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the outbreak of Ebola virus disease in West Africa, 2014–2016
Alexandre Hassanin, Nicolas Nesi, Julie Marin, Blaise Kadjo, Xavier Pourrut, Éric Leroy, Guy-Crispin Gembu, Prescott Musaba Akawa, Carine Ngoagouni,
Emmanuel Nakouné, et al.
To cite this version:
Alexandre Hassanin, Nicolas Nesi, Julie Marin, Blaise Kadjo, Xavier Pourrut, et al.. Comparative phylogeography of African fruit bats (Chiroptera, Pteropodidae) provide new insights into the out- break of Ebola virus disease in West Africa, 2014–2016. Comptes Rendus Biologies, Elsevier, 2016,
�10.1016/j.crvi.2016.09.005�. �hal-01382796�
Evolution/E´volution
Comparative phylogeography of African fruit bats (Chiroptera, Pteropodidae) provide new insights into the outbreak of Ebola virus disease in West Africa, 2014–2016
Alexandre Hassanin
a,b,*, Nicolas Nesi
a,b, Julie Marin
a, Blaise Kadjo
c, Xavier Pourrut
d, E´ric Leroy
d, Guy-Crispin Gembu
e,
Prescott Musaba Akawa
e, Carine Ngoagouni
f, Emmanuel Nakoune´
f, Manuel Ruedi
g, Didier Tshikung
h, Ce´lestin Pongombo Shongo
h, Ce´line Bonillo
baInstitutdesyste´matique,e´volution,biodiversite´,ISYEB–UMR7205CNRS,MNHN,universite´ Paris-6(UPMC),SorbonneUniversite´s, Muse´umnationald’histoirenaturelle,75005Paris,France
bMuse´umnationald’histoirenaturelle,UMS2700,75005Paris,France
cUniversite´ Fe´lix-Houphoue¨t-Boigny, UFRbiosciences,22BP582,Abidjan22,Coˆted’Ivoire
dCentreinternationalderecherchesme´dicalesdeFranceville,BP769,Franceville,Gabon
eFaculte´ dessciences,universite´ deKisangani,BP2012,Kisangani,DemocraticRepublicoftheCongo
fInstitutPasteurdeBangui,BP923,Bangui,CentralAfricanRepublic
gDe´partementdemammalogieetd’ornithologie,muse´umd’histoirenaturelle,Gene`ve,Switzerland
hFaculte´ deme´dicineve´te´rinaire,universite´ deLubumbashi,Lubumbashi,DemocraticRepublicoftheCongo
ARTICLE INFO
Articlehistory:
Received1stJuly2016
Acceptedafterrevision13September2016 Availableonlinexxx
Keywords:
Filovirus Ebolavirus Sub-SaharanAfrica Guinea
Migration Rainforests Megachiroptera
ABSTRACT
BothEbolavirusandMarburgvirusweredetectedinseveralfruitbatspeciesofthefamily Pteropodidae,suggestingthatthistaxonplaysakeyroleinthelifecycleoffiloviruses.
AfterfourdecadesofZaireEbolavirus(ZEBOV)outbreaksinCentralAfrica,theviruswas detectedforthefirsttimeinWestAfricain2014.Tobetterunderstandtheroleoffruitbats aspotentialreservoirsandcirculatinghostsbetweenCentralandWestAfrica,weexamine herethephylogenyandcomparativephylogeographyofPteropodidae.Ourphylogenetic resultsconfirmtheexistenceoffourindependentlineagesofAfricanfruitbats:thegenera EidolonandRousettus,andthetribesEpomophoriniandScotonycterini,andindicatethat thethree species suspected to represent ZEBOV reservoir hosts (Epomops franqueti, Hypsignathus monstrosus,and Myonycteris torquata) belong to an African clade that diversifiedrapidlyaround8–7Mya.Totestforphylogeographicstructureandforrecent gene flow from Central to West Africa, we analysed the nucleotide variation of 675cytochromebgene(Cytb)sequences,representingeightfruitbatspeciescollected in48geographiclocalities.WithinEpomophorina,ourmitochondrialdatadonotsupport themonophylyoftwogenera(EpomopsandEpomophorus)andfourspecies(Epomophorus gambianus, Epomops franqueti, Epomops buettikoferi, and Micropteropus pusillus). In Epomops,however,wefoundtwogeographichaplogroupscorrespondingtotheCongo Basin and Upper Guinea forests, respectively. By contrast, we found no genetic
* Correspondingauthorat:Institutdesyste´matique,e´volution,biodiversite´,ISYEB–UMR7205CNRS,MNHN,universite´ Paris-6(UPMC),Sorbonne universite´s,Paris,France.
E-mailaddress:hassanin@mnhn.fr(A.Hassanin).
ContentslistsavailableatScienceDirect
Comptes Rendus Biologies
w ww . sc i e nce d i re ct . co m
http://dx.doi.org/10.1016/j.crvi.2016.09.005
1631-0691/ß2016Acade´miedessciences.PublishedbyElsevierMassonSAS.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Filoviruses contain Ebolaviruses and Marburgviruses that have caused many hemorrhagicfever outbreaksin sub-SaharanAfricasinceafewdecades,resultinginhigh case-fatalityrates(25–90%)inhumansandotherprimates, suchaschimpanzeesandgorillas.Todate,sixfiloviruses were described in Africa, including four Ebolaviruses (Zaire, Sudan, Taı¨ Forest, and Bundibugyo) and two Marburgviruses (Marburg and Ravn) [1,2] (Fig. 1). Two filoviruseswerealsodetectedoutsideofAfrica:theReston Ebolavirusinhealthyhumansandillanimals(macaques anddomesticpigs)inthePhilippinesandChina[3,4],and theLloviuCuevavirus,which causedmassive die-offsin cavecoloniesofSchreiber’sbat(Miniopterusschreibersii)in France,Spain,andPortugalin2002[5].
Sincethe1970s,researchershavesampledthousandsof arthropodsandvertebratestodetectthepresenceofanti- filovirusantibodiesoradirectevidenceoffiloviruses(RT- PCRorisolation)[2,6,7].In2005,Leroyetal.[8]provided thefirst molecular evidence that fruit bats maybe the reservoirhostsforZaireEbolavirus(ZEBOV):theviruswas detectedbyRT-PCRinseveralwild-caughtandapparently healthyfruitbatsbelongingtothreespeciesofthefamily Pteropodidae: Epomops franqueti (Franquet’s epauletted
fruitbat),Hypsignathusmonstrosus(hammer-headedfruit bat), and Myonycteris torquata (littlecollared fruit bat).
Subsequently,batshavebeenintensivelystudiedtobetter understand their role in themaintenance,transmission, andevolutionoffiloviruses.Twoyearsafter,Marburgvirus wasdetected,usingbothspecificantibodiesandRT-PCR,in Egyptian fruit bats (Rousettus aegyptiacus) collected in northeasternDemocraticRepublicoftheCongo(DRC)and Gabon[9].In 2009,Towneretal.[10]isolatedthevirus fromfiveR.aegyptiacusfoundinKitakaCave(Uganda),and detected highly divergent viral genomes (21%) corres- pondingtobothMarburgvirusandRavnvirusinthesame colony, lending additional support to the idea that R. aegyptiacus represents a major reservoir host for Marburgviruses. The geographic distribution of R. aegyptiacus overlaps withthat of Marburgand Ravn outbreaks.
ThegeographicrangeofthethreespeciesofPteropo- didae identified as potential host reservoirs of ZEBOV coincideswiththatofEbolaoutbreaks(Fig.1),buttodate, no live Ebolavirus has been isolated from any bat.
Therefore,it isdifficult toknowiftheyaretheprimary sourceofinfectionforthisvirusoriftheyareonlyinvolved withsecondarytransmissionofinfectiontootherspecies.
Fruit bats seem, however,to play an important role as differentiationbetweenCentralandWestAfricanpopulationsforallspeciesknownto makeseasonal movements, Eidolon helvum,E.gambianus,H.monstrosus,M.pusillus, Nanonycterisveldkampii,andRousettusaegyptiacus.Ourresultssuggestthatonlythree fruitbatspecieswereabletodispersedirectlyZEBOVfromtheCongoBasintoUpper Guinea:E.helvum,H.monstrosus,andR.aegyptiacus.
ß2016Acade´miedessciences.PublishedbyElsevierMassonSAS.Thisisanopenaccess articleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Fig.1.LocationofEbolahemorrhagicfever(circles)andMarburghemorrhagicfever(greensquares)outbreaks.ThefourspeciesofEbolavirusesare distinguishedbycolours:redforZaire(ZEBOV),blueforSudan,whiteforTaı¨ Forest,andyellowforBundibugyo.Thetropicalandsubtropicalmoistbroadleaf forestsarehighlightedingreen(http://www.worldwildlife.org/science/wildfinder).
reservoir hostofboth Marburgvirusesand Ebolaviruses.
Particularly relevant is the fact that anti-Ebolavirus antibodies have been detected in five other fruit bat speciesinAfrica:Eidolonhelvum(Africanstraw-coloured fruit bat), Epomophorusgambianus (Gambian epauletted fruitbat),Micropteropuspusillus(Peter’sdwarfepauletted fruitBat),Nanonycterisveldkampii (Veldkamp’sbat),and R. aegyptiacus [11,12], as well as in the Asiatic R. leschenaultii (Leschenault’s rousette) in Bangladesh [13](taxahighlightedinFig.2).
BecauseofthemassiveoutbreakinWestAfrica(Guinea, Liberia and Sierra Leone) in 2014–2016,it is crucial to know which fruit bat species might serve as potential reservoirs and circulating hosts of ZEBOV from Central Africa to West Africa or vice versa. Indeed, after four decadesofZEBOVoutbreaksinCentralAfrica,itwasonlyin 2014thatZEBOVwasdetectedinacountryotherthanDRC, GabonandRepublicoftheCongo(Congo).Ofthe39fruit batspeciescurrentlydescribedontheAfricanmainland, only13arepresentinWestAfrica,wheretheycanbefound in rainforestsand forest-savannah mosaics[14,15].Fur- thermore, phylogeographic analyses have shown that three species are in fact endemic to West Africa, i.e.
Megaloglossus azagnyi (western Woermann’s fruit bat), Myonycterisleptodon(westernlittlecollaredfruitbat),and Scotonycteris occidentalis (Hayman’s tear-drop fruit bat) [16,17]. The species Epomops buettikoferi (Buettikofer’s epaulettedfruitbat)isalsoconsideredendemictoWest Africa [14,15], but no molecular data are currently available for this taxon. Ofthe nine species distributed in both WestandCentral Africa, onlyfourspecies have beenanalysedforphylogeographystructure,includingthe veryrareCasinycterisophiodon(Pohle’sfruitbat)[17],as well as E. helvum [18], Myonycteris angolensis (Angolan fruitbat)[16],andR.aegyptiacus[19].
Here, we analysed the molecular phylogeography of African fruit bats of the family Pteropodidae to better understandtheirpotentialroleasreservoirsandcirculat- inghostsbetweenCentralandWestAfrica.Thecomplete mitochondrial Cytb gene was sequenced for different populations of the following eight species to test for phylogeographic structure and recent gene flow from Central to West Africa: E. helvum, E. gambianus, E. buettikoferi, E. franqueti, H. monstrosus, M. pusillus, N.veldkampii,andR.aegyptiacus.Amongthesetaxa,two are supposed to be the reservoir hosts of ZEBOV, i.e.
E.franquetiandH.monstrosus[8],whereasR.aegyptiacusis consideredtobethemajorreservoirhostforMarburgvi- ruses[10].
2. Materialsandmethods 2.1. Taxonomicsampling
Most ofthefruit bat samplesanalysed in this study werecollected bytheauthorsusingmist-nets(Ecotone, Gdynia, Poland) during field trips to Cameroon (AH), CentralAfricanRepublic(AH,CN,ENandNN),Gabon(AH, ELandXP),IvoryCoast(BKandNN),KatangaProvinceof the DRC (AH, CPS, DT and NN), Liberia (BK), Orientale
ProvinceoftheDRC(AH,GCGandPMA),Republicofthe Congo(ELandXP),andSenegal(XP).Fruitbatspecieswere identified morphologically using the key of Bergmans [20]. In addition, several tissuesamples were obtained from specimens housed in the following museums:
‘Muse´um national d’histoire naturelle’ (MNHN; Paris, France), ‘Muse´um d’histoire naturelle’ of the City of Geneva (MHNG;Switzerland),and ‘NaturmuseumSenc- kenberg’ (SMF; Frankfurt, Germany). Names and geo- graphicalcoordinatesofallsampledlocalitiesareprovided inAppendixA(Supportinginformation).
Thenumberofindividualssequencedperspecieswas 50forE.helvum,59forE.buettikoferi,14forEpomopssp.
(buettikoferi or franqueti) from West Africa, 146 for E.franqueti fromCentral Africa, 1 for Epomops dobsonii, 1 for Epomophorus anselli, 1 for Epomophorus crypturus, 12 for E. gambianus, 1 for Epomophorus labiatus, 1 for Epomophorus minimus, 76 for H. monstrosus, 79 for M.pusillus,26 forN.veldkampii,1 forPlerotesanchietae, and47forR.aegyptiacus(AppendicesAandB).EightAsian specieswerealsosequencedforphylogeneticandmolec- ulardatinganalyses(Fig.2).
2.2. Molecularmethods
Total DNA was extracted from muscle or patagium samples using DNeasy Tissue Kit (Qiagen, Hilden, Germany). The complete Cytb gene was amplified and sequencedusingtheprimersdetailedinNesietal.[21]and Hassanin[22].Thepolymerasechainreactions(PCR)were carriedout ina volumeof 20
m
lcontaining3m
lof PCR buffer10XwithMgCl2,2m
lofdNTPs(6.6mM),1m
lofeach of two primers (10m
M) and 0.1m
l of Taq polymerase (2.5U,Qiagen,Hilden,Germany).ThePCRswererunusing the C1000 Touch thermal cycler (BIO-RAD) as follows:4min at 948C; the denaturation/annealing/elongation processwassetwithfivecyclesof30sat948C,60sat 608C,and60sat728C,followedby30cyclesof30sat 948C, 45 sat 508C, and 60 sat 728C. Final elongation followedfor7minat728C.PCRproductsweresequenced inbothdirectionsbythe‘Centrenationaldese´quenc¸age’
(Genoscope, Evry, France) or Eurofins MWG Operon (Ebersberg,Germany).Sequenceswereeditedandassem- bledusingSequencher5.1(GeneCodesCorporation,Ann Arbor,Michigan,USA).The523sequencesgeneratedfor thisstudyweredepositedintheGenBankdatabaseunder accessionnumbersKX822797–KX823319.
2.3. Phylogeneticanddatinganalyses
The Cytb dataset used for phylogenetic analyses contains 1140 nucleotides and 92 taxa. Our new Cytb sequenceswerecomparedtothoseavailableinGenBank forotherspeciesofthefamilyPteropodidae(77additional sequences;AppendixB).Tenoutgroupspecieswereused to root the pteropodid tree, representing three other mammalorders,i.e.Pholidota(Manisjavanica),Cetartio- dactyla (Bos javanicus), Perissodactyla (Ceratotherium simum),andsevenotherbatfamilies,i.e.Emballonuridae (Taphozous melanopogon), Hipposideridae (Hipposideros armiger), Megadermatidae (Megaderma lyra), Natalidae
Fig.2.ChronogramofthefamilyPteropodidaebasedoncompletemitochondrialcytochromebgenesequences.Phylogeneticrelationshipsanddivergence timeswerefirstestimatedusingBEAST.ThephylogenywasalsoinferredusingMrBayes(seetextfordetails).Thetenoutgrouptaxaarenotshown.Foreach
(Natalusmajor),Nycteridae(Nycterisjavanica),Phyllosto- midae(Artibeusjamaicensis)andRhinolophidae(Rhinolo- Rhinolophusluctus).DNAsequenceswerealignedonSe–
Alv2.0a11(http://tree.bio.ed.ac.uk/software/seal/).
Divergence timeswereestimatedusing theBayesian approach implemented in BEAST v.2.1.3 [23]. As no sufficiently accurate calibration point (fossil record or biogeographicevent)isavailableforthefamilyPteropo- didae,divergencetimeswereestimatedusingamolecular calibration point corresponding to theage of themost recent common ancestor of Nyctimene and Pteropus (subfamily Pteropodinae in Fig. 2) estimated at 16.51.5MainMeredithetal.[24].WeappliedaGTR+I+G modelofevolutionforeachofthethreecodonpositions(as selectedunderjModelTest2.1.7usingtheAkaikeinformation criterion[25])andarelaxed-clockmodelwithuncorrelated lognormaldistributionforsubstitutionrates.Nodeageswere estimatedusinga calibratedYule speciationpriorand108 generations,withtreesamplingevery2000generations,and aburn-inof10%.AdequacyofchainmixingandMCMCchain convergencewere assessedusingtheESSvalues inTracer v.1.6.ThechronogramwasreconstructedwithTreeAnnota- torv.1.7.5andvisualizedwithFigTreev.1.4.1(http://www.
tree.bio.ed.ac.uk/software/).
For comparison,phylogeneticrelationships werealso inferredusingMrBayes3.2.1[26].Theposteriorprobabili- ties(PP)werecalculatedusingfourindependentMarkov chains run for 10,000,000 Metropolis-coupled MCMC generations,withtreesamplingevery1000generations, andaburn-inof25%.
2.4. Populationgeneticanalyses
Phylogeographic analyses were performed on eight speciesusingmitochondrialCytbsequences:E.buettikoferi, E. franqueti, E. gambianus, E. helvum, H. monstrosus, M.pusillus,N.veldkampii,andR.aegyptiacus.Mitochondrial DNA is particularly suitable for phylogeographic studies because itevolves withhigh ratesofsubstitutionand is transmittedmaternallywithoutrecombination.However, discordantpatternsbetweenmtDNAandnuDNAmarkers canarisewhenmitochondrialintrogressionoccurreddueto secondarycontactbetweencloselyrelatedspecies,orwhen dispersalrateswerehigherinmalesthaninfemales[17,21].
Foreachdataset,populationgeneticindices,including number of haplotypes (H), haplotype diversity (h) and nucleotide diversity (
p
), were calculated from Cytb sequences using DNASP v5.10 [27]. Mean, minimum, andmaximumK2PdistanceswerecalculatedwithPAUP4 [28].NetworksofCytbhaplotypeswereconstructedwith themedianjoiningmethodavailableinPopART1.5(http://www.popart.otago.ac.nz/) using equal weights for all mutations.
Thegeneticdifferentiationbetweenpairsofpopula- tions (e.g., Central versus West Africa) was measured using both Fst and GammaST calculated with DNASP v5.10[27].
3. Results
3.1. AMoleculartimescaleforpteropodidevolution
TheBayesianchronogramofFig.2showsthatspeciesof thefamilyPteropodidaecan bedividedinto fourmajor clades(PP=0.9–1)thatweconsiderhereascorrespond- ingtothesubfamiliesPteropodinae,Cynopterinae,Macro- glossinae, and Rousettinae. According to our molecular datingestimates,thesefourgroupsdivergedrapidlyfrom each otherduring theEarlyMiocene,between19.5and 17.8Mya.
AllfruitbatspeciesofAfricabelongtothesubfamily Rousettinae, except Eidolon helvum, a member of the subfamily Pteropodinae that is closely related to E.dupreanum(PP=1),itscongenericspeciesinMadagas- car.TheAfricanspeciesofthesubfamilyRousettinaeare furthersubdividedintothreerobustclades(PP=1),here treatedasthreedifferenttribes:(1)thetribeScotonycte- rini,whichcontainssixAfricanspeciesarrangedintotwo genera, Scotonycteris and Casinycteris; (2) the tribe Rousettini,whichincludesasinglegenus,Rousettus,with onlyoneAfricanspecies,R.aegyptiacus,andseveralspecies fromAsia(R.amplexicaudatusandR.leschenaultii),Mada- gascar (R. madagascariensis), and the Comoro Islands (R. obliviosus); (3) thetribe Epomophorini sensu lato, a large African clade composed of the four subtribes Epomophorina (with species of Epomophorus, Epomops, Hypsignathus,Micropteropus,andNanonycteris), Myonyc- terina (with species of Myonycteris and Megaloglossus), Plerotina(Plerotesanchietae),and Stenonycterina(Steno- Stenonycterislanosus).Our molecularestimatessuggest that the three tribes Scotonycterini, Rousettini, and Epomophorinidiversifiedsynchronously duringtheLate Miocene,ataround8–7Mya.
3.2. Phylogeographicnetworks
We analysed the nucleotide variation of 675 Cytb sequences(AppendixA)toexplorethephylogeographyof eightspeciesofPteropodidae.Thenumberofsequences, segregating sites, haplotypes, as well as haplotype diversity, nucleotide diversity, and K2P distances are describedforeachtaxoninAppendixC.Finally,anetwork ofCytbhaplotypeswasconstructedforonlysixdifferent datasets (Fig. 3). Indeed, preliminary analyses revealed thattwopairsofspeciescannotbedistinguishedonthe
node,thevalueinboldisthemeandivergencetimeinmillionsyears(greybarsindicatehighestposteriordensity[HPD]intervalsat95%),whereasother valuescorrespondtoposteriorprobabilitiescalculatedusingeitherBEAST(attheleftoftheslash)orMrBayes(attherightoftheslash).Thesymbol‘‘–’’
indicatesthatthenodewasnotfoundintheMrBayesanalysis,butnoalternativehypothesiswassupportedbyPP>0.8.Theasterisk(*)showsthatthenode wassupportedbyPP>0.95inbothBayesiananalyses.Adottedbranchindicatesthatthenodewasnothighlysupported(PP<0.8)inthetwoBayesian analyses.Africanspeciesarehighlightedwithredbranches,andthosefound(evenoccasionally)intherainforests,UG(UpperGuinea)and/orGabon-DRC, areindicatedwithgreentext.Redimmunoglobulinsymbolsindicatetaxainwhichanti-ZEBOVantibodiesweredetectedinpreviousstudies(seemaintext forreferences).PicturesofEbolavirusshowthethreespeciesfromwhichZEBOVRNApolymerasewassequenced[8].
Fig.3. PhylogeographicpatternsoffruitbatspeciesdistributedinbothWestandCentralAfrica.Medianjoiningnetworksbasedoncytochromeb haplotypeswerereconstructedforthesixfollowingdatasets:(A)MicropteropuspusillusandEpomophorusgambianus(212individuals);(B)Nanonycteris veldkampii(27individuals);(C)EpomopsbuettikoferiandEpomopsfranqueti(221individuals);(D)Hypsignathusmonstrosus(76individuals);(E)Eidolon helvum(88individuals);(F)Rousettusaegyptiacus(51individuals).ThegeographicdistributionmapswereextractedfromtheIUCN[14].Localitiesofthe haplotypeswereclassifiedinninebiogeographicregions(seedetailsinAppendixA):Senegal(orange),UpperGuineanrainforest(redorpink),Cameroon (yellow),westernEquatorialAfricanrainforest(green),CentralAfricanRepublic(lightblue),easternEquatorialAfricanrainforest(navyblue),Uganda (pink),southeasternAfrica(purple),andMediterraneanregion(beige).In(C),whitehaplotypeswereobtainedfromWestAfricanindividualsthatcannotbe assignedtoeitherE.franquetiorE.buettikoferionthebasisofthethirdpalatalridge.
Fig.0015. (Continued).
basis of their mitochondrial sequences: E. gambianus/
M.pusillus[21]andE.franqueti/E.buettikoferi.
The network reconstructed from the E. gambianus/
M.pusillusdataset(Fig.3A)revealedhighgeneticdiversity, with 21 Cytb haplotypes for Epomophorus and 94 Cytb haplotypes for Micropteropus. There is no taxonomic coherence,asthehaplotypesdonotclusteraccordingto species names, and there is no geographical structure amongsampledpopulations. In addition,wefoundtwo Cytb haplotypes shared between the two species. In Micropteropus, we detected higher levels of nucleotide diversity(0.0171versus0.0094),withtheexistenceoftwo highlydivergentCytbhaplogroupsdifferingby44muta- tions(namedH1andH2inFig.3A):H1wasfoundinboth northern populations (Ivory Coast and Central African Republic)andsouthernpopulations(GabonandKatanga ProvinceoftheDRC);H2wasdetectedonlyinsouthern populations (Gabonand Katanga).All the fourH2 Cytb haplotypesshare99–100%ofidentitywithCytbhaplotypes ofEpomophoruscrypturusandE.wahlbergicollectedinDRC (Katanga)andSouthAfrica(unpublisheddata).
ThedatasetforE.franquetiandE.buettikoferi(Fig.3C) also revealed high genetic diversity, with 91 Cytb haplotypes for E. franqueti and 35 Cytb haplotypes for E.buettikoferi.Itwasnotpossibletoassign14individuals collected in West Africa to either E. franqueti or E. buettikoferi. The network of Epomops Cytb sequences revealed the existence of two divergent haplogroups separated by 21 mutations and geographically highly segregated: the first one includes all individuals of E.franqueticollectedinCentralAfrica(Cameroon,Central AfricanRepublic, Gabon,Congo, and DRC), whereas the secondonecontainsallindividualsidentifiedasE.franqueti orE.buettikoferifromWestAfrica(LiberiaandIvoryCoast), withthreehaplotypessharedbythetwospecies.
Inallotherspecies,thereisnostronggeneticstructure betweenCentral andWestAfrica, suggestingthatpopu- lationswereabletomigrateextensivelybetweenthetwo regions.ForN.veldkampii,themtDNAanalysisshoweda star-likenetwork(Fig.3B),typicalof recentlyexpanded populations.ThetwoCytbhaplotypesfromCentralAfrica (CameroonandCentralAfricanRepublic)werefoundtobe identical tothe most common Cytb haplotype of West Africa. InR.aegyptiacus, theindividualscollected in the easternMediterraneanregion(CyprusandEgypt)differby 13mutationsfrompopulationsofsub-SaharanAfrica.In addition, some Cytb haplotypes of southeastern Africa were found to be highly divergent from the others (19mutations)(Fig.3F).Bycontrast,thepopulationfrom West Africa does not differ significantly from that of CentralAfrica,asindicatedbythelowvalues ofFstand GammaST(<0.1)(AppendixC).InbothH.monstrosusand E. helvum (Fig. 3D and E), we found no evidence of geographic structure, and Fst and GammaST values confirmedtheabsenceof differentiationbetweenpopu- lationsfromCentralandWestAfrica.Thisobservationwas corroboratedbythediscoveryofsharedCytbhaplotypes foreachofthetwospeciesbetweenthesetworegionsof Africa. In Eidolon, one Cytb haplotype is shared by individualsfromSenegalandKatanga(DRC).InHypsigna- thus,oneCytbhaplotypeissharedbetweentwoindividuals
fromIvoryCoast(collectedin2009)andanindividualfrom Gabon(G5CHA068),fromwhichLeroyetal.[8]sequenced aZEBOVRNApolymerase.
4. Discussion
4.1. OutofAsia:multipleoriginsoffruitbatsinAfrica
The fruit bats of the family Pteropodidae are only distributedintheOldWorld,andmostofthespeciesare foundintheequatorialregions,wherefruitsareabundant throughouttheseasons(AppendixD).Threedifferentlines ofevidencesupportaSoutheastAsianoriginofthefamily:
(1)94speciesofPteropodidaearefoundinthisregion[14], whichrepresentsmorethan50%ofthetotaldiversity;(2) Sumatra,BorneoandSulawesicontainthehighestspecies density(>15)(AppendixD);and (3)theoldestfossilof fruit bats has been described in the Late Eocene/Early OligoceneofThailand[29].
Withourexpandedtaxonomiccoveragewithrespectto previousstudies[30,31],themolecularanalysespresented hereinconfirmtheexistenceoffourindependentlineages of African fruit bats, all of which occupying a derived positionwithrespecttospeciesfromAsiaandOceania(see red branches in Fig. 2): E. helvum is the sole African representativeofthesubfamilyPteropodinae,whereasthe three other African lineages, i.e. R. aegyptiacus, Epomo- phorini and Scotonycterini, belong to the subfamily Rousettinae.Thechronogramin Fig.2suggeststhatthe African continent was colonized by at least four Asian ancestors:between2.0and1.7MyaforRousettus,between 11.6and3.4MyaforEidolon,between11.2and7.6Myafor Epomophorini, and between 16.6 and 6.8 Mya for Scotonycterini.Anti-Ebolavirusantibodieshavebeenpre- viouslydetectedineightAfricanspeciesofPteropodidae, representing three of these four lineages: E. franqueti, E. helvum, E. gambianus, H. monstrosus, M. pusillus, M. torquata, N. veldkampii, and R. aegyptiacus [8,11,12].
These results suggest therefore that several unrelated pteropodidtaxahavedevelopedaspecificimmunological response to Ebolavirus, and that the transmission of Ebolavirus between fruitbat speciesmayhaveoccurred throughcontactsinfruittrees.
4.2. Whicharethefruitbatspeciesabletodispersebetween CentralandWestAfrica?
All species ofPteropodidae are highly dependent on plantsforfood.Thisexplainswhythespeciesrichnessof fruit bats is higher in equatorial regions (Appendix D), wherefruitsandflowersaremorediverse andavailable mostoftheyear.Sincepteropodidsdonothibernate,they need abundantfood all yearround. To exploitseasonal foodresources,somespecieshavedevelopedtheabilityfor migrations ornomadicmovements. AmongAfrican pte- ropodids,onlyE.helvumandN.veldkampiiareconsidered asbeingmigratoryspecies,whichmeansthattheytravel seasonallyfromonehabitattoanotherusingpredetermi- nedroutes[32].PopulationsofN.veldkampiiareresidentof the rainforest during the dry season, and both sexes
migrate northwards towards savannah habitat types duringthewetseason.Thissmallspeciesseemstomake an annual round-trip migration of 300–1100km [32].Males ofE.helvumare known tomakevery long- range migrations across Central Africa (2500km over five months), but the routes used by the populations during their annual migration remain tobe discovered [33].
ThefollowingpteropodidspeciesofAfricaareconsid- eredtobenomadicratherthanmigratory,whichmeans that their seasonalmovementsareirregular andunpre- dictable: most species of Epomophorus (including E. gambianus), M. pusillus, M. leptodon, M. woermanni, and R. aegyptiacus [32,34]. In addition, local hunters of Luebo and Mweka villages (DRC) have reported that E. franqueti and H. monstrosushave annual movements awayfromtheseregions[35].Althoughfieldstudieshave indicatedseasonalmovementsforH.monstrosus[36,37],to ourknowledge,nothingwaspublishedonthemigratory behaviour of E. franqueti. Only two species of African Pteropodidaehavebeenthesubjectsoftelemetrystudies, i.e.E.helvumand R.aegyptiacus[12,33,38],andevenfor thesetaxa,seasonalmovementsremainpoorlydocumen- ted,asthemarkedindividualscouldnotbetrackedforat leastoneannualcycle.
Phylogeographic analyses therefore are particularly importanttoprovideinsightintowhichspeciesdisperse between Central and West Africa. Previous molecular studiesonMyonycterina[16]andScotonycterini[17]have identifiedastrong separationbetweenpopulationsfrom the two main blocks of African rainforest, the Upper Guineaforest inWestAfricaand thelarge CongoBasin forestinCentralAfrica.Accordingly,severalspecieswere described as endemic to each of these forest blocks:
M.azagnyi,M.leptodon,andS.occidentalisinWestAfrica, andtheirsister-speciesinCentralAfrica,i.e.M.woermanni, M.torquata,and S.zenkeri/S.bergmansi,respectively.All these sistertaxa have divergedin allopatry during two majorglacialperiodsofthePleistocene,at2.8–2.5Myaand 1.8–1.6 Mya. By contrast, a greater capacity for long- distance dispersalsbetween thesetwo rainforestblocks was proposed for the largest species of Scotonycterini, C. ophiodon, and for the sole cave-dwelling species of Myonycterina,M.angolensis[16,17].
Here,weusedmitochondrialCytbsequencestocompare the phylogeographyof E.helvum, R.aegyptiacus,and six species of the subtribe Epomophorina. For the two migratoryspecies,E.helvumandN.veldkampii,weobtained a typical star-like network pattern, with no genetic structure across their geographic range (Fig. 3Band E).
These results confirm that these two species have high dispersal capacity. Similar phylogeographic patterns, with nogeographic structurebetweenCentraland West Africa,werealsofoundformostspeciesconsideredtobe nomadic,i.e.E.gambianus,H.monstrosus,M.pusillus,and R. aegyptiacus. In R. aegyptiacus, we detected, however, divergent Cytb haplotypes in all individuals from the eastern Mediterranean, and in some individuals from Katanga(DRC)andMalawi,suggesting thatthese distant populationstendtobemoreisolatedfromtheothers.For severalindividualsfromsouthernpopulationsofM.pusillus
(GabonandKatanga;Fig.3A),wedetectedverydivergent H2Cytbhaplotypes(4.68–6.04%),whichshare99–100%of identitywith individualsofE.crypturusandE.wahlbergi collectedinDRC(Katanga)andSouthAfrica(unpublished data).CombinedwiththefactthattheH1Cytbhaplotypes of M. pusillus cannot be differentiated from those of E.gambianus(Fig.3A;seealsoNesietal.[21]),wesuggest that several events of inter-specific hybridization have occurredbetweenthedifferentspeciesoftheEpomopho- rus–Micropteropuscomplex.Weplantofurtherexplorethis issuewith RADSeq data. Taxonomically,we recommend placingM.pusillusinthegenusEpomophorus,asoriginally proposedbyPetersin1867[39].Ourphylogenetictreein Fig.2alsoindicatesthatthespeciesE.dobsonishouldbe excludedfromEpomopsandrathertreatedasaspeciesof Epomophorus.Morphologically,itisimportanttonotethat bothM.pusillusandE.dobsonihavesixthickpalatalridges (consideringthatthesecondandthirdridgesarepartially fusedinE.dobsoni),asinallotherspeciesofEpomophorus, whereas the species of Epomops (E. buettikoferi and E.franqueti)haveonlythreethickpalatalridges[20].Eco- logically,bothM.pusillusandE.dobsoniaremorecommon in woodland savannahs, as all species of Epomophorus, whereas other species of Epomops (E. buettikoferi and E.franqueti)occurmainlyinrainforesthabitats.
WithinEpomopssensustricto,ourCytbanalysesdonot supportthemonophylyofthetwospecies,E.franquetiand E.buettikoferii:allindividualsfromWestAfrica,identified aseitherE.franquetiorE.buettikoferii,shareverysimilaror identical haplotypes, which are divergent from those sequencedfor allindividualsof E. franqueti collectedin CentralAfrica(K2Pdistances:2.24–3.63%).Taxonomically, thisresultshowsthatthesinglediscretecharacterusedin thekeyofBergmans[20]fordistinguishingE.buettikoferi fromE.franqueti,i.e.themedialdivisionofthethirdridge inthepalate,isnotdependable.Inagreementwiththat,it must be noted that the palatal pattern supposed to characterizeE.buettikoferihasbeenpreviouslydescribed insomeindividualsofE.franqueticollectedinCameroon and Gabon [39]. Tobetter understand thetaxonomyof Epomops,wesequencedallthe12nuclearintronsanalysed in Hassanin et al. [17]for two individualsrepresenting each putative species and each geographic group, i.e.
E. buettikoferi from West Africa and E. franqueti from Central Africa(datanotshown).Thenucleardivergence was only 0.08%, which is in the range of intraspecific distancesfoundinothergroupsofLaurasiatheria,suchas Myonycterina (<0.21%) [16], Scotonycterini (<0.13%) [17], or cattle and bison of the tribe Bovini (<0.18%) [40]. The analyses of mitochondrial and nuclear data suggesttherefore thatthegenusEpomops contains only onespecies, which canbedivided intotwo subspecies:
E.franquetifranquetiinCentralAfricaandE.f.buettikoferiin WestAfrica[32].
To summarize, our phylogeographic results suggest that all migratory or nomadic species known to be common in savannah woodland habitats, can disperse betweenCentralandWestAfrica:E.gambianus,E.helvum, M.pusillus,N.veldkampii,andR.aegyptiacus.Bycontrast,all species restrictedorlargely restrictedtotherainforests (C. argynnis, E. franqueti, M. azagnyi, M. leptodon,
M. torquata, M. woermanni, S. bergmansi, and S. occidentalis), withthe exception of H. monstrosus, do not disperse long distances, specifically across the DahomeyGap,i.e.thesavannahcorridorinGhana,Togo andBeninthatseparatestherainforestblocksofWestand Central Africa [16,17]. Because of its larger body size, muscular strength, and higher wing loading [41], H.monstrosuscan fly muchfaster and thereforefarther thanotherfruitbatspresentinAfricanrainforests,which mayexplainitsgreaterdispersalcapacity.
4.3. ConsequencesfortheoriginofZEBOVinWestAfrica
On21March2014,acirculatingvirusinhumanswas identifiedbytheInstitutPasteurinLyon(France)asZEBOV, astrainpreviouslydetectedinonlythreeCentralAfrican countries(DRC,GabonandCongo)between1976and2014 [42].Epidemiologicinvestigationshavesuggestedthatthe firstcaseoftheWestAfricanoutbreakwasa2-year-old childwhodiedon6December2013inMeliandou,asmall villageinGuinea[43].Subsequently,Guinea,Liberia,and SierraLeoneexperiencedthelargestoutbreakofEbolaever recorded, with widespread and intense transmission between August 2014and December 2014, afterwhich case incidence declined. In total, 28,603 cases were identified,withdeaths(asof10June2016)[44].
Phylogenetic analyses of Ebola virus genomes have suggestedthat thenewZEBOVvariantfromWestAfrica diverged from lineages of Central Africa around 2003–
2004[45,46].AnintroductionintoGuineafromahuman travellerseemsunlikely,becausetheepicentreregionof Gue´cke´dou is a remote area of the Upper Guinean rainforest,whichis farfromtheCongorainforestwhere allpreviousZEBOVoutbreaksoccurred[47].Bycontrast, migratory bats may have carried ZEBOV from Central Africa to Guinea. Among fruit bats, eight species are commonly found in the Congo rainforest [14,15]:
C. argynnis, E. helvum, E. franqueti, H. monstrosus, M. woermanni, M. torquata, R. aegyptiacus, and S. bergmansi. In 2005, Leroy et al. [8] obtained ZEBOV RNApolymerasesequencesfromliverandspleensamples ofH.monstrosus(19%;4/21),E.franqueti(4.3%;5/117),and M. torquata (2.8%; 4/141), suggesting that these three speciesrepresentnaturalreservoirhostsofEbolaviruses.
In Gabon and Congo, ZEBOV-specific antibodies were detected in all fruit bat species tested for a sample size>125 individuals: R. aegyptiacus (7.8%; 24/307), H.monstrosus (7.2%; 9/125),E. franqueti (4.5%; 36/805), M.torquata(3.3%;19/573),andM.pusillus(2.0%;4/197).In addition, two insectivore bat species werefound to be ZEBOVpositive:Hipposideros gigasand Mopscondylurus [11].In WestAfrica, ZEBOVantibodiesweredetectedin four pteropodid species: E. gambianus (13.5%; 5/37), H. monstrosus (12.5%; 2/16), E. franqueti (10.7%; 3/28), andE.helvum(0.4%;1/262)[12,48].Allthesedatasuggest thatZEBOVisacirculatingvirusacrossAfricaandthatit wastransmittednotonlyinbatsendemictorainforests, butalsotothosegenerallyfoundinsavannahwoodlands, suchasE.gambianusandM.condylurus.Transmissionof ZEBOVbetweenbat speciesmayhaveoccurred through contactsinfruit trees,involving eitherfightingfor food
resources, or indirect contamination via infected body fluids (saliva, blood, faeces, and urine) deposited on branchesandfruits[6].
Ourphylogeographicanalysesrevealedthatonlythree fruitbatspecieswereabletodispersedirectlyZEBOVfrom the rainforests of Central Africa (where ZEBOV was endemic until sometimebefore 2014) tothose ofWest Africa (where ZEBOV suddenly appeared in 2014):
E.helvum,H.monstrosus,andR.aegyptiacus (Fig.3).The speciesE.helvumseems,however,tobeassociatedwith secondary transmissions from a zoonotic reservoir: it showed a very low ZEBOV prevalence (0.4%); and its implicationinhumanoutbreaksappearsunlikely,because it typically lives in large urban colonies (e.g., Bangui, Yaounde´)and is a sourceof bushmeatin many African regions[49].Bycontrast,H.monstrosusandR.aegyptiacus representthetwo leadingcandidatesfor explainingthe dispersalofZEBOVfromCentraltoWestAfrica.Firstofall, H.monstrosuswasidentifiedasoneofthethreereservoir hostsofZEBOVintherainforestsofCentralAfrica[8,35], whereasR.aegyptiacuswasidentifiedasthemainreservoir hostofMarburgviruses[10].Thehighestseroprevalences against ZEBOV were found for R. aegyptiacus and H.monstrosus(7.8and7.2%,respectively)[11].InUganda, thestudyofalargepopulationofMarburg-virus-infected R. aegyptiacus fruit bats has evidenced that the two biannual birthing seasons represent times of increased infectionamongolderjuvenilebats(sixmonthsofage) that roughly coincide with historical dates of Marburg virusspilloverintohumans[50].InGabonandCongo,most humanZEBOVoutbreakshave occurredthroughcontact withinfectedanimalcarcasses,especiallygreatapesand duikers. Ithasbeen suggestedthat the transmissionto great apes and duikers have been initiated by the consumption of fruits contaminated with blood and placentas during parturition of infected fruit bats [11]. Allthese elements suggest that theparturition of infectedfruitbatsmayhaveincreasedZEBOVprevalence infruitbatpopulations,andthereforethecontaminationof otherspecies,includinghumans. Thishypothesis isalso supportedbythefactthatthefirstcaseoftheWestAfrican ZEBOVoutbreakwasinfectedinNovemberorDecember,a periodthatcoincidesperfectlywithoneofthetwobirthing seasonsofH.monstrosusandR.aegyptiacusinWestAfrica [32,51].
5. Conclusion
Our geneticanalyseshaveshownthat threefruitbat species havedispersal movements between Central and WestAfrica,and,hence,arecapableofactingasdispersal agentsforthisvirus.Amongthem,onlyH.monstrosusis restricted to rainforest habitats, where all ZEBOV out- breakshaveoccurred,whileE.helvumandR.aegyptiacus arealsocommonlyfoundinsavannahwoodlands.Since fruit bats often eat on the same trees, inter-species infections of ZEBOV are expected to be frequent, in particular during the two biannual birthing seasons of themainreservoirhostspecies.Becauseofthat,wecannot completelyruleoutthehypothesisinvolvingthatZEBOV was dispersed indirectly in West Africa, i.e. through
nomadicormigratoryspeciesofsavannahwoodlands,i.e.
E.gambianus,M.pusillusandN.veldkampii,whichmayhave encounteredthemainreservoirhostspeciesoftheCongo Basinintherainforest–savannahmosaics.
Acknowledgments
Wethankthenumerousindividualswhohelpedusto collecttissuesamplesduringfieldmissions:PhilippeBlot, Andre´ De´licat, and Jean-Pierre Hugot in Gabon; Je´roˆme Fuchs, Philippe Gaubert, Flobert Njiokou, and Anne Ropiquetin Cameroon;AlainLeFaou inCentral African Republic; Christiane Denys, Mireille Dosso, Franc¸ois Jacquet, and Ste´phane Kan Kouassi in Ivory Coast; and Raphae¨lColombo,LaurentDaudet,BenjaminDudu,Tamas Go¨rfo¨l, Keunen Hilde, Ros Kiri Ing, Jean-Franc¸ois Julien, VuongTanTuandPeterValloinDRC.Weareverygrateful tocollectionmanagersandcuratorswhoprovidedsamples frommuseumspecimens:KatrinKrohmannandVirginie Volpato(NaturmuseumSenckenberg),Anne-MarieOhler andJean-MarcPons(MNHN).AHwouldliketothankKevin RacineandtheWorldBatLibrary(Geneva,Switzerland)for bibliographic assistance. We also acknowledge the two anonymousreviewersfortheirhelpfulcommentsonthe manuscript.ThisworkwassupportedbytheMNHN,CNRS,
‘PPF Taxonomie mole´culaire, DNA Barcode and gestion durable des collections’, ‘PPF Biodiversite´ actuelle et fossile’,‘Socie´te´ desamisduMuse´umnationald’histoire naturelleetduJardindesplantes’,LabExBCDiv2012-2013, and‘Consortiumnationalderechercheenge´nomique’.Itis partof agreement No. 2005/67between theGenoscope andtheMNHNontheprojects‘SpeedID’and‘Bibliothe`que duVivant’.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbe found,intheonlineversion,athttp://dx.doi.org/10.1016/j.
crvi.2016.09.005.
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