Analysis of heteroplasmy in bank voles inhabiting the Chernobyl exclusion zone : A commentary on Baker et al. (2017) "Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent."

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Analysis of heteroplasmy in bank voles inhabiting the Chernobyl exclusion zone : A commentary on Baker et al. (2017) "Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent." © 2017 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd Published version Kesäniemi, Jenni; Boratyński, Zbyszek; Danforth, John; Itam, Prince; Jernfors, Toni; Lavrinienko, Anton; Mappes, Tapio; Møller, Anders Pape; Mousseau, Timothy A.; Watts, Phillip C. Kesäniemi, J., Boratyński, Z., Danforth, J., Itam, P., Jernfors, T., Lavrinienko, A., Mappes, T., Møller, A. P., Mousseau, T. A., & Watts, P. C. (2018). Analysis of heteroplasmy in bank voles inhabiting the Chernobyl exclusion zone : A commentary on Baker et al. (2017) "Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent.". Evolutionary Applications, 11(5), 820-826. https://doi.org/10.1111/eva.12578 2018 Evolutionary Applications. 2017;1–7.   | 1 wileyonlinelibrary.com/journal/eva Received:9October2017 | Accepted:3November2017 DOI: 10.1111/eva.12578 COMMENTARY Analysis of heteroplasmy in bank voles inhabiting the Chernobyl exclusion zone: A commentary on Baker et al. (2017) “Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent.” Jenni Kesäniemi1 | Zbyszek Boratyński2 | John Danforth1 | Prince Itam1 | Toni Jernfors1 | Anton Lavrinienko1 | Tapio Mappes3 | Anders Pape Møller4 | Timothy A. Mousseau5 | Phillip C. Watts1 1DepartmentofEcologyandGenetics,UniversityofOulu,Oulu,Finland 2CIBIO/InBIO,ResearchCenterinBiodiversityandGeneticResources,UniversityofPorto,Vairão,Portugal 3DepartmentofBiologicalandEnvironmentalScience,UniversityofJyväskylä,Jyväskylä,Finland 4EcologieSystmatiqueEvolution,UniversitParis-Sud,CNRS,AgroParisTech,UniversitParis-Saclay,OrsayCedex,France 5DepartmentofBiologicalSciences,UniversityofSouthCarolina,Columbia,SC,USA Correspondence JenniKesäniemi,DepartmentofEcologyandGenetics,UniversityofOulu,Oulu,Finland. Email:jenni.k[email protected] Funding information AcademyofFinland,Grant/AwardNumber:PCW:287153andTM:268670 KEYWORDS:ecologicalgenetics,molecularevolution,populationecology 1 | INTRODUCTION Exposuretoionizingradiationisawell-establishedcauseofmutation. Giventheglobalproblemofaccidentalreleaseofradionuclidesinto theenvironment(Loureno,Mendo,& Pereira,2016), it is essential tofullyunderstandthegeneticconsequencesofexposuretoradionuclides.On26April1986,afireandexplosioninReactor4ofthe former nuclear power plant at Chernobyl (CNPP), Ukraine, released morethan9millionterabecquerels(TBq)ofradionuclidesovermuch (>200,000km2)ofEuropeandeasternRussia(seereviewsontheeffects,e.g.,Mousseau&Møller,2012;Møller&Mousseau,2006).The ChernobylExclusionZone(CEZ)wasestablishedatabouta30-kmradiusaroundtheaccidentsitetolimithumanexposuretoradioactive fallout.TheCEZcontainselevatedlevelsofpersistentradioisotopes, notably strontium-90 (90S), caesium-137 (137Cs) and plutonium-239 (239Pu) that have half-lives of 28.8, 30.2 and 24,100years, respectively. Wildlife inhabiting the CEZ provide clear models of the biological consequences of exposure to environmental radionuclides, with many reports of elevated levels of developmental instability, genetic damage and mutation rate associatedwith inhabiting areas contaminated by radionuclides. Hence, a meta-analysis revealed a strongeffectofradiationuponmutationrateinorganismsaffectedby Chernobylfallout(datafor30speciesin45publishedstudies)(Møller &Mousseau,2015).Withthisinmind,thereportbyBakeretal.(2017) in Evolutionary Applicationsofelevatedlevelsofgeneticdiversityrates inbankvolesinhabitingtheCEZappearsconsistentwiththeputative mutageniceffectofexposuretoradionuclides. TheanalysisbyBakeretal.(2017)ispromisingfortwoprincipal reasons:(i)theyhavedatafromtwotimepointsand(ii)theyusenext- generation sequencing (NGS) to identify polymorphisms and thus bringstudiesofChernobylwildlifeintothegenomicsera.Bakeretal. (2017) sequencedwhole mitochondrial genomes of samples of the bankvoleMyodes glareolustodeterminewhetherthebankvolesinhabitingtheCEZhaveaccumulatedmutationsasaconsequenceof exposuretoelevatedlevelsofradionuclides.Thebankvoleisasmall rodentthatiscommoninforesthabitatsinnorthernEurope.Asthis speciesiscommonwithinandaroundtheCEZ,thebankvolehasbeen widelystudiedasamodelofthemammalianresponsetoradionuclides ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium, providedtheoriginalworkisproperlycited. ©2017TheAuthors.Evolutionary ApplicationspublishedbyJohnWiley&SonsLtd 2 | KESÄNIEMI et al. (Boratyński,Lehmann,Mappes,Mousseau,&Møller,2014;Chesser etal., 2000; Lehmann, Boratyński, Mappes, Mousseau, & Møller, 2016;Meeks,Chesser,Rodgers,Gaschak,&Baker,2009;Meeksetal., 2007;Rodgers&Baker,2000;Rodgers,Wickliffe,Phillips,Chesser,& Baker,2001).Bakeretal.(2017)foundgreatermitochondrialdiversityinsamplesfromtwocontaminatedareas(RedForestandGlyboke Lake)thaninsamplesofbankvolesfromthreeuncontaminated(control)areas(Nedanchychy,NezamozhnyaandOranoe)(seeFigure1for samplelocations).Intheabstract,theauthorsstate[thattheirdata are] “consistent with the possibility that chronic, continuous irradiation resulting from the Chernobyl disaster has produced an accelerated mutation rate in this species over the last 25 years.”However,Bakeretal., 2017didnotfullydiscussthreeimportantissuesrelatingtotheirdata: (i)sampling,(ii)bankvolepopulationdynamicsand(iii)heteroplasmy. 2 | SAMPLING: CORRECTION FOR VARIATION IN SAMPLE SIZES SamplesizesusedbyBakeretal.(2017)varybetween11and20(and onesampleofthreebankvolesatGlybokeLake),withthetwolargest samplesfromtheRedForest(i.e.,contaminatedsite).Bakeretal.(2017) discusspossibleeffectsofunbalancedsamplesizeontheirconclusions andlargelyattemptedtocorrectforunevensamplesizesbydividingestimatesofgeneticdiversitybythesamplesize(Table3inBakeretal. (2017)). This method of correction is not appropriate (e.g., as genetic diversityandsamplesizehaveanonlinearrelationship,seeFigure2),and rarefactionismoretypicallyusedtocompareestimatesofgeneticdiversityamongsamplesthatdifferinsize(Petit,Mousadik,&Pons,1998; Szpiech,Jakobsson,&Rosenberg,2008).Asanillustration,weobtained dataforBakeretal.’s(2017)samplesofbankvolesforthemitochondrial locusND4(1,378bpthathad60variablesites)fromDRYAD(https:// doi.org/10.5061/dryad.j11s7)andcalculatedthenumberofhaplotypes persample,correctedforsamplesizeusingrarefactionimplementedby ADZE(Szpiechetal.,2008).Wealsofoundthatmitochondrialdiversity (asmeasuredbythenumberofhaplotypes)ishigherincontaminated thaninuncontaminatedsites(Figure2),reinforcingBakeretal.’s(2017) conclusions; moreover, high diversity is apparent in the small (n = 3) GlybokeLake2011samplealthoughthesedatawerenotincludedinthe statisticalcomparisonofpopulationgeneticdiversity.Hence,thelevel ofmitochondrialdiversityisassociatedwiththelevelofenvironmental radioactivity.But,dothesedataindicateahighmutationrate? 3 | SAMPLING: UNCLEAR CHOICE OF CONTROL SITES Severalstudieshavequantifiedmitochondrialdiversity(ata291-bp fragmentofthecontrolregionandsomeadjacenttRNA)inbankvoles inhabitingtheCEZandinuncontaminatedsitesinUkraine(Matson, Rodgers,Chesser,&Baker,2000;Meeksetal.,2007,2009;Wickliffe etal.,2006):noneofthesestudiesconcludedthattherewasarobust association between mutation rate and the level of environmental FIGURE1 LocationofsiteswithinandoutsidetheChernobyl ExclusionZone(CEZ)fromwhichbankvoleswerecollected,as describedbyMeeksetal.(2007)(filledcircles)andbyBakeretal. (2017)(opencircles).CNPPreferstothelocationoftheformer ChernobylNuclearPowerPlant(filledsquare)andcirclesindicate10- kmand30-kmradiusfromtheCNPP.Contaminatedsamplesitesare CL—GlybokeLakeandRF—RedForest.Uncontaminated(references) samplesitesareNZ—Nezamozhnya,ND—Nedanchychy,OR— Oranoe,PA—Paryshev,ST—StupnikovoandKR—Krasnoye.ND2017 referstotheNedanchychylocationreportedbyBakeretal.(2017), whileND2007referstothelocationofthesiteusedbyMeeksetal. (2007):notethatthecoordinatesfortheRedForestcontaminated siteinBakeretal.(2017)are~12kmfromtheoriginalRedForest site.MapwascreatedinRv.3.3.3(RDevelopmentCoreTeam,2014) usingGGMAPv.2.6.1(Kahle&Wickham,2016) FIGURE2 Rarefactioncurvesofmitochondrialgeneticdiversity (numberofhaplotypesattheND4locus)againstsamplesizefor tensamplesofbankvolesfromsitesthatwerecontaminated(filled circles,solidlines)oruncontaminated(opencircles,dashedlines)by environmentalradioactivity.SamplecodesareCL—GlybokeLake,RF— RedForest,NZ—Nezamozhnya,ND—NedanchychyandOR—Oranoe 0 2 4 6 8 10 12 0510 15 20 Sample size sepytolpahforebmuN RF 1998 RF 2011 GL 1998 GL 2011 OR 1998 NZ 1998 & 2011 OR 2011 ND 2011 ND 1998 | 3 Kesäniemi et al radionuclides. Rather, studies have highlighted the need for additionalsampling(Matsonetal.,2000;Wickliffeetal.,2006)orfound mitochondrialdiversitytobecomparablebetweencontaminatedand uncontaminated sites that were located close to the CNPP (Meeks etal.,2007);moreover,geneticdiversitywasheterogeneousamong samples of bank voles collected over a large area of Ukraine, with uncontaminated locations containing more unique haplotypes and ahigherratioofuniquetototalhaplotypes(Table1inMeeksetal., 2009). Variation in mitochondrial diversity in bank voles has been explainedbydemographicandecologicalprocesses,ratherthanexposure to environmental radionuclides (e.g., Meeks etal., 2009). These studies on bank vole mitochondrial genetic diversity were notaddressedindetailbyBakeretal.(2017)despitetheconclusion byMeeksetal.(2009)that“genetic diversity in radioactive regions of Ukraine is probably a function of natural geographic variation rather than increased mutational pressure from radiation exposure and underscore the importance of adequate geographic sampling.” Thepriorinformationaboutlevelsofmitochondrialdiversityfor bankvolepopulationswithinandaroundtheCEZisimportantwhen choosingsamplinglocations.Ofthethreecontrolsamplesselectedby Bakeretal.(2017),NedanchychyhadnotablylessmitochondrialdiversitythanotherbankvolesamplesfromUkraine(Meeksetal.,2007, 2009;Wickliffeetal.,2006):haplotypediversity(h)atNedanchychy (h = 0.19) is less than half that of the samplewith the next lowest diversity (Ezyaslav, h = 0.44) (Table 1 and Figure 2 in Meeks etal. (2009)). Reasons for low genetic diversity at Nedanchychy are not known,buthighlightmarkedsite-specificvariationingeneticdiversityinwildbankvolepopulations.Althoughmitochondrialdiversity atNedanchychyappearsatypicalofbankvolepopulationsinUkraine, theimpactofthissampleontheconclusionaboutlevelsofmitochondrialdiversityappearslimitedastheotherreferencesamplesareless diversethanthecontaminatedsamples(Figure2).Also,Meeksetal. (2007) sampled bank voles from six potential control (uncontaminated)locationsthatliewithinabout40kmoftheCNPP(Figure1).A comparisonofanythesesites,ratherthansamplesfromdistantareas, withcontaminatedsampleswouldreducetheeffectofhistoricdemographicprocessedonpatternsofgeneticdiversity.However,Baker etal.(2017) provide noreasonfortheir analysisof thethree more distant(>30kmfromCNPP)controlsratherthanobtainingdatafrom oneormoreoftheuncontaminated sites (Stupnikovo, Krasnoye or Paryshev)thatarecloser(1–15km)tothecontaminatedareas(see Figure1).LocationswithinorclosetotheCEZarethelikelysources of the bank voles that recolonized the contaminated areas within the CEZ after the nuclear accident. As such, the sites Stupnikovo, KrasnoyeorParyshevmightbethemostappropriatecontrolsforan assessmentoftheimpactofenvironmentalradionuclidesongenetic diversity.Alternatively,useoftheseproximatesitesmightconfound analysesofdiversityduetoongoingdispersalofbankvolesamong contaminatedanduncontaminatedareas.Bankvolesarefairlymobile andcanmove1kmwithinabreedingseason(Kozakiewicz,Chołuj,& Kozakiewicz,2007).Choiceofappropriatecontrolsitesisanimportantissuethat,here,iscomplicatedbyacombinationofpriorinformationaboutvariationinmitochondrialdiversityinpotentialsamples andalackofknowledgeaboutthepopulationdynamicsofwildbank voles. 4 | BANK VOLE POPULATION DYNAMICS Thepossibleinfluenceofpopulationhistoryonspatialpatternsofmitochondrialdiversityshouldbereconsidered.Bakeretal.(2017)imply thattheirdataareinconsistentwiththehypothesisofrecolonization explainingtheobservedhighmitochondrialdiversityincontaminated sites because the surrounding areas (potential sources) have lower mitochondrial diversity. However, an area recolonized by several, genetically different, sources could exhibit an increase in genetic diversity(asdiscussedby(Matsonetal.,2000)).Theuncontaminated areastotheeastofChernobyl(NedanchychyandNezamozhynya)are geneticallydifferentfromadistantcontrolsite(Korostychev)thatis southofChernobyl(Meeks etal., 2009). Also, bank voles from uncontaminatedsitesNedanchychy,KrasnoveandParyshev(Figure1), aswellasthecontaminatedsiteswithinCEZ,haveunique(notfound atanyothersite)mitochondrialhaplotypes(Meeksetal.,2007,2009; Wickliffe etal., 2006). Indeed, there were no shared mitochondrial haplotypes among the CEZ and control regions (Figure1 in Baker etal.(2017)).Thispatternofgeneticdiversityindicatesthatgeneflow amongCEZandcontrolsitesislimited,butitdoesnotruleoutrecolonizationoftheCEZfromseveralsurroundingsourcesitesasother controlsareaswerenotanalysedbyBakeretal.(2017)(Figure1).As bankvolepopulationswithinandaroundtheCEZaregeneticallydifferent,recolonizationprocessesremainapossiblereasonforthehigh mitochondrialdiversityofbankvolesinhabitingtheCEZ.Thishighlightsthedifficultiesinassociatingasignatureofgeneticvariationto theeffectsofionizingradiationratherthanotherdemographicpopulationprocesses,anissuealsopointedoutbyBakeretal.(2017). AstrengthoftheanalysisbyBakeretal.(2017)istheirdatafrom 1998and2011.The13-yeardifferencebetweensamplingperiodsis comparabletothetimeintervalbetweentheaccident(1986)andthe firsttimepoint(1998)andrepresentsabout26generations(ofexposuretoradionuclides).Onecorollaryofthemutageniceffectsofexposuretoenvironmentalradionuclidesisthatmutationsaccumulate withtime.Bycontrast,notemporalchangeinmitochondrialdiversity attheRedForest(contaminated)samplewasapparent(Tables3and4 inBakeretal.(2017),alsoseeFigure2),althoughthereweregreater nucleotide differences between temporal samples at the contaminatedsites,butsmallsamplesizepreventedanystatisticalinference (TableS4inBakeretal.(2017)).Alackoftemporaleffectweakensthe argumentthatexposuretolow-doseradionuclidessimplyincreases mutation. One implication is that most of the mitochondrial diversity(viamutation)mayhaveaccumulatedatsomepointtoaffectthe 1998sample,butnotsubsequently.Forexample,mostofthefallout fromtheChernobylaccidentwasiodine-131.Exposureto131I conceivablymighthavehadsomeinitial,butnotcontemporary,impact onwildlifeasthisisotopedissipatedrapidly(half-lifeof8days).Initial exposuretoradiationcantriggerasuiteofcellulareffectsthatpersistforsometime(Mothersill&Seymour,2006).Exposureto131Iis 4 | KESÄNIEMI et al. associatedwithelevatedincidenceofhumanthyroidcancers(Cardis etal., 2006), but its long-term effects on wildlife are not known. Otherprincipalradionuclides(90S,137Csand239Pu—seeIntroduction) withintheCEZaremorepersistent,andtheireffectsarelesslikely tohavedissipatedrecently.Wemightspeculatethatbankvolesexhibitsomeadaptiveresponsetoradionuclidecontamination,forexample,viaimprovedantioxidativemeasuresand/orDNArepairthat preventsfurtheraccumulationofmutations.Fibroblastsofbankvoles fromtheCEZexhibitgreaterantioxidativecapacitythandobankvole fibroblastsfromcontrolareasnearKiev(V.Mustonen,J.Kesäniemi, A.Lavrinienko,E.Tukalenko,T.Mappes,P.C.Watts,&J.Jurvansuu, unpublishedresults).Wecouldalsoarguethatanytemporalgenetic patterninthesebankvoledataisconfoundedbystochasticrecolonizationoftheCEZfromdifferentsourcepopulations.Withthisin mind,Wickliffeetal.(2006)foundmarkedfluctuationsinmitochondrialdiversity(haplotypediversityhvariedfrom0.67to0.82)among samplesofbankvolesfromthecontaminatedRedForestsiteover a three-year period (1998–2001). The discussion above does not excludearoleformutation,buthighlightsthatotherprocessescan explainthecontemporarypatternofgeneticdiversityinbankvoles withinandaroundtheCEZ. 5 | HETEROPLASMY AS A MARKER OF MUTATION? Inferringmutationratefrompopulationgeneticdiversityitselfiscomplicatedbyprocessesthatdeterminewhether(ornot)amutationis incorporatedintothepopulationatadetectablefrequency:theprobabilitythatamutationisretained withina population,for example, depends on strength of selection, population size, recombination (e.g.,fornuclearDNA)andsamplesize.Acomplementaryanalysisof mutationcouldfocusonmutationsoccurringwithinindividualsindependent of demography. One solution is to quantify heteroplasmy, theoccurrenceofmorethanonemitochondrialhaplotypewithinan individual(Li,Schröder,Ni,Madea,&Stoneking,2015;Lietal.,2010). HeteroplasmymaybecausedbypaternaltransmissionofmitochondrialDNAorreflectmutationsproducedbyDNAreplicationerrors, inefficientDNArepairoroxidativedamage(Kmiec,Woloszynska,& Janska,2006):anincreaseinheteroplasmythereforecouldbeapotentialsignal ofexposureto mutagens.Heteroplasmyhas been exploredasabiomarkerofexposuretoenvironmentalradioactivityin bankvolesfromtheCEZ,whereexposuretoradionuclideseliciteda nonsignificantincreaseinheteroplasmy(Wickliffe,Chesser,Rodgers, & Baker, 2002). Next-generation sequencing (NGS) is well suited fordetectingheteroplasmyasthehighdepth of coverage that can bereadilyachievedwhen(re)sequencingasmallgenome(e.g.,mitochondrialDNA)allowsforrobustdetectionofintra-individualpolymorphisms(Lietal.,2010;Tang&Huang,2010;Wachsmuth,Hübner, Li,Madea,&Stoneking,2016);forexample,ata1,000×coverage,a heteroplasmyoccurringat1%frequencyisexpectedtobevisiblein about10reads,asignalthatshouldbedistinctfromthenumbersof mismatchesderivedfroma~0.1%errorrateassociatedwithIllumina HiSeq2000chemistry (Glenn,2011). Bakeretal.’s(2017) NGSdata presentapowerfulopportunitytoexaminethepotentialassociation betweenexposuretoenvironmentalradionuclidesandheteroplasmy as(i)heteroplasmyiscommoninmuscle(Lietal.,2015),thesource ofbankvolegeneticmaterial,and(ii)theauthorsachievedhighdepth ofcoverage(averagecoverage=3,974,range=64–7,841;tableS1in Bakeretal.(2017))overmostmitochondrialgenomes. WeobtainedBakeretal.’s(2017)NGSdatafromNCBI’ssequence read archive (https://www.ncbi.nlm.nih.gov/sra/, project accession SRX2515630).Only122ofthe131bankvolesamplesdescribedby Bakeretal.(2017)werearchived.Sampleinformationwasnotprovided withtherawreaddata,soweassignedaputativeoriginbymatching thecountofrawreadsineachfiletothereaddatainformationprovidedintableS1byBakeretal.(2017).Potentialadaptorsandpoor qualityreadswereremovedfromtherawdatausingTRIMMOMATIC v.0.35(Bolger,Lohse,&Usadel,2014)(minimumlength=90,quality score=20,slidingwindowsize=5).Paired readswere mappedtoa bank vole mitochondrial genome (GenBank accession NC_024538) using BOWTIE2v.2.2.9 (Langmead & Salzberg, 2012) (mapping options: -D 5 -R 1 -N 0 -L 22 -i S,0,2.50), as the mitochondrial referenceformappingbyBakeretal.(2017)wasnotpublicatthetimeof analysis.MappingdataweresortedandconvertedtoaMPILEUPfile using SAMtools v.1.4 (http://samtools.github.io/hts-specs/SAMv1. pdf).PotentialheteroplasmicsiteswerecalledusingVARSCANv.2.3.9 (Koboldtetal.,2012)onthebasisofaminimumreadfrequencyof1%, butonlywhenaminimumreaddepthof500wasachievedandwhenat least10%ofthereadsmappedtothealternatestrand(toreducenumbersoffalse-positivesitesarisingfromPCRartefacts(Scarcellietal., 2016)).Variablesiteswerecalledbetweenpositions220and15,793of thereferencegenomeduetolowcoverageatthebeginningandendof thereference.Thisanalysisallowedustoquantifyheteroplasmyonthe basisof(i)whetheranindividualcontainedatleastoneheteroplasmy or not and (ii) the total number of heteroplasmic sites foundwithin anindividual’smitochondrialgenome.Assevensampleshadlowsequencingcoverageacrosstheentire mitochondrial genome,we had afinal sample of 115individuals (n = 46 and 69 fromcontaminated anduncontaminatedsites,respectively)foranalysisofheteroplasmyin bankvoles(Table1). Asitisinmanyotheranimals(Kmiecetal.,2006),heteroplasmy appearstobecommoninbankvolesasweidentified72(63%)individualswithatleastoneheteroplasmicsite,representedby28(61%) and44(64%)individualsfromthecontaminatedanduncontaminated sites,respectively(Table1).Mostindividualswithheteroplasmycontainedonly1or2heteroplasmicsites(1site=60%;2sites=22%; ≥3sites=18%ofdata).Theaveragenumberofheteroplasmicsites perindividualwas0.91inthecontaminatedsamplesand1.02inthe uncontaminatedsamples(or1.64withthewholedatasetofuncontaminatedsamples,seebelowfordiscussionaboutoutlierindividuals).Additionally,theaveragefrequencyofheteroplasmywithinthe variablesiteswassimilarincontaminatedanduncontaminatedareas (Table1).Heteroplasmiesweredetectedinbankvolemitochondria at131positions(at90positionswithoutthetwooutlierindividuals). Twobankvolescontainedapparentlymanyheteroplasmicsites,both | 5 Kesäniemi et al ofwhichweretakenfromcontrolareasin1998:oneindividualfrom Nezamozhnyawith26sitesandoneindividualfromOranoewith19 sites.Readmappingforthesetwoindividualswasvisuallyinspected inTABLETv.1.14.11.07 (Milne etal., 2009). The potential heteroplasmicsiteswerescatteredaroundthemitochondrialgenomeand representedbypairsofreadsthathaddifferentinsertsizes,implying thattheheteroplasmydetectionwasnotsimplyanartefactofPCR bias. Nonetheless, statistical analyses ofvariation in heteroplasmy with radionuclide contamination were made with andwithout the two“outlier”samples(notethatbotharefromuncontaminatedsites in1998).Weestimatedwhetherlevelsofheteroplasmydifferedbetweencontaminatedanduncontaminatedsitesusingthegeneralized linear mixed model (GLMM) implemented by the GLMER function inLME4(Bates,Mächler,Bolker,&Walker,2015)runinRv.3.1.1(R DevelopmentCoreTeam,2014).Modelsexaminingwhetheranindividualcontainedaheteroplasmy(Hp)ornot(ProportionofHpindividuals)weretreatedwithabinomialerrordistribution,whilemodels usingthenumberofheteroplasmiespresentwithineachindividual (Hpsites/individual)usedaPoissondistribution.Contamination(yes, no)andyear(1998,2011)wererepresentedasfixedfactors,andthe fivesamplesites(Figure1)wereincludedasarandomfactor(Seefull resultsinTableS1).Withalldata(n = 115),theproportionofindividualswithaheteroplasmywaslowerinthecontaminatedsitesand alsoin2011,althoughneithereffectwassignificant(p = .63and.31, respectively;Table2).Thenumbersofheteroplasmiesinindividuals weresignificantlylowerincontaminatedsitesandin2011(p < .001 for both predictors; Table2).The qualitative pattern of lower heteroplasmyinthecontaminatedareasandapossibletemporalreductionin heteroplasmy(between1998 and 2011)remainswhen the twooutlierindividualsareremoved,butwithnosignificantpredictor for either measure (presence/absence or count) of heteroplasmy (Table2).Hence,analysisofBakeretal.’s(2017)NGSdatayieldno evidence that exposure to environmental radiation is associated withthelevelofheteroplasmy.Qualitatively,bankvolesinhabiting theCEZhavelowerlevelsofheteroplasmyandexhibitadecreasein thelevelofheteroplasmybetween1998and2011intheRedForest (Tables1 and 2). Neither of these spatial nor temporal patterns is anexpectedconsequenceofasimple,positiveassociationbetween chronic exposure to environmental radionuclides and the rate of mutation. TABLE1 Heteroplasmy(Hp)estimatesineachofthesamplesseparately Locality Year NBNHp Proportion of Hp individuals Hp sites/individual Average frequency of heteroplasmies Uncontaminated Nedanchychy 1998 11 11 0.727 1.182 0.134 Nedanchychy 2011 12 12 0.750 1.250 0.076 Nezamozhnya 1998 12 11(12) 0.636(0.667) 1.000(3.083) 0.031 Nezamozhnya 2011 12 10 0.500 0.900 0.110 Oranoe 1998 15 12(13) 0.583(0.615) 0.833(2.231) 0.083 Oranoe 2011 14 11 0.545 0.909 0.134 In total 76 67(69) 0.627(0.638) 1.015(1.638) 0.078 Contaminated GlybokeLake 1998 15 14 0.571 0.929 0.032 GlybokeLake 2011 3 3 0.667 1.000 0.070 RedForest 1998 20 18 0.722 1.167 0.055 RedForest 2011 17 11 0.455 0.455 0.062 In total 55 46 0.609 0.913 0.050 Fortheaveragefrequencyofallheteroplasmies,frequencyoftheheteroplasmicallelewascalculatedseparatelyforeachheteroplasmysitewithinindividuals.Forthetwosampleswith“outlier”individuals,NezamozhnyaandOranoe(both1998),valuesinparenthesesaretheestimatesincludingtheoutliers. NBrepresenttheoriginalsamplesizesinBakeretal.(2017),whileNHpistheamountofindividualsusedinthepresentheteroplasmyanalysis. TABLE2 Summarizedresultsofgeneralizedlinearmixedmodels (GLMM)testingtheeffectsofcontaminationandsamplingyearon thelevelsofheteroplasmy(Hp)ofbankvolesfromcontaminatedand uncontaminatedsites n = 115 n = 113 ProportionofHpindividuals Effect Estimate pEstimate p Intercept 0.765 .018 0.697 .033 Contaminatedsite −0.196 .625 −0.145 .719 Year2011 −0.406 .308 −0.353 .377 Hpsites/individual Effect Estimate pEstimate p Intercept 0.777 <.001 0.094 .534 Contaminatedsite −0.697 <.001 −0.137 .493 Year2011 −0.727 <.001 −0.167 .401 GLMMwasrunwithallavailableindividuals(n=115)andareduceddata setwiththetwooutlierindividualsfromuncontaminatedsitesremoved (n=113). 6 | KESÄNIEMI et al. 6 | DISCUSSION AND CONCLUSIONS Understanding the biological effects of exposure to low-dose radiationisanimportantissuegiventhatnumeroushumanactivities haveleftsubstantialamountsofradionuclidesintheenvironment (Lourenoetal.,2016):reportsofacceleratedmutationratehave clearpolicyimplications.Whileahighrateofmutationischaracteristic of diverse taxa affected by Chernobyl fallout (Geras’kin, Fesenko,&Alexakhin,2008),thespecificresponsestoradionuclide exposurevarybetweentaxa(Møller&Mousseau,2015)andmammals are comparatively understudied. Application of NGS techniquesrepresentsamuchneededscientificadvanceforstudiesof wildlifeinhabitingtheCEZ.However,sequencedataforwholemitochondrialgenomes(fromBakeretal.,2017)arealsoconsistentwith theresultsofpreviousstudiesofbankvolemitochondrialdiversity atthecontrolregionwiththeresultsbeingexplainedbyprocesses otherthanmutation(e.g.,Matsonetal.,2000;Meeksetal.,2007, 2009;Wickliffeetal.,2006).Analysisofheteroplasmyinbankvoles offers high power to detect low-frequency intra-individual mutationsandcancircumventtheuncertaintyassociatedwithinferring mutation from populations whose demographic histories are unknown.Alackofassociationbetweenheteroplasmyandcontaminationbyenvironmentalradionuclidesisimportantasoccurrence oflow-frequency,intra-individualmutationsispresumablyneeded togeneratethe“rawmaterial”formutationsthatarelatervisibleas “populationgeneticdiversity.”Arecentmeta-analysishasrevealed anassociationbetweenmutationrateandenvironmentalradiation exposure in many species from Chernobyl (Møller & Mousseau, 2015). However, given our discussion about sampling, bank vole populationhistoryandheteroplasmy,wesuggestthatinadditionto thereportofhighmitochondrialdiversityinsamplesofbankvoles inhabitingtheCEZ,furtherstudiesareneededtodemonstratean acceleratedmutationrateinthisspecies. ACKNOWLEDGEMENTS PCWandTMarefundedbyAcademy of Finland (project numbers PCW:287153 and TM:268670) and TJ has received a grant from KuopioNaturalists’Society.TheauthorswishtoacknowledgeCSC– ITCenterforScience,Finland,forcomputationalresources. ORCID Jenni Kesäniemi http://orcid.org/0000-0001-8328-558X Anton Lavrinienko http://orcid.org/0000-0002-9524-8054 Tapio Mappes http://orcid.org/0000-0002-5936-7355 Phillip C. Watts http://orcid.org/0000-0001-7755-187X REFERENCES Baker, R. J., Dickins, B., Wickliffe, J. K., Khan, F. A. A., Gaschak, S., Makova, K. D., & Phillips, C. D. (2017). Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent. 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