48CorrespondenceAnalyticalflawsinacontinenta

Correspondence:Analyticalflawsinacontinental-scaleforestsoilmicrobialdiversitystudyLehoTedersoo1NatureCommunications8:15572doi:10.1038/ncomms15572(2017);Published6Jun2017Zhouetal.1recentlyaddressedcontinental-scalebiogeographicpatternsofsoilmicroorganisms.TheauthorssuggestedthatthemeanannualtemperatureratherthanotherenvironmentalpredictorsdrivethelatitudinalgradientofdiversityinsoilbacteriaandfungisampledfromsixpermanentstudyareasacrossNorthAmerica.HereIarguethattheirapproachisflawedbecauseofpseudoreplicateddesign,incorrectchoiceofstatisticaltoolsandoveroptimisticinterpretation.Fromeachofthesixsites,theauthorscollected21compositesoilcores(located1–400mapart)thatweretreatedasindependentsamplingunitstoinfercontinental-scalepatterns.Theauthorsthenusedsimpleregressionanalysestoaddressthedifferencesamongsamplesinsteadofnestingsampleswithinsitesorusingsitesascategoricalpredictors,thusoverestimatingtheresidualdegreesoffreedomby31-fold.Further,theauthorscalculatedChao1minimumrichnessestimatesforeachsiteasproxiesofdiversitytotesttheresponseofrichnesstoenvironmentalvariables.However,theminimumrichnessestimatesarebasedonrelativeabundanceoftheraretaxa2.Mostofthis‘rarebiosphere’inmetabarcodingstudies(suchasZhouetal.1)mayrepresentunfilteredanalyticalartefacts3,4andmagnifythedifferencesinrichnessamongtaxon-poorandtaxon-richsites4.AfterintegratingrichnessintoChao1estimates,theauthorsconductedsimpleandmultipleregressionanalysesformanyexplanatoryvariables,determiningthatinverselytransformedtemperaturerepresentsthebestlinearpredictorformicrobialrichness.Whilethisholdstrueforthecurrentverylimitedsamplingonacontinentalscale,inclusionofsoilsfrombroaderrangeofpHandprecipitationarelikelytoalterthispattern.Previousresearchhasindicatedthatonglobalandcontinentalscales,soilpHandprecipitationconstitutethestrongestpredictorsofrichnessandcompositionofterrestrialmicroorgan-isms5–10.Severalofthesestudieshavealsoidentifiedtemperatureasoneofthekeypredictors.Furthermore,twoglobalstudiespreviouslyreportedthattemperatureisthestrongestpredictorofbacterial11andfungal12richness.Zhouetal.1neglectedtocitethesestudies,andthusover-emphasizedthenoveltyofconnectingtemperaturetothelatitudinalgradientofdiversityinmicrobes.Ithasbeenwellestablishedthattruereplicationisacornerstoneofexperimentaldesigninecology13andmicrobiology4,14tosecurecorrectdataanalysisandinterpretation.Properreplicationaswellasspatiallyindependentandrepresentativesamplesisrequiredtoinferbothsmall-scaleandlarge-scalebiogeographicpatternsandprocesses.Samplesthatserveasgoodreplicatesonafinescalemaybe,however,unsuitedforthispurposeoverlargegeographicscaleswithoutintroducingnesteddesignoraccountingforspatialautocorrelation.Therefore,Iurgeresearcherstocarefullyconsiderreplicationonarelevantgeographicscaleinthedisciplineofbiogeography.References1.Zhou,J.etal.Temperaturemediatescontinental-scalediversityofmicrobesinforestsoils.Nat.Commun.7,12083(2016).2.Gotelli,N.&Chao,A.inEncyclopediaofBiodiversity(ed.Levin,S.A.)195-210(Elsevier,2013).3.Lynch,M.D.&Neufeld,J.D.Ecologyandexplorationoftherarebiosphere.Nat.Rev.Microbiol.13,217–229(2015).4.Ba´lint,M.etal.Millionsofreads,thousandsoftaxa:microbialcommunitystructureandassociationsanalyzedviamarkergenes.FEMSMicrobiol.Rev.40,686–700(2016).5.Bates,S.T.etal.Globalbiogeographyofhighlydiverseprotistancommunitiesinsoil.ISMEJ.7,652–659(2013).6.Lauber,C.,Hamady,M.,Knight,R.&Fierer,N.Pyrosequencing-basedassessmentofsoilpHasapredictorofsoilbacterialcommunitystructureatthecontinentalscale.Appl.Environ.Microbiol.75,5111–5120(2009).7.Maestre,F.T.etal.Increasingaridityreducessoilmicrobialdiversityandabundanceinglobaldrylands.Proc.NatlAcad.Sci.USA112,15684–15689(2015).8.Po˜lme,S.etal.Biogeographyofectomycorrhizalfungiassociatedwithalders(Alnusspp.)inrelationtobioticandabioticvariablesattheglobalscale.NewPhytol.198,1239–1249(2013).9.Talbot,J.M.etal.EndemismandfunctionalconvergenceacrosstheNorthAmericanmycobiome.Proc.NatlAcad.Sci.USA111,6341–6346(2014).10.Tedersoo,L.etal.Globaldiversityandgeographyofsoilfungi.Science346,1078(2014).11.Prober,S.M.Plantdiversitypredictsbetabutnotalphadiversityofsoilmicrobesacrossgrasslandsworldwide.Ecol.Lett.18,85–95(2015).12.Tedersoo,L.etal.Towardsglobalpatternsinthediversityandcommunitystructureofectomycorrhizalfungi.Mol.Ecol.21,4160–4170(2012).13.Hurlbert,S.H.Pseudoreplicationandthedesignofecologicalfieldexperiments.Ecol.Monogr.54,187–211(1984).14.Prosser,J.I.Replicateorlie.Environ.Microbiol.12,1806–1810(2010).DOI:10.1038/ncomms15572OPEN1NaturalHistoryMuseum,UniversityofTartu,14aRavila,50411Tartu,Estonia.CorrespondenceandrequestsformaterialsshouldbeaddressedtoL.T.(email:[e-mail]).NATURECOMMUNICATIONS|8:15572|DOI:10.1038/ncomms15572|:Theauthorsdeclarenocompetingfinancialinterests.Reprintsandpermissioninformationisavailableonlineat:Tedersoo,L.Analytic