenceofM-

fluorophoredidnotcorrespondwithpigmentationpatternofinvestigatedearthworms.Ingeneral,i nter-segmentalgrooveswerehardlyvisibleinrelativelyuniformlycolored,lighterordarker,M- positiveEaspecimens(aAA41MpandaAA45Mp,respectively).Inter-specifichybrids,bothM- positiveaAF101MpandfFA143/159Mp,andM-

negativefFA67/149Mn,hadslightlybandedposteriorpartsofthebody.BandingwasdistinctinEfs pecimens,bothM-positive(fFF158Mp)andM-

negative(fFF42Mn,fFF61Mn,andfFF112Mn),withlighterordarkercolorationandsharplydemar -catedmuchlighterinter-segmentalgrooves(Fig6).

Discussion

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AsymmetricalhybridizationbetweenEaandEfresultedinawidespectrumofnewpheno- types,includingEa-likeearthwormswithEf-likebandedposteriorbodyparts,andthe

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Fig4.Hypotheticalgenotypesoftheoffspringoftest-crossesofthehybrids(aAFMm)withparentalspecimens.Punnettsquaresofa) (aAFMm+aAAMM);b)(aAFMm+fFFmm)pairs.Self-

fertilizationwithinaAFMmhybridisshownonS1Figthusisomittedhere.SymbolsarethesameasonFig3.

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Fig5.HypotheticalgenotypesoftheoffspringofM-positiveEfspecimenswithinEfMpculture.Punnettsquaresofa)(fFFMn +fFFMn)pair;self-fertilizationshowninonepartneronly;b)(fFFMn+fFFmm)pair.SymbolsarethesameasonFig3.

existenceofEa-specificM-fluorophoreincoelomicfluidofmosthybridsandsomeEfearth- worms;inotherwords,hybridizationenrichedthegeneticpoolofbothspecies(Fig7).

Adaptivevalueofuniformorbandedbodypigmentationpatternsmightbeexperimentallytested,e.g .throughmeasurementsofattractivenessforpotentialpredators.Sofar,wemaycon-

cludethatpigmentationpatternsmightberelevantforpreliminaryspecies/hybriddelimita- tion,anddonotcorrespondwithpresence/absenceofM-fluorophoreincoelomicfluid.TheM- fluorophorewasconsideredasmolecularmarkerofE.andreiwhileitturnedoutthatisalso

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Fig 6. PigmentationpatternsofEa,EfandtheirhybridswithandwithouttheM-fluorophore.PhotosofM-positive(Mp)andM- negative(Mn)codedspecimensofEiseniaandrei(aAA),E.fetida(fFF)andtheirhybrids(aAFandfFA).SymbolsarethesameasonFigs1and2.

presentinmajorityofhybridsandsomespecimensofE.fetida.Thus,hybridizationofEawithEfres ultedinbi-directionalgeneflow;Ef-specificgenes/allelesresponsibleforstripedpigmen- tationaretransferredfromEftoEa.Incontrast,hypotheticaldominantMallelesofgene/sresponsi bleformetabolicpathwayleadingtoproductionofM-fluorophoreflowfromM-posi-

tiveEa,throughM-positiveEa-derivedhybridstoEf,resultinginsomeM-positiveEfspeci- mens.EvenoneM-positiveEfearthwormmatedwithM-

negativepartnerpropagatestheMalleleswithinEfculture;amongoffspringof‘atypical’M- positiveEf,thetypicalM-negativeEfspecimensmayreappear(Fig7).

TheM-negativeEaandM-negativeEa-

derivedhybridswereabsentamong46investigatedearthwormsalthoughsuchphenotypes/genotyp esaretheoreticallypossible;theymightbedetectedifthenumberofinvestigatedearthwormswouldbe increased.Ontheotherhand,theirviabilitymightbeimpairediftheM-

factorplaysanimportantbiologicalroleinEiseniaspecies.

BetterviabilityandhigherfecundityofE.andreithanthoseofE.fetidaweredescribedbyseveralscie ntificteams[23–25]includingours[18].ThesameconcernsEa-

ovaderivedhybridsthatarefertile,incontrasttorareandsterilehybridsofEf-ovaorigin.Thus,theques- tionappearswhetheritisthisdependentonthepresenceofM-fluorophoreorsomeundiscov- eredmetabolicpathwaysleadingtoitsproduction?

CharacteristicfluorescencespectraofcoelomicfluidofE.andreiandE.fetidawereforthefirsttimeus edasspecificfingerprintsfortaxonomyofthesespeciesin2003,andauthorsstated

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Fig7.SchemeofmatingandmainresultsconcerningMp/MnphenotypesofEa,Efandtheirhybrids.Combinedsummaryof previous[18]andpresentexperiments.SymbolsarethesameasonFig3.

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thattheuniquefluorescencepropertiesofE.andreimolecularmarkerarecharacteristicforthe4- methylumbelliferylβ-D-glucoronide(MUGlcU)[11],calledheretheM-

fluorophore.Infact,fluorescencespectrasimilartoM-

fluorophorehavebeenshownin2008asthosederivedfrommethanolsolutionof4- methylumbelliferone,amemberofcoumarinfamilyCoumarinsare

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naturalproductspresentinetherealoilsofmanyplants,e.g.cinnamon(Cinnamonumzeylani-cum) [26].Biologicaleffectsofnaturalandsyntheticcoumarinderivativesincludeanti-plas-

modialandantimalarian[27],anti-fungal[28],anti-tuberculosis[29],anti-coagulant[30],andanti- cancer[31]activities.In2017,anotheraromaticmetaboliteuniqueforcoelomicfluidof

E.andreiwasidentifiedasthecompoundSP-8203,consistingtwoquinazoline-2,4- dionesjoinedbyanN-

acetylsperminelinkerbutitsfluorescencespectrahavenotbeenanalyzedinthispaper[32].Compound SP-

8203ispharmacologicallypotentinmammaliancellsshowingneuroprotectiveactivity[33;34].Thepre cisechemicalcharacteristicoftheM-

fluorophorerequiresfurtheranalysisbutthatisnotourcurrentconcern.Nevertheless,duetoitshypothet i-calconnectionswithpharmacologicallypotentfactors,wemayassumethattheM-

fluorophoremightbesomehowresponsibleforhigherviabilityofM-positiveE.andreiandM- positiveEa-derivedhybridsthanM-negativeE.fetidaandrareinfertileEf-

ovaderivedhybrids.FurtherstudiesontheselectedM- positiveE.fetidamightbefruitfulintestingsuchsupposition.

SpeculationsonhypotheticalgenewiththedominantM-alleleareconsistentwithassump- tionoftheintrinsicoriginofM-

fluorophore,beingentirelydependentontheearthwormownmetabolicpathways.Keepinginmin dthepeculiarcopulatorybehavioroflumbricidearth-

worms(Fig7),hypothesisofmicrobialoriginofM-

fluorescencecannotbeneglected.Almostalllumbricidearthwormsharborextracellularspecies- specificbacterialsymbiontsofthegenusVerminephrobacterlocalizedintheirexcetorynephridia [35;36].Thesesymbiontsareverti-

callytransmittedviathecocoonscontainingdevelopingembryosandpersistinspecificloca- tionthroughoutthewholelifespanofcolonizedearthworms[37;38].Recentlyithasbeenshownth atbacterialsymbiontshavebeneficialeffectsonmaturationandreproductionofE.andrei[39].So meproductsofbacterialmetabolisms,includinghypotheticalM-

fluorophore,mightaccumulateinearthwormcoelomicfluid.Hypothetically,someoftheseextrac ellularsymbiontsmaybereleasedduringcopulationtotheseminalfluid,andmayreachspermath e-

casofthepartnersofcopulation.Thentheyarereleasedtococoonstogetherwithsperm,andwithi ncocoonsinfectovaordevelopingembryosresultedfromself-orcross-

fertilization.InsuchcasesnotonlyEaandEa-derivedhybridsbutalsosomeEfandEf- derivedembryoscanbeinfectedandbecameM-positiveadults.Inconclusion,theM- positivityofsomeearthwormsmightbeconsideredasaresultof‘sexually-

derivedinfection’bysomebacterialsymbiontsspecificforE.andrei,responsibleformetabolicpat hwaysleadingtoproductionofM-fluoro-phore.Itisalsopossiblethatbothearthworm-

derivedandbacteria-derivedfactorsmustcoop-

eratetogivethefinalfluorescentproduct,thatiseitheraccumulatedbreakdownproductbeingsign ificantonlyasmolecularbiomarker,ormayhaveunrecognizedyetcrucialbiologicalsignificance.

Onthebasisofourpreviousresultswemayassumethatcoelomocytesarenotthemaincel- lularsourceofM-

fluorophoreinE.andrei,asitsamountcamebackrapidlytotheinitiallevelafterexperimentalexpulsionof coelomicfluid[13,19].ThismakesM-fluorescenceareliablemolecularmarkerfortrackingtheM- positivityamongspecimensofE.andrei/E.fetidacom-

plex,butothertechniquesshallbeusedtoshowconclusivelyitspresenceinvariousearthwormcelltype sliningcoelomiccavityand/orother(bacterial?)sources.

Conclusion

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AsymmetricalhybridizationbetweenEaandEfresultedinbi-

directionalgeneflowresultingintwophenomenarecognizedinourlaboratory.First,Ef- likebodypigmentationpatternappearedinposteriorbodysegmentsofhybrids,bothEf-andEa- derived;second,Ea-specificM-

fluorophorewastransferredtomajorityofhybridsandsomeEfearthworms.Thechemi- calnatureandbiologicalsignificanceofthisfluorophoreisstillanopenquestion,butits

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fluorescencespectraarereliablemarkersfortrackingthegeneflowbetweenE.andreiandE.fetid a.IfM-

fluorophoreisgeneticallycontrolledbyhypotheticalgeneofEiseniasp.withthedominantMallele,th ensuchallelemaybeinheritedbyEa-derivedhybridfromM-

positiveEaparent,andthentransferredduringmatingwithM-negativeEfearthwormintosome E.fetidaandsomeEf-derivedhybrids.Evenone‘atypical’M-

positiveEfmightpropagatethisallelebycrossingwith‘typical’M-

negativeEf.Viceversa,inculturesofM-positiveEfearthwormsmightreappear‘typical’M- negativespecimens.However,hypothesisofthemicrobialoriginofF-

fluorescencederivedfromE.andreispecificbacterialsymbiontscannotbeneglected.Moreover, boththeintrinsicandexternalfactorsmightcooperatetoproducetheM-

fluorophore.TheexistenceofEaandEfhybridizationmakethesecommonspecieseasilymaintaine dinlaboratorytheattractivemodelsforstudiesoninterspeciesgeneflow,inter-

specifictransmissionofbacterialsymbionts,andhypotheticaleffectsofexternalfactorsonthesep henomena.

Supportinginformation

S1Fig.Hypotheticalgenotypesoftheoffspringofself-fertilizinghybrid(aAFMm)earth- worm.a)SchemeofaAFMmparentalcell,gametes(ova,spermatozoa)andzygotes;b)Punnettsquar e.Shadowedpartsofpart‘a’andcrossedoutpartsofpart‘b’indicatemitochon-drial-

nuclearconflicts.Framedgenotypeswereabsentamonginvestigatedearthworms.Assumptionistha tM-

fluorescencemightbeencoded/controlledbythenucleargenewiththedominant‘M’alleleandther ecessive‘m’allelesegregatingindependentlyfromthenuclearA/Fsequencesof28srRNAgene.T he‘MM’and‘Mm/mM’determinestheM-positive(Mp)phe-

notype(inorange)while‘mm’genotypedeterminestheM-

negative(Mn)phenotype(inblue).Punnettsquareisadaptedtopairsofhermaphroditicearthwor msabletoself-

fertilization;ovainyellow,spermatozoaingreen.Ineachpairthefirstalleleisthatofmaternalorigin.

Framedgenotypeswereapparentlyabsentamonginvestigatedearthworms.Ovaandresultedoffspri ngwithmito-nuclearincompatibilityarecrossedout.

(TIF)

Acknowledgments

ManythankstoDr.SebastianHofmanforphylogrampreparation.

AuthorContributions

Concept ualization:BarbaraPlytycz.Formala nalysis:BarbaraPlytycz.

Investigation:BarbaraPlytycz,JanuszBigaj,TomaszPanz.

Methodology:BarbaraPlytycz.

Projectadministration:BarbaraPlytycz.

Supervision:BarbaraPlytycz.Validation:

TomaszPanz,PawełGrzmil.

Writing–originaldraft:BarbaraPlytycz.

(19)

Writing–review&editing:BarbaraPlytycz.

(20)

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