Molecular phylogeny and systematics of native North American lumbricid earthworms

36 Pages·2017·1.46 MB·English

RESEARCHARTICLE
Molecular phylogeny and systematics of
native North American lumbricid earthworms
(Clitellata: Megadrili)
CsabaCsuzdi1☯,Chih-HanChang2☯*,Toma´sPavl´ıcek3,T´ımeaSzederjesi4,DavidEsopi5,
KatalinSzla´vecz2
1 DepartmentofZoology,Eszterha´zyKa´rolyUniversity,Eger,Hungary,2 DepartmentofEarthand
PlanetarySciences,JohnsHopkinsUniversity,Baltimore,MD,UnitedStatesofAmerica,3 Instituteof
Evolution,UniversityofHaifa,Haifa,Israel,4 DepartmentofZoologyandAnimalEcology,PlantProtection
a1111111111
Institute,CentreforAgriculturalResearch,HungarianAcademyofSciences,Budapest,Hungary,5 Sidney
a1111111111 KimmelComprehensiveCancerCenter,JohnsHopkinsUniversity,Baltimore,MD,UnitedStatesofAmerica
a1111111111
a1111111111 ☯Theseauthorscontributedequallytothiswork.
a1111111111 *[email protected],[email protected]
Abstract
OPENACCESS ThefamilyLumbricidaeisarguablythemostwell-knownandwell-studiedearthwormgroup
Citation:CsuzdiC,ChangC-H,Pavl´ıcekT, duetoitsdominanceintheEuropeanearthwormfaunaanditsinvasionintemperateregions
SzederjesiT,EsopiD,Szla´veczK(2017)Molecular worldwide.However,itsNorthAmericanmembers,especiallythegenusBimastosMoore,
phylogenyandsystematicsofnativeNorth
1893,arepoorlyunderstood.WerevisedthesystematicsofthegenusBimastosandtested
Americanlumbricidearthworms(Clitellata:
thehypothesisofthemonophylyofNorthAmericanlumbricidsusingmorphologicalcharac-
Megadrili).PLoSONE12(8):e0181504.https://doi.
org/10.1371/journal.pone.0181504 tersandeightmolecularmarkers.Phylogeneticanalysesbasedonourextensivesampling
ofBimastosandinclusionofDendrodrilusandAllolobophoridellaindicatedawell-supported
Editor:MichaelSchubert,LaboratoiredeBiologie
duDe´veloppementdeVillefranche-sur-Mer, cladecontainingBimastosandEisenoidesGates,1969,andprovidedthefirstevidence
FRANCE supportingthatNorthAmericanlumbricidsaremonophyletic.Assumingtheavailablediver-
Received:August25,2016 gencetimeestimationsanddatingoflandbridgesarecorrect,itwouldsuggestthatthe
ancestorofthiscladearrivedNorthAmericathroughBeringiaortheDeGeerrouteduring
Accepted:June28,2017
LateCretaceous,andsincethenthecladehasdivergedfromitsEurasiansistergroup,
Published:August9,2017
Eisenia.TheperegrinegeneraDendrodrilusandAllolobophoridellaarenestedwithinthe
Copyright:©2017Csuzdietal.Thisisanopen
Bimastosclade;weproposetotreatthemasjuniorsynonymsofthegenusBimastos,and,
accessarticledistributedunderthetermsofthe
contradictorytothecommonlyheldbeliefofbeingEuropean,theyareindeedpartofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and indigenousNorthAmericanearthwormfauna.Morphologicalcharacters,suchasred-violet
reproductioninanymedium,providedtheoriginal pigmentation,proclinateU-shapednephridialbladdersandcalciferousdiverticulainsegment
authorandsourcearecredited.
10furthersupportthisplacement.TheEastMediterranean–LevantineSpermophorodrilus
DataAvailabilityStatement:DNAsequences Bouche´,1975andHealyellaOmodeo&Rota,1989arenestedwithintheDendrobaena
reportedinthisstudyareavailablefromGenBank
sensulatoclade;thereforetheircloserelationshipwiththeNorthAmericanBimastosis
underaccessionnumbersKX651115-KX651415.
refuted.SpeciesfitthereviseddiagnosisofBimastosarereviewedandkeyed,andanew
Allotherrelevantdataarewithinthepaperandits
SupportingInformationfiles. species,Bimastosschwertisp.nov.,isdescribed.
Funding:Fundingtothisstudywaspartially
providedbytheNationalScienceFoundation(DEB
9714835,EAGERNEON1550795,ACI1244820)to
KSandbytheHungarianScientificResearchFund
(OTKAK100369)toCsC.Thefundershadnorole
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RevisionoftheearthwormgenusBimastos
instudydesign,datacollectionandanalysis, Introduction
decisiontopublish,orpreparationofthe
EarthwormsofthefamilyLumbricidaearenativetotheHolarctic.Theyrepresentakeystone
manuscript.
groupofmacrofaunaintemperatesoils,withabout30commonspeciesspreadgloballyby
Competinginterests:Theauthorshavedeclared
humanactivity[1].Thefamilycurrentlyhasaround750describedspecieswithinapproxi-
thatnocompetinginterestsexist.
mately40–60genera[2–4].WhilethemajorityofthespeciesandgeneraarenativetothePale-
arcticregion,twogenera,BimastosMoore,1895andEisenoidesGates,1969,aregenerally
believedtobenativetoNorthAmerica[5],withnineandtwodescribedspecies,respectively.
IntheUSA,speciesbelongingtothetwogenerafrequentlyco-occurwithintroducedEuro-
peanlumbricids,suchasLumbricusrubellus,Aporrectodeacaliginosa,andOctolasioncyaneum
[6–8],butusuallyatlowerabundance.
Thevalidity,taxonomicboundaryandoriginofBimastos,aptlycalleda“systematicwaste-
basket”byGates[9],havebeenwidelydebated[5,10–16].Takingintoaccountpreviously
overlookedmorphologicalcharacters,includingtheshapeandorientationofnephridialblad-
dersandthestructureandpositionofcalciferousglands,Gates[5]arguedthatthename
BimastosshouldberestrictedonlyforNorthAmericanspecies.ThisconceptofBimastoswas
latersupportedbyOmodeoandRota[14],whoseparatedtheBalkanicandAnatolianSpermo-
phorodrilusBouche´,1975andHealyellaOmodeo&Rota1989fromNorthAmericanBimastos.
However,OmodeoandRota’sconceptsofthethreegenerasufferedseverelyfromambiguous
morphologicaldescriptionsandoverlappingdiagnosis.Forthisreason,Zicsi[13,15]con-
cludedthatthethreegeneraformahomogenousgroup,andbothSpermophorodrilusandHea-
lyellaarejuniorsynonymsofBimastos.IncontrasttoGates’restricteddefinitionofBimastos
totheNearctic,Zicsi’sconceptofBimastosencompassesspeciesnotonlyfromNorthAmerica
butalsofromtheBalkansandtheAnatolia,thuscreatingabiogeographicpuzzlewithques-
tionsonhowthisgenusachieveditscurrentnativerangeofdistribution.
ThebiogeographicpuzzleconcerningthetwocompetinghypothesesofBimastosisfurther
complicatedbythecloseaffinityamongBimastosandtwomonotypicgenera,Allolobophoridella
andDendrodrilus[17,18].Thisaffinityhasbeensuggestedinpreliminarymolecularanalyses
[19,20],andwasrecentlyconfirmedinthemultigenemolecularphylogenyofLumbricidae[21].
ThegenusAllolobophoridellawascreatedtohosttwoenigmaticspecies,LumbricuseiseniLevin-
sen,1884andAllolobophoraparvaEisen,1874[17].ThelatterwassoontransferredtoBimastos
[3],makingAllolobophoridellamonotypic.Inthepastfewdecades,Allolobophoridellaeisenihas
beenmovedaroundamongAllolobophora[22],Eisenia[15],andBimastos[18]andhasalso
beensuggestedtoshowaffinitieswithseveralDendrobaenaspecies[23].Clearly,aphylogenetic
re-evaluationofthespeciesandthestatusofthegenusAllolobophoridellawereurgentlyneeded.
Whilemorphologicalsimilarities,suchasproclinateU-shapednephridialbladders,and
NearcticdistributionsimplythatBimastosandEisenoidesmaybecloselyrelated,ahypothe-
sizedNorthAmericancladecomposedofonlyBimastosandEisenoideswereputthroughafor-
maltestonlyrecently.MolecularphylogenyofLumbricidaeconstructedbyDominguezetal.
[21]showedthattheNorthAmericanBimastosismonophyletic,andisnestedwithinaclade
consistingBimastos,Dendrodrilus,andAllolobophoridella,withtheformertwogenerabeing
thesistergroupsofeachother.WhilethehypothesisthatEisenoidesiscloselyrelatedtothe
aforementionedcladegainedsomesupportinDominguezetal.[21],theinferredphylogeny
didnotsupportastrictBimastos-Eisenoidesmonophyly.Moreover,theresultwasinsufficient
formakingdefinitiveinferencesregardingthestatusofBimastos,Healyella,andSpermophoro-
drilusasonlythreeoutoftheninenominalspeciesofBimastoswereincluded,andthegenus
SpermophorodrilusandthetypespeciesofHealyellaweremissingfromtheanalyses.Neverthe-
less,themolecularphylogeneticstudyofLumbricidaebyDominguezetal.[21]provideda
solidbasisforfurtheranalysis.
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RevisionoftheearthwormgenusBimastos
HerewereportonadetailedmorphologicalandmolecularanalysisofBimastosandrelated
genera.Ourobjectiveswereto(1)evaluatethemonophylyofNorthAmericanlumbricids,(2)
testthetwocompetinghypothesesofBimastosoutlinedbyGates(1969)andbyZicsi(1981),
(3)investigatethephylogeneticpositionofthegeneraAllolobophoridella,Dendrodrilus,Sper-
mophorodrilus,andHealyella,and(4)conductafullrevisionofthegenusBimastos.Weinte-
gratedmorphologywiththemultigene-phylogenyapproach[24–28],usingbothnuclearand
mitochondrialgenestoacquireaconcatenatedsequencelengthof5715bp.Weexpandedsam-
plingofBimastosfromthreetoeightspeciesandincludedthetypespeciesofallofthecon-
cernedgenera,includingAllolobophoridellaeiseni,Bimastospalustris,Dendrodrilusrubidus
rubidus,Eisenoideslonnbergi,Healyellasyriaca,andSpermophorodrilusantiquus.
Materialsandmethods
Specimens
Between2000and2015wecollectedinTurkey,theLevant,MiddleEast,andinthemid-Atlantic
regionofNorthAmerica.WealsoexaminedtheBimastoscollectionintheNationalMuseumof
NaturalHistoryinWashingtonD.C.,whereseveraltypespecimensarekept,aswellasthemateri-
alsintheHungarianNaturalHistoryMuseumwhereseveralHealyellaandSpermophorodrilus
speciesarehoused.Earthwormswerecollectedbyboththedilutedformalinmethod[29]andby
diggingandhandsearching.Specimenswerekilledin75%ethanolandfixedin4%formalinthen
transferredto75%ethanolafterseveraldays.Specimensusedformolecularanalysiswerepre-
servedin96%ethanolwithoutformalinfixation.Allofthespecimenscollectedand/orexamined
arepermanentlyarchivedateithertheNationalMuseumofNaturalHistory,SmithsonianInsti-
tution(USNM),WashingtonD.C.,USAortheSoilZoologyCollectionoftheHungarianNatural
HistoryMuseum(HNHM),Budapest,Hungary.Thedetailedcatalognumbersofspecificspeci-
menssequencedcanbefoundinTable1.Formorphologicalanalysisthefollowingspecimens
wereexamined:Allolobophoridellaeiseni17specimens(HNHM/12484,HNHM/14565,HNHM/
14743);Dendrodrilusrubidus15specimens(HNHM/14185,HNHM/14228,HNHM/14445,
HNHM/15283,HNHM/16384,HNHM/6519);Spermophorodrilusantiquus10specimens(HN
HM/8857,HNHM/9247,HNHM/15756,HNHM/15819,HNHM/15840);Healyellasyriaca16
specimens(HNHM/11131,HNHM/12169,HNHM/14045,HNHM/15141,HNHM/16507);
Healyellajordanis14specimens(HNHM/12914,HNHM/12915,HNHM/12918,HNHM/14620);
Bimastosgieseleri11specimens(USNM25848);Bimastosheimburgeri44specimens(USNM
123883,USNM123879,HNHM/14186,HNHM/14906,HNHM/16498,HNHM/16502);Bimas-
toslongicinctus64specimens(USNM24599,USNM25871,USNM47867,HNHM/17151);
Bimastospalustris34specimens(HNHM/13039,HNHM/14183,HNHM/14189,HNHM/14215,
HNHM/14224,HNHM/14227);Bimastosparvus10specimens(HNHM/14301,HNHM/14886,
HNHM/15170,HNHM/16067,HNHM16464);BimastosschwertiHolotype(HNHM/16614)
Paratypes39specimens(HNHM/16615,HNHM/14184,HNHM/4188,HNHM/16500,HNHM/
16510,HNHM/16566,HNHM/16567,HNHM/17158);Bimastostumidus55specimens(USNM
1164,USNM123878,USNM123889,USNM25848,USNM19683,HNHM14193,HNHM/
14198,.HNHM/16503,HNHM/16497);Bimastoswelchionespecimen(USNM16782);Bimastos
zetekieightspecimens(USNM16782,USNM26214,USNM123897,USNM123898).
Ethicsstatement
PermissiontocollectearthwormsamplesatJugBaywasissuedbytheJugBayWetlandsSanc-
tuaryunderDirectorChrisSwarth.Noneoftheotherlocationsfromwhichsampleswerecol-
lectedrequiredspecificpermissions.Noneoftheearthwormscollectedinthisstudyarelisted
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RevisionoftheearthwormgenusBimastos
Table1. SpecimensnewlycollectedforphylogeneticanalysesandtheirHungarianNaturalHistoryMuseumcatalognumbers(HNHM).
Species HNHM Locality
Allolobophoridellaeiseni 15811 KoulaMts.,Greece
Allolobophoridellaeiseni 16448 Cold’Aspin,France
Aporrectodeacaliginosa 17163 MayoBeachPark,MD,USA
Aporrectodeatuberculata 17164 MayoBeachPark,MD,USA
Bimastosheimburgeri 16498 GunpowderFalls,USA
Bimastosheimburgeri 16502 SmithsonianEnvironmentalResearchCenter,MD,USA
Bimastoslongicinctus 17157 Gameland242,SiddonsburgPA,USA
Bimastospalustris 16565 JugBayWetlandsSanctuary,MD,USA
Bimastosparvus 16357 WadiKelt,Israel
Bimastosschwerti 16500 JugBayWetlandsSanctuary,MD,USA
Bimastosschwerti 16566 JugBayWetlandsSanctuary,MD,USA
Bimastosschwerti 17158 Gameland242,Siddonsburg,PA,USA
Bimastostumidus 16497 GunpowderFalls,USA
Bimastostumidus 16503 SmithsonianEnvironmentalResearchCenter,MD,USA
Dendrobaenaalpina 16077 RadjuvaPlanina,Bulgaria
Dendrobaenaattemsi 16299 SocolauValley,Maramures,Romania
Dendrobaenaattemsi 16468 PalmeiradeFaro,Portugal
Dendrobaenabyblica 16660 Kakopetros,Crete,Greece
Dendrobaenabyblicaolympiaca 15835 Peristeri,Greece
Dendrobaenaoctaedra 16212 TreskovacMts.,Montenegro
Dendrobaenaoctaedra 16528 PayolleValley,France
Dendrodrilusrubidusrubidus 15657 Cerova´Highlands,Slovakia
Dendrodrilusrubidusrubidus 15816 IstranchaMts.,Turkey
Dendrodrilusrubidussubrubicundus 15283 Borşa,Romania
Eiseniafetida 17161 Baltimore,MD,USA
Eisenoidescarolinensis 17160 HawkMountain,PA,USA
Eisenoideslonnbergi 17159 PlummersIsland,MD,USA
Fitzingeriaplatyuraplatyura 16439 Velem,Hungary
Healyellajordanis 16369 Rehaniya,Israel
Healyellasyriaca 16273 NahalTabor,Israel
Healyellasyriaca 16507 Samandog,Turkey
Lumbricusrubellus 17165 SmithsonianEnvironmentalResearchCenter,MD,USA
Octolasionlacteum 17162 SmithsonianEnvironmentalResearchCenter,MD,USA
Spermophorodrilusantiquus 15756 SapkaMts.,Greece
https://doi.org/10.1371/journal.pone.0181504.t001
asendangeredorprotected.Allofthespecimensincludedinthisstudyarearchivedinthe
institutionsstatedaboveandarepubliclyaccessible.
Histologicalmethods
Forhistologicalstudyofthelongitudinalmusculature,severalpostclitellarsegmentswere
embeddedinparaffin,slicedto10μmthincross-sectionsusingaMicromrotary-microtome,
andstainedwithhematoxylinandeosin[30].Forcomparisonofthestructureofcalciferous
glands,severallongitudinalsectionsofthepreclitellarregionswerealsoslicedandtreatedas
above.ThemicroscopicslideswereexaminedandphotographedusingaNikonEclipse660
DICmicroscope.
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RevisionoftheearthwormgenusBimastos
Taxonsamplingforphylogeneticanalysis
TounravelthephylogeneticrelationshipsamongBimastos,Healyella,Spermophorodrilus,Dendro-
drilusandAllolobophoridella,andtotestthehypothesisthatNorthAmericanlumbricidsaremono-
phyletic,wesampledatotalof14taxafromtheabovegenera,includingtwoBimastosspecies
reportedinDom´ınguezetal.[21],andbothoftheknownspeciesbelongingtotheNorthAmeri-
cannativegenusEisenoides(Table1).Ouroveralltaxonsamplingcompriseseightofthe10valid
speciesofBimastos,includinganewspeciesdescribedinthepresentstudy,andfullyrepresentsthe
monotypicgeneraAllolobophoridellaandDendrodrilus.Wealsoincludedninespecies(threefrom
GenBank;S1Table)fromDendrobaenaandFitzingeriaasthetwogeneraarecloselyrelatedand
speciesinHealyellaandSpermophorodrilushavebeenclassifiedintoDendrobaena[11].Whileour
samplingofthelatterfourgenerawasfarfromexhaustive,Healyella,SpermophorodrilusandFitzin-
geriahaveonly10,three,andthreevalidspecies,respectively[4].Furthermore,oursamples
encompassedthetypespeciesofallofthetargetedgenera(Allolobophoridellaeiseni,Bimastospalus-
tris,Dendrobaenaoctaedra,Dendrodrilusrubidusrubidus,Eisenoideslonnbergi,Fitzingeriaplatyura
platyura,Healyellasyriaca,andSpermophorodrilusantiquus,respectively),providingastrongtaxo-
nomicbasisfordrawingunequivocalconclusions.SamplesofthelumbricidspeciesEiseniafetida,
Lumbricusrubellus,Octolasionlacteum,AporrectodeacaliginosaandAporrectodeatuberculatawere
alsoincluded.Overall,sequencesfrom34specimensrepresenting25species/subspecieswere
newlyacquired,andwerecombinedwithselectedtaxafromtheLumbricidaedatasetreportedin
Dom´ınguezetal.[21]andPe´rez-Losadaetal.[31]forphylogeneticanalyses.
DNAextraction,polymerasechainreactions,andsequencing
GenomicDNAwasextractedfromearthwormtissuesusingtheQiagenDNeasyBloodandTissue
Kit(QIAGEN,Valencia,CA,USA).Regionsofeightmolecularmarkers,includingthreenuclear
rRNAs(18S,5.8Sand28S),twomitochondrialrRNAs(12Sand16S),andthreemitochondrialpro-
teincodinggenes(cytochromecoxidasesubunit1and2(COI,COII)andNADHdehydrogenase
subunit1(ND1)),wereacquiredusingpolymerasechainreaction(PCR)withprimerslistedinS2
Table.PCRwasconductedina50ultotalvolumewith1xreactionbuffer,1.25unitsJumpStartTaq
Polymerase(Sigma,StLouis,MO,USA),200uMofeachdNTP,1.5mMMgCl ,0.24mg/mLBSA,
2
5%DMSO,200nMofeachprimer,and10or20ngtemplateDNA.Cyclingconditionsweresetto
onecycleof94˚Cfor2min,followedby35cyclesof94˚Cfor15s,45˚C(forCOI),47˚C(forCOII
andND1),49˚C(for16SrRNA)or50˚C(for12S,18S,5.8Sand28SrRNAs)for15s,and72˚Cfor
90s,withafinalcycleof72˚Cfor5min.TheamplifiedproductsweresequencedatBeckmanCoul-
terGenomicsusingBigDyeTerminatorv3.1CycleSequencingKit(AppliedBiosystems,Foster,
CA,USA)andanalyzedonanABIPRISM3730XL(AppliedBiosystems).Chromatogramswere
visualizedandassembledinDNABaserv4.31.0(HeracleBioSoft,Romania).Allnewsequences
havebeendepositedinGenBankundertheaccessionnumbersKX651115-KX651415.
Phylogeneticanalysis
Twodatasetswereanalyzed.First,sequencesfromselectedspeciesrepresentingthemajor
cladesofLumbricidaeandtheoutgroupHormogasterreportedinDom´ınguezetal.[21]and
Pe´rez-Losadaetal.[31]werecombinedwithourdata(S1Table).Thecombineddataset,com-
posedof12S,16S,18SrRNAs,COI,COII,andND1,contains64samplesrepresenting57spe-
cies/subspeciesandis3940bpafteralignment(the‘shortdataset’hereafter).Second,data
acquiredinthisstudywereanalyzed.Octolasionlacteumwasusedastheoutgroupbasedon
theinferredphylogenyinDom´ınguezetal.[21].Thisdataset,5715bpafteralignment,islon-
ger(duetobothlongersequencesandsomelongeralignments)andallowsustofocuson
unravellingthephylogenywithinBimastos(the‘longdataset’hereafter).
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RevisionoftheearthwormgenusBimastos
NucleotidesequencesfromeachgenewerealignedusingMAFFTv7[32]underthedefaultset-
tings.ThealignedsequenceswereconcatenatedusingDAMBE5[33].Fortheshortdataset,the
alignedsequencesare3940bpinlength,including18SrRNA(768bp),12SrRNA(392bp),16S
rRNA(516bp),COI(651bp),COII(681bp),andND1(932bp).Forthelongdataset,thealigned
sequencesare5715bpinlength,including18SrRNA(1578bp),5.8SrRNA(122bp),28SrRNA
(867bp),12SrRNA(388bp),16SrRNA(496bp),COI(651bp),COII(681bp),andND1(932
bp).ThemostappropriatemodelsofevolutionwereselectedusingjModelTest2[34]underthe
Akaikeinformationcriterion(AIC)foreachgenepartitionineachdataset.Foreachprotein-coding
gene,thethirdcodonwasfurthertreatedasaseparatepartition.Differentpartitionsweretreatedas
unlinkedandmodelparameterswereestimatedindependentlyforeachpartitioninallanalyses.
Phylogenieswereinferredusingmaximumlikelihood(ML)analysesandBayesianinfer-
ences.MLanalyseswereconductedusingRAxMLv8[35]asimplementedintheCIPRESSci-
enceGateway3.3webportal[36](www.phylo.org)usingthegeneraltimereversiblemodelwith
proportionofinvariablesitesandgammadistribution(GTR+I+G)estimatedforeachindi-
vidualgenepartition.Cladesupportwasevaluatedusingthenon-parametricbootstrappingpro-
cedurewith1000bootstrappingreplicates.ThebestMLtreewascomparedtoalternativetree
topologiesusingtheShimodaira–Hasegawa(SH)testasimplementedinRAxMLv8.Bayesian
inferencescoupledwithMarkochainMonteCarlo(MCMC)wereconductedusingMrBayes
v3.2.6[37]withdefaultpriorsandrandomstartingtrees.ThreeindependentMCMCsearches,
eachwiththreeheatedandonecoldchains,wererunfor2x107generations.Theresulting
treesweresampledevery1000generationsafterdiscardingthefirst20%treesasburn-in.The
posteriorprobabilitiesandthetopologiesoftheresultingconsensustreesfromseparateanalyses
werecomparedforcongruencyandcombinedina50%majority-ruleconsensustree.
Weoriginallyconsideredestimatingdivergencetimeandconductingancestralarearecon-
structionbutdecidednottodosoforthreereasons.First,therearenoearthwormfossilsavail-
ableforcalibration.Molecularclockestimationinearthwormshasbeenconductedexclusively
usinggeologicalevents[21,24,26].However,doingsowouldimplyvicariance,anassumption
thathasbeenrepeatedlyquestioned[28].Second,externalcalibrationpointsarenotavailable
inourphylogenetictrees.Third,withthelackofnativelumbricidsamplesfromEastAsiaand
aprioriknowledgeabouttheoriginofAllolobophoridellaaeiseniandDendrodrilusrubidus,
ancestralareareconstructioncannotbeproperlyconducted.
Nomenclaturalacts
TheelectroniceditionofthisarticleconformstotherequirementsoftheamendedInterna-
tionalCodeofZoologicalNomenclature,andhencethenewnamescontainedhereinareavail-
ableunderthatCodefromtheelectroniceditionofthisarticle.Thispublishedworkandthe
nomenclaturalactsitcontainshavebeenregisteredinZooBank,theonlineregistrationsystem
fortheICZN.TheZooBankLSIDs(LifeScienceIdentifiers)canberesolvedandtheassociated
informationviewedthroughanystandardwebbrowserbyappendingtheLSIDtotheprefix
"http://zoobank.org/".TheLSIDforthispublicationis:urn:lsid:zoobank.org:pub:236DD001-
3F6B-4D1C-AAB2-D3E08FFEC0B3.Theelectroniceditionofthisworkwaspublishedina
journalwithanISSN,andhasbeenarchivedandisavailablefromthefollowingdigitalreposi-
tories:PubMedCentral,LOCKSS.
Results
Phylogeny
Inbothoftheshort(3940bp)andthelong(5715bp)datasets,themaximumlikelihood(ML)
analysesandBayesianinferencesgeneratesimilartopologiesandhavenoincongruence
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RevisionoftheearthwormgenusBimastos
regardingsupportedclades(bootstrapsupportvalues(cid:21)50orposteriorprobabilities(cid:21)0.90).
Therefore,theMLandBayesiantreesareconsideredtogetherandonlytheMLtreesare
shown.
Thetreesinferredfromtheshortdataset(Fig1)providedlittlesupportforsomeofthe
basalinternalbranches,whichiswhatweexpectedasthegenesincludedintheshortdataset
areonlyapartofthoseusedinDom´ınguezetal.[21].Asourgoalistounderstandthephylog-
eniesofBimastos,Allolobophoridella,Dendrodrilus,SpermophorodrilusandHealyellawithan
extendedsampling,thelackofphylogeneticresolutionamongotherlumbricidgeneradoes
notaffectourabilitytodrawmeaningfulconclusions,andcanreasonablybecompensated
withourcurrentunderstandingonLumbricidaephylogeny[21,31].
Thephylogeniesinferredfrombothdatasetsstronglysupportamonophyleticgroup(the
Bimastoscladehereafter)composedofBimastos,Allolobophoridella,andDendrodrilus(boot-
strap/posteriorprobabilityvalues=100/1.0forbothdatasets).However,thegenusBimastos
sensuGates[5],isparaphyleticduetotheexclusionofAllolobophoridellaorDendrodrilus.
WithintheBimastosclade,Bimastospalustrisisbasalrelativetoalltheotherspecies.Bimastos
parvusandDendrodrilusrubidusaresisterspecies;thetwotogetherarethesistergroupofAllo-
lobophoridellaeiseni(Fig2).WecomparedthebestMLtree(Fig2)withtwoalternativetopolo-
giesusingtheSHtestwiththefollowingconstraints:(1)monophylyoftheBimastosgenus;
and(2)monophylyoftheBimastosgenusexceptB.parvus.Bothcomparisonssuggestedthat
thebestMLtreeandthetwoalternativetopologiesarenotsignificantlydifferentfromeach
other(P>0.05).
ThesistergroupoftheBimastoscladeisEisenoides(bootstrap/posteriorprobabilityval-
ues=95/1.0and97/1.0fortheshortandlongdatasets,respectively).Together,thetwogenera
formacladethatincludesalllumbricidspeciesofNorthAmericanorigin(theNorthAmerican
cladehereafter)(Figs1and2).Thephylogenetictreesinferredfromthelongdatasetalsosug-
gestedthatEiseniaisthesistergroupoftheNorthAmericanclade(bootstrap/posteriorproba-
bilityvalues=64/1.0).
ThetreetopologiesdonotsupportthehypothesisthatSpermophorodrilusandHealyellaare
associatedwithBimastos.Instead,thetwogeneraformaweaklysupportedclade(bootstrap/
posteriorprobabilityvalues=59/0.85and56/0.72fortheshortandlongdatasets,respectively)
nestedwithinaclade(bootstrap/posteriorprobabilityvalues=86/0.99and94/1.0fortheshort
andlongdatasets,respectively)thatalsoincludesFitzingeriaplatyuraplatyuraandnineDen-
drobaenataxa.TheseninetaxaencompassallDendrobaenaspeciesincludedinouranalyses
exceptDendrobaenabyblicabyblica.Accordingly,Dendrobaena,ascurrentlydefined,is
polyphyletic.
Taxonomictreatment
OneofourmainresearchgoalswastorevisethesystematicsofthegenusBimastos.The
inferredphylogeniesandthenon-significantSHtestresultsuggesttwopossiblerelationships
amongBimastos,DendrodrilusandAllolobophoridella:(1)Bimastosisparaphyleticduetothe
existenceofDendrodrilusrubidusandAllolobophoridellaeiseni,or(2)Bimastosismonophy-
letic.Regardlessoftherelationship,thethreegroupsformedahighlysupportedclade(Figs1
and2).Giventheweakmorphologicaldistinctionamongthesethreegenera[38],wepreferan
unambiguouslysupported,moreinclusiveBimastos,andhereinproposetotreatDendrodrilus
andAllolobophoridellaasjuniorsynonymsofBimastos.Thistaxonomictreatmentaccommo-
datedbothscenariosofphylogeneticrelationships.Furthermore,Mrˇsič[38],inhisoriginal
descriptionofAllolobophoridella,noted:"Shoulditbestated,thatinthespeciesofthegenus
BimastosfromNorthAmericatheglandularpartofthenephridialbladderisorientedinthe
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RevisionoftheearthwormgenusBimastos
Fig1.MaximumlikelihoodtreebasedontheshortdatasetforBimastos,Dendrodrilus,Allolobophoridella,
Healyella,Spermophorodrilus,Dendrobaena,andotherlumbricids.Bootstrapsupportvalues(if(cid:21)50)andBayesian
posteriorprobabilities(if(cid:21)0.90)areshownaboveandbelowthebranches,respectively.Thefourgenerathatwere
previouslyhypothesizedtoberelatedtoBimastosarecolored.Theannotated-and-shadedareasontherightcorrespond
tothetwomajorcladescharacterizedinResults.Specimensnewlyreportedinthisstudyweremarkedwiththeirfive-digit
HNHMcatalognumbers.
https://doi.org/10.1371/journal.pone.0181504.g001
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RevisionoftheearthwormgenusBimastos
Fig2.MaximumlikelihoodtreebasedonthelongdatasetforBimastos,Dendrodrilus,Allolobophoridella,Healyella,Spermophorodrilus,
Dendrobaena,andotherlumbricids.Bootstrapsupportvalues(if(cid:21)50)andBayesianposteriorprobabilities(if(cid:21)0.90)areshownaboveandbelowthe
branches,respectively.ThefourgenerathatwerepreviouslyhypothesizedtoberelatedtoBimastosarecolored.Theannotated-and-shadedareasonthe
rightcorrespondtothetwomajorcladescharacterizedinResults.Specimensweremarkedwiththeirfive-digitHNHMcatalognumbers.
https://doi.org/10.1371/journal.pone.0181504.g002
samewayasineiseniandparvus,thegenusAllolobophoridellawillbejustasynonymofthe
genusBimastos."
GenusSpermophorodrilusBouche´,1975. EophilaRosa,1893[39]:Černosvitov1938[40]:
198(partim).
BimastosMoore,1893[41]:Zicsi1981[13]:432(partim);Zicsi&Michalis1981[42]:244
(partim);Blakemore2008b[43]:536(partim).
SpermophorodrilusBouche´,1975[44]:2;Omodeo&Rota,1989[14]:169;1991[45]:172;
Csuzdietal.2006[46]:26;Pavl´ıčeketal.2010[47]:2000.
Diagnosis.Setaestrictlypaired,pigmentationlacking(Fig3).Prostomiumepilobous,first
dorsalporearound5/6.Maleporeon15large,justabovesetallineb,facingventrad.Female
poressmallon14justabovesetaeb.Clitellumannular,evenlydeveloped,spermathecaeand
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RevisionoftheearthwormgenusBimastos
Fig3.SetalratiosinthegenusBimastos.Lettersab,bc,cdreferstosetalintervals.
https://doi.org/10.1371/journal.pone.0181504.g003
tubercleslacking.Nephridialporesirregularlyalternatebetweenbandaboved.Twopairsof
testesin10,11,andtwopairsofseminalvesiclesin11,12.Calciferousglandsinsegments10–
12,withsmalldiverticulainsegment10.Excretorysystemholoic,nephridialbladderssausage-
shapedthroughout.Typhlosolebifid,thecross-sectionoflongitudinalmusclelayerisofpin-
natetype.
Typespecies:EophilaantiquaČernosvitov,1938(=SpermophorodrilusalbanianusBouche´,
1975)
Distribution.FromtheBalkanPeninsulatoNorthAnatolia.
Remarks.Omodeo&Rota[14]notedthatthespeciesofSpermophorodrilusdifferedfrom
thetypespeciesofthegenusBimastos,B.palustris,“inmanyrelevantpoints”(p.169).However,
theydidnotdiscussitindetails,andtheonlydifferencementionedistheonesegmentlonger
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