Geobiology. 2019;1–14. wileyonlinelibrary.com/journal/gbi  | 1© 2019 John Wiley & Sons Ltd

Received:24August2018  |  Revised:24October2018  |  Accepted:27November2018DOI:10.1111/gbi.12329

O R I G I N A L A R T I C L E

Absence of biomarker evidence for early eukaryotic life from the Mesoproterozoic Roper Group: Searching across a marine redox gradient in mid- Proterozoic habitability

Kevin Nguyen1 | Gordon D. Love1  | J. Alex Zumberge1 | Amy E. Kelly2 |  Jeremy D. Owens3 | Megan K. Rohrssen4 | Steven M. Bates1 | Chunfang Cai5 |  Timothy W. Lyons1

1DepartmentofEarthSciences,UniversityofCalifornia,Riverside,California2ShellInternationalExplorationandProduction,Houston,Texas3DepartmentofEarth,Ocean&AtmosphericSciences,FloridaStateUniversity,Tallahassee,Florida4DepartmentofEarth&AtmosphericSciences,CentralMichiganUniversity,MountPleasant,Michigan5InstituteofGeologyandGeophysics,ChineseAcademyofSciences,Beijing,China

CorrespondenceGordonD.LoveandTimothyW.Lyons,DepartmentofEarthSciences,UniversityofCalifornia,Riverside,CA.Emails:glove@ucr.edu(GDL)andtimothyl@ucr.edu(TWL)

Funding informationDirectorateforGeosciences;NASAAstrobiologyInstitute,Grant/AwardNumber:NNA15BB03A

AbstractByabout2.0billionyearsago(Ga),thereisevidenceforaperiodbestknownforitsextended,apparentgeochemicalstabilityexpressedfamouslyinthecarbonate–car-bonisotopedata.Despitethefirstappearanceandearlyinnovationamongeukary-otic organisms, this period is also known for a rarity of eukaryotic fossils and anabsenceoforganicbiomarkerfingerprintsforthoseorganisms,suggestinglowdiver-sity and relatively small populations compared to the Neoproterozoic era.Nevertheless, the search for diagnostic biomarkers has not been performedwithguidance from paleoenvironmental redox constrains from inorganic geochemistrythat should reveal the facies thatweremost likelyhospitable to theseorganisms.Siltstonesandshalesobtainedfromdrillcoreoftheca.1.3–1.4GaRoperGroupfromtheMcArthurBasinofnorthernAustraliaprovideoneofourbestwindowsintothemid-Proterozoicredoxlandscape.Thegroupiswelldatedandminimallymetamor-phosed(ofoil windowmaturity),andpreviousgeochemicaldatasuggestarelativelystrongconnection to theopenoceancompared toothermid-Proterozoic records.Here, we present one of the first integrated investigations of Mesoproterozoic biomarkerrecordsperformedinparallelwithestablishedinorganicredoxproxyindi-cators.ResultsrevealatemporallyvariablepaleoredoxstructurethroughtheVelkerriFormation as gauged from ironmineral speciation and trace-metal geochemistry,vacillating between oxic and anoxic.Our combined lipid biomarker and inorganicgeochemicalrecordsindicateatleastepisodiceuxinicconditionssustainedpredomi-nantlybelowthephoticzoneduringthedepositionoforganic-richshalesfoundinthemiddleVelkerriFormation.Themoststrikingresultisanabsenceofeukaryoticsteranes(4-desmethylsteranes)andonlytracesofgammaceraneinsomesamples—despiteoursearchacrossoxic,aswellasanoxic,faciesthatshouldfavoreukaryotichabitabilityand in lowmaturityrocksthatallowthepreservationofbiomarkeral-kanes.ThedearthofMesoproterozoiceukaryoticsteranebiomarkers,evenwithinthemoreoxicfacies,issomewhatsurprisingbutsuggeststhatcontrolssuchasthelong-termnutrientbalanceandotherenvironmentalfactorsmayhavethrottledtheabundancesanddiversityofearlyeukaryoticliferelativetobacteriawithinmarine

www.wileyonlinelibrary.com/journal/gbihttps://orcid.org/0000-0002-6516-014Xmailto:glove@ucr.edumailto:timothyl@ucr.edumailto:timothyl@ucr.edu

2  |     NGUYEN Et al.

1  | INTRODUC TION

The mid-Proterozoic (2.0–1.0Ga) is well known for its long-livedgeochemicalstasis,showingonlysubtlevariationincarbonateδ13Cspanningthis interval (Brasier&Lindsay,1998).Bytheendofthistime interval, themajor clades of crown eukaryotes had diverged(Parfrey, Lahr, Knoll, & Katz, 2011), but their evolutionary devel-opmentwasapparentlystifledsothatdiversitypatternsandabun-danceswerepersistentlylowcomparedtothelaterNeoproterozoic(Knoll, Javaux, Hewitt, & Cohen, 2006). Pastwork has suggestedthat low-oxygencontentsandrelated limitedavailabilityofcriticalnutrientsintheoceanmayhavecontributedtotheapparentbiogeo-chemicalandpaleobiologicalstasis (Derry,2015;Laakso&Schrag,2017; Lyons, Reinhard, & Planavsky, 2014; Reinhard etal., 2013,2016).Despite important innovationsduring this interval, this ap-parentmonotonyrelativetotheeventsthatfollowedhasledtothecommonreferraloftheperiod2.0and1.0Gaasthe“dullesttimeinEarth’shistory”(Brasier&Lindsay,1998;Buick,DesMarais,&Knoll,1995).

Ourcurrentunderstandingof the redoxchemistryof themid-Proterozoicoceanisthatthesurfacewatersweremostlikelyoxygen-ated,whilethedeeperlow-oxygenwatersweremarkedbysulfidic(euxinic)zonesatintermediatedepths,particularlyalongthehighlyproductiveoceanmargins (Canfield,1998). Iron-richanoxic (ferru-ginous)conditionsdominatedatgreaterdepth (Lyons,Reinhard,&Scott,2009;Planavskyetal.,2011;Poulton&Canfield,2011).Theredoxstructureoftheoceanatthattimemayhaveinfluencedthepatterns and rates of evolution bymodulating the spatiotemporaldistributionofessentialmacro-andmicronutrients,aswellasoverallavailabilityofoxygen(Anbar&Knoll,2002;Derry,2015;Laakso&Schrag,2017;Lyonsetal.,2014;Reinhardetal.,2013,2016).Ithasbeen suggested that biological feedbacks, including sulfide-basedanoxygenic photosynthesis (Johnston, Wolfe-Simon, Pearson, &Knoll, 2009),may have helpedmaintain aworldwith low-oxygenlevels and sulfide pervasive enough to limit the development andproliferationofearlycomplexlife.However,variousfeedbacksmakethepersistenceofwidespreadanoxia, particularly euxinia, equallychallenging.Specifically,nutrientlimitationscoupledtowidespreadanoxiaandeuxinia(e.g.,lossoffixednitrogenthroughdenitrificationandperhaps limitedavailabilityoftrace-metalmicronutrientssuchasmolybdenum) can result in reduced availability of organicmat-terrequiredtosustainanoxiaviaaerobicrespirationandtosupport

sulfide production viamicrobial sulfate reduction (Anbar & Knoll,2002;Scottetal.,2008).Theapparentbalanceamongthesefeed-backsthatkeptthemid-Proterozoicocean“dull”remainsatopicripeforfurtherstudy.

Itiswidelyacceptedthatthechemistryoftheearlyoceanandlifeco-evolved.However,thetruegeochemicalnatureofEarth’s“boringbillion”remainspoorlyconstrained.TherearemanylinesofevidencethatdocumentanincreaseinEarth’ssurfaceoxidationstateapproxi-mately2.4–2.3Ga(Bekkeretal.,2004;Luoetal.,2016),andanotherbiospheric oxygenation estimated between 800 and 550Ma (re-viewedinOch&Shields-Zhou,2012)thatwascoincidentwithaglobalexpansionofeukaryoticabundanceinthemarinerealm(Brocksetal.,2017;Issonetal.,2018).Conditionsintheinterveningmid-Proterozoicarelessstudiedandcorrespondinglylessknown,withthelikelihoodofintermediateredoxstatesintheoceanandatmosphere.Manyofthemost-citeddatahavecomefromtheMcArthurBasinofnorthernAustralia.Specifically,shalesoftheRoperGroupinthisregionhaveemergedasoneofourbestarchivesofmid-Proterozoicoceanredoxbecausethegroupiswelldated,minimallymetamorphosed,andlieinthemiddleofthiskeyinterval(Jackson,Sweet,&Powell,1988).Mostimportantly, sedimentological andgeochemical data suggest a rela-tivelystrongconnectiontotheopenoceaninmainlyanoutershelfsetting(Abbott&Sweet,2000;Jackson,Powell,Summons,&Sweet,1986;Jackson&Raiswell,1991;Jacksonetal.,1988).Asaresult,con-ditionsintheRoperGroupmayreflecttheredoxstateoftheglobaloceanandspecificallytheconditionsthatfavored,orchallenged,thedevelopmentandexpansionofcomplexlifeduringthisinterval.Giventhispotential,wehereinreportonthefirsthigh-resolution,fullyinte-gratedgeochemicalstudyoftheRoperGroup—withthegoalofper-formingorganicbiomarkerworkwithinatightinorganicgeochemicalcontextthatdefinesagradientinhabitabilityandthuspotentialeu-karyoticinhabitation.Ourresultssuggestthatcontrolsotherthanjustlocalredoxmodulatedthedistributionofsteroid-producingeukary-otesinthemid-Proterozoicocean.

1.1 | Sampling location

Much of the attention drawn to theMcArthur Basinwas initiallythrough the discovery of “live oil”, originally reported by Jacksonetal. (1986). Their findings garnered significant interest in thearea,andsubsequent investigationshavebeendesigned todefineits depositional environment. Concisely, the Roper Group within

microbial communities. Given that molecular clocks predict that sterol synthesisevolvedearly in eukaryotichistory, and (bacterial) fossil steroidshavebeen foundpreviouslyin1.64Garocks,thenaverylowenvironmentalabundanceofeukaryotesrelativetobacteriaisourpreferredexplanationforthelackofregularsteranesandonlytracesofgammaceraneinafewsamples.ItisalsopossiblethatearlyeukaryotesadaptedtoMesoproterozoicmarineenvironmentsdidnotmakeabundantsteroidli-pidsortetrahymanolintheircellmembranes.

     |  3NGUYEN Et al.

theMcArthur Basin is located at the tip of northern Territory ofAustralia,neartheGulfofCarpentaria,andoccursoveranareaofat least 145,000km2with amaximum thickness of approximately5,000m(Abbott&Sweet,2000).ThesamplesinthisstudyarefromtheVelkerriFormationsampledintheAltree-2drillcore;theVelkerrihasaknownextentofatleast80,000km2(Jackson&Raiswell,1991)and iswithin the oilwindow, as reflected by its thermalmaturity(Crick,1992;Summons,Taylor,&Boreham,1994).Jackson,Sweet,Page, and Bradshaw (1999) reported a U-Pb age of 1,492±4Mafrom the lowermost member of the Roper Group, the MainoruFormation,implyingyoungeragesforthedepositionoftheVelkerriFormation.Re-Osageconstraintsyieldedageestimatesforthebase(1,417±29Ma) and top (1,361±21Ma) of theVelkerri Formation(Kendall,Creaser,Gordon,&Anbar,2009),althoughdetritalzirconU-PbdatessuggestthattheVelkerriFormationwasdepositedbe-tween 1,349 and 1,320Ma (Yang etal., 2017). The Altree-2 drillcorewassampledoveradepth intervalof440–1,250m,coveringthe full thickness of the Velkerri Formation. The formation is di-vided into three informal subunits—the upper (392–672m), mid-dle (672–948m), and lower Velkerri (948–1,242m). The VelkerriFormationstratawerelikelydepositedinadistalmarineshelfsetting(Abbott&Sweet,2000).Rock-Evalpyrolysisdata(HydrogenIndex,Table1)independentlyconfirmedthatthesecoresamplesgenerallyspan a thermalmaturity range spanning from early oilwindow tojustbeyondpeakoilwindowandthusaresuitablefordetailedor-ganicgeochemicalinvestigationsincetheserocksarethermallywellpreserved.

2  | METHODS

2.1 | Inorganic geochemistry

Samplingspecificallytargeteddrillcoreshalestoexplorethedegreeofoxygenationwithinthelocalwatercolumnandtoprovideaddedperspectiveonoxygenintheglobalocean.Totalcarbon(TC),totalinorganic carbon (TIC), and total sulfur (TS)weremeasuredby in-fraredabsorptionofCO2/SO2usinganEltraCS-500equippedwithafurnaceandacidificationmodule.TCandTSweredeterminedviacombustion at1,400°C,whileTICwas liberated via reactionwith4MHCl.Totalorganiccarbon(TOC)wascalculatedbydifference.

SequentialextractionofreactiveFephaseswascarriedoutusingthepreviouslydescribedmethod(Poulton&Canfield,2005)andan-alyzedonanAgilent7500cequadrupoleinductivelycoupledplasmamassspectrometer(ICP-MS).ReproducibilityofFespeciationmea-surements, based on replicate analyseswithin and between sam-plebatches,wasbetterthan93%.Pyriteiron(FePy)wasquantifiedusingchromouschloridedistillationfollowedbytitrationwithiodineasoutlined inRef. (Canfield,Raiswell,Westrich,Reaves,&Berner,1986).Apyritestandardwas included ineverysamplebatch. Ironrelationshipswerealsoassessedusing theconventionaldegreeofpyritizationtechnique(DOP;Raiswell,Buckley,Berner,&Anderson,1988), whereby residual (unpyritized) “reactive” Fe is extractedusingaboiling,12MHClstep.ThisDOPtechniquewill,ifanything,

overestimatethetrulyreactivephases.Assuch,falsepositivesforeuxiniaareunlikely,andhighDOPvaluesaretakenasaparticularlyrobust indicatorofthatcondition intheancientwatercolumn(re-viewedLyons&Severmann,2006).Onceextracted,theamountofironwasquantifiedspectrophotometricallyusingthestandardfer-rozinemethod. Replicate analysis yielded a standard deviation of0.25wt.%.

PyritesulfurinthesampleswasalsoextractedusingtheCrre-ductionmethodforsulfur isotopeanalysisviaasilvernitratetrap,ratherthanthezincacetatesolutionusedforthepyrite-Squantifica-tionbyiodometrictitration.Theresultingsilversulfidewashomoge-nized,weighedwithexcessV2O5,andcombustedonlineforanalysesof34S/32SratiosusingaThermoDeltaVmassspectrometer,whicharereportedusingthedeltanotationaspermil(‰)deviationsfromtheViennaCanyonDiabloTroilite(VCDT)standard.Reproducibilitywasbetterthan0.14‰basedonsingle-runandlong-termstandardmonitoring(additionalanalyticaldetailsfortheDOPandδ34Sdeter-minationsareprovidedintheSupportingInformation).

Amulti-aciddigestiontechniquewasemployedfortrace-metalanalysis. Samples were ashed at 900°C for 24hr, and mass lostthroughignitionwasrecordedpriortoundergoingaciddigestion(HF,HNO3,andHCl).AnalyseswereperformedonsameICP-MSwiththeDevonianOhioShale(SDO-1)asareference,withreproducibilityinindividualrunsofbetterthan95%forthepresentedelements.

2.2 | Organic geochemistry

2.2.1 | Extraction of free (extractable) biomarker lipids

Fororganicbiomarkeranalysis,coresamplesweretrimmedwithawater-cooled rock saw to removeouterweathered surfaces (typi-cally5–10mmthicknesswasremoved)toexposeasolidinnerpor-tion, which was fragmented. The resulting chips from this innerportionweresolvent-rinsedpriortopowderinginazirconiaceramicpuckmillwithinaSPEX8515shatterbox.Lipidbiomarkerswereex-tractedat100°Cin9:1dichloromethane/methanol(v/v)mixturefor15minsinaCEMMicrowaveAcceleratedReactionSystem(MARS).Wholerockbitumenswereseparatedintosaturates,aromatics,andpolar (N, S, O compounds) fractions by silica column chromatog-raphy. Typically 10–20g of rock powderwas used for extraction,yielding from 0.3 to 7.5mg of bitumen per gram of rock powder(mean=3.5mg per gram, Supporting Information Table S5). Fullprocedural blanks with pre-combusted quartz sand (850°C) wereperformed inparallelwitheachbatchof rocks toensure that anybackgroundhydrocarboncompoundswerenegligibleincomparisonwithbiomarkerabundances.

2.2.2 | Catalytic hydropyrolysis (HyPy) of pre- extracted rocks/kerogens

Pre-extracted rock residues, containing predominantly kerogen,weresubjectedtocontinuous-flowcatalytichydropyrolysis (HyPy)

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TABLE 1 SelectedgeochemicaldataandlipidbiomarkerratiosforAltree-2rocksusedfororganicgeochemicalinvestigation

Dep

th (m

)TO

C (w

t.%)

HI

S (w

t.%)

MPI

- 1%

RcTs

/(T

s + T

m)

C 29α

β/C 3

0αβ

C 30H

βα

/αβ

C 31α

βS/

(S +

R)

2αM

eH

(%)

G/H

C 27 S

t/C 3

0αβ

C 21 +

C22

St

/C 3

0αβ

Tota

l st

eran

es

(ppm

sat

s)

Tota

l ho

pane

s (p

pm s

ats)

452.

14–4

52.2

21.

5–

1.23

0.45

0.67

0.59

0.98

0.06

0.57

5.9

0.04

0.00

0.00

     |  5NGUYEN Et al.

tocleavecovalentbondsandfragmentthekerogenmatrixintosolu-bleproducts(Love,Snape,Carr,&Houghton,1995;Loveetal.,2009)for subsequent GC-MS and MRM-GC-MS analysis. The parallelanalysesofcovalentlyboundbiomarkersandotherboundmolecularconstituents,alongsidetheanalysisofrockbitumens,giveusconfi-dencethatwehavecorrectlyidentifiedsyngeneticcompoundsandallowsustoscreenforanysignificantcontaminationcontributionsinanimportantself-consistencycheck(Loveetal.,2009).Extractedsamplepowders(~1.0–1.8g)weresecuredintheHyPyreactortubeatopa steelwoolplug,whichwasSoxhlet-extracted (DCM,48hr)andbaked(450°C,2hr)toensurethatnocontaminateswereintro-ducedtothesample.FullproceduralHyPyblankswereconductedusingcleansilicagelasasubstrate.BlankHyPyrunswereconfirmedtobecleanandfreeofcontamination,asdescribedinFrenchetal.(2015).HyPyisatemperatureprogrammedpyrolysismethodusingtwo temperature ramps: 0–250°C at 100°C/min immediately fol-lowedby250–520°Cat8°C/minwhilemaintainingconstanthydro-genpressureof~15MPawithahydrogen sweepgas flow rateof6dm3/min.Thepyrolysatewascollected inadry ice-cooledprod-ucttrapontosilicagel(35–70mesh,activatedat450°Covernight).AftereachHyPyrun,thesilicagelplugfromthetrapcontainingtheadsorbedpyrolysateproductwasseparatedusingthesamecolumnchromatographicandGC-MSmethodologyasdescribedfortherockbitumens.

2.2.3 | Lipid biomarker analysis and quantification

Gas chromatography–mass spectrometry (GC-MS)was performedon saturated and aromatic hydrocarbon fractions from rock bitu-mensandhydropyrolysatesinfullscanmodeoveramassrangeof

50–600DaonanAgilent5975CinertMassSelectiveDetector(MSD)massspectrometerinterfacedtoanAgilent7890AGCequippedwithaDB-1MScapillarycolumn(60m×0.32mm,0.25μmfilm)andruninsplitlessinjectionmodewithHeascarriergas.ThetemperatureprogramforGC-MSfullscanwas60°C(2min),rampedto150°Cat20°C/min,thento325°Cat2°C/min,andheldat325°Cfor20min.200ngofd14- p-terphenylstandardwasaddedtobetween1.4and12.1mgofaromatichydrocarbonfractionasastandardforquantifi-cation.MPI-1indexvalueswerecalculatedusing178Dapeakareasforphenanthreneand192Dapeakareasformethylphenanthrenes.

Toprobepolycyclicalkanebiomarkerdistributionsindetail,meta-stablereactionmonitoring–gaschromatography–massspectrometry(MRM-GC-MS)was conducted on the saturated hydrocarbon frac-tionsfromrockbitumensandkerogenhydropyrolysateswithaWatersAutoSpecPremiermassspectrometer.ThespectrometerisequippedwithanAgilent7890AgaschromatographandDB-1MScoatedcap-illarycolumn(60m×0.25mm,0.25μmfilmthickness)usingsplitlessinjectionandHeforcarriergas.TheGCtemperatureprogramusedforcompoundseparationconsistedofaninitialholdat60°Cfor2min,heatingto150°Cat10°C/min,followedbyheatingto320°Cat3°C/min,andafinalholdat320°Cfor22min.MRMtransitionsforC27–C35 hopanes,C31–C36methylhopanes,C21–C22andC26–C30steranes,C30 methylsteranes, and C19–C26 tricyclics were monitored. Biomarkercompoundswere identified based on retention time and publishedmassspectraandquantified inMRM-GC-MSbycomparisonwithadeuteratedC29steraneinternalstandard(d4- ααα-24-ethylcholestane(20R);ChironLaboratories,AS)assumingequalresponsefactorsbe-tweensamplecompoundsandtheinternalstandard.Individualyieldsofhopaneandsteranediastereoisomersfoundinfulllaboratorypro-ceduralblanksweretypically0.4reflectanoxicdeposition.FePy/FeHRratiosabove0.8reflecteuxinicdeposition(Poulton&Canfield,2011).FeT/Al ratios below 0.4–0.5typicallyreflectoxicsettings,whilearatioof0.6–1.0likelyindicatesanoxia/euxinia.DashedlinerepresentsaverageoxicmarinevaluesfortheFespeciationdata.%Rc:vitrinitereflectanceequivalent;DOP:degreeofpyritization;FeHR:highlyreactiveiron;FePy:pyriteiron;FeT:totaliron;TIC:totalinorganiccarbon;TOC:totalorganiccarbon;TS:totalsulfur

6  |     NGUYEN Et al.

3  | RESULTS AND DISCUSSION

3.1 | Total organic carbon, inorganic carbon, and total sulfur

Totalorganiccarbon(TOC)content isappreciable inmostofthecoresamples(Figure1),withvaluesreachingashighas8.8wt.%(Supporting Information Table S1). The lower Velkerri ismarkedbytwoepisodeswhereTOCcontenthoversaround3.5wt.%onaverageforafewmetersrelativetoabaselinewithnear-zeroval-ues.ThemiddleVelkerrishowshigherTOCcontentsrangingfromroughly 5.5wt.% at 932m to amaximumof 8.8wt.% at 707m,withageneralup-core increase,beforeoscillatingbetweenverylowandvaluesashighas6.2wt.%intheupperVelkerri.Highor-ganic content observed in the Velkerri Formation is interpretedto reflecthighbut varyingorganic carbonburial against aback-dropofrelativelylowclasticdilution(Warren,George,Hamilton,& Tingate, 1998). TIC content for the majority of the sampleswasconsistentlylowthroughoutthesequence,rangingfrom0to1.2wt.%,withthelatterequatingtoabout10%calciumcarbonate.TSbehavedsimilarlytoTOC,rangingfromvaluesbelowdetectiontoamaximumof9.9wt.%at707minthemiddleVelkerribeforetaperingofftolowconcentrationsrangingfromvaluesbelowde-tection to1wt.% in theupperVelkerri (Figure1andSupportingInformationTableS1).

3.2 | The iron paleoredox proxies and δ34S

Highlyreactiveiron(FeHR)iscomprisedofpyriteiron(FePy)andotheriron-bearingphasesthatarereactivetowardsulfideinthewatercol-umnand/orduringearlydiagenesis—that is, ferric (oxyhydr)oxides(FeOx),magnetite(FeMag),andironassociatedwithcarbonates(FeCarb; Poulton&Canfield,2005).Modernmarinesedimentsdepositedbe-neathoxicwatersexhibitFeHR/FeTratiosthataverageabout0.2andseldomexceed0.38,whichisoftentreatedastheupperlimitforoxicdeposition(reviewedinPoulton&Canfield,2011).Valueswellabovethis threshold implyanoxicdeposition (Raiswell&Canfield,1998);cautionmustbeexercisedwheninterpretingdatathatfallnearthethresholdvalue.Theratiofortotalirontoaluminum,FeT/Al,canalsobeusedasanindicatorforwatercolumnanoxia ifFeenrichmentsare significantly above the average for continental crust (Lyons&Severmann,2006;Taylor&McLennan,1985).IfanoxiaisindicatedbyhighFeHR/FeTandFeT/Alratios,theFePy/FeHRratiocanfurtherdistinguishbetweenanoxiaandeuxinia.Undereuxinic conditions,appreciableFe2+andsolidhighlyreactiveFephasescannotcoexistsimultaneouslywithfreeH2Sinthewatercolumn—withtheresultofnear-completeconversionofFeHRtopyriteandthushighFePy/FeHR ratios.

Values for FeHR/FeT and FeT/Al in the lower Velkerri are con-sistent with oxic deposition (Figure1 and Supporting InformationTables),withratiosfallingbelow0.2andslightlyabove0.4,respec-tively.However,oxicdepositioncanbedifficult todelineateusingtheironproxiesbecauserapidsedimentationcandilutethereactive

Fepool(aswellasredox-sensitivetracemetals)—thatis,theenrich-mentsthatarediagnosticofanoxicconditions—viaextremedetritalinputs(Lyons&Severmann,2006).However,FeHR/FeTvaluesinthemiddleandupperunitsareoftenelevated,and thepersistenceofshales throughout all three subunitmakes it difficult to argue forsignificant temporalchanges in ratesorpatternsof sedimentationasrelated,forexample,tovaryingwaterdepth.Moreover,previouswork has shown that comprehensive dilution of the FeHR enrich-mentsthatarediagnosticofanoxiaseemstorequireextremelyhighratesofsedimentation(Lyons&Severmann,2006).Althoughfalsepositivesforoxicdepositionarepossible,weshouldbeabletorec-ognize theseextremesusingstandardsedimentologicalandstrati-graphiccriteria.Finally,thepresenceofsomehighvaluesforFePy/FeHR in the lower interval, despite lowFeHR/FeT, suggests sulfidicbuildupinporefluidsbeneathoxicbottomwaters,asisobservedinmarine sediments today (Canfield,Raiswell,&Bottrell, 1992).Thehigherobservedorganiccontentsofthesethinintervals,relativetothoseimmediatelyaboveandbelow,wouldhavefavoredintensedi-ageneticpyriteformation.

Awater column transition to euxinia during deposition of themiddleVelkerri is suggestedby simultaneously elevatedFeHR/FeT,FeT/Al,andFePy/FeHR.Thesevaluestaperoffgraduallyupsectionataround850mandbecomepronounceagainat725m,suggestingatransienteuxinicphasefollowedbypersistenteuxinia.Euxiniaisalsoconvincingly indicatedfor thismiddle intervalbyhighDOPvaluesthatapproach1.0nearthetop(Figure1).Thesameupperportionofthemiddleintervalisalsomarkedbyanobviousdecreaseinδ34Svalues. Such isotopically light values can be interpreted to reflecta predominance ofwater column (euxinic) pyrite formation underopen-systemconditions (Lyons,1997).The remainingvaluesshowthe34Senrichmentsthataretypicalofthemid-Proterozoic,whichareoftenattributedtogenerallylowsulfateavailabilityintheoceanatthistime(Canfield,Farquhar,&Zerkle,2010;Kah,Lyons,&Frank,2004;Shen,Knoll,&Walter,2003).Theheaviestvalue,+25.8‰,oc-curs in theupperVelkerriandcloselymatchesapastestimate fortheisotopiccompositionofcoevalseawatersulfate(Muir,Donnelly,Wilkins,&Armstrong,1985).TowardtheupperVelkerri,valuesforFeT/Alremainelevated,whilethoseofFeHR/FeToscillate—suggest-ingferruginousandoxicconditions.FerruginousratherthaneuxinicconditionsareconfirmedbyintermediateFePy/FeHRratios.Overall,theironproxiessuggestatexturedpaleoredoxstructureforVelkerriFormation—fromoxicgradingtowardeuxinic inthelowerVelkerri,thentransientlyeuxinictopersistentlyeuxinicinthemiddleVelkerri,andfinallyoscillatingbetweenferruginousandoxicconditionsintheupperVelkerri.

3.3 | Trace- metal proxies

Molybdenum(Mo),uranium(U),andvanadium(V)concentrations(Figure2andTablesinSupportingInformation)areatcrustallev-els in the lowerVelkerribut reachamaximaof105ppmforMoand21ppmforUinthemiddleVelkerri.SimilarvaluesforMoandUwerepreviouslyobservedfromthesameunitsbyKendalletal.

     |  7NGUYEN Et al.

(2009) and Partin etal. (2013), respectively.Mo, U, and V con-centrations return to crustal levels in theupperVelkerri. Strongenrichment inMoconvincinglyarguesforeuxinicdepositionbe-causeMoburial is favoredunderreducingconditionsmarkedbyappreciableH2Sinthewatercolumn,withthelowerenrichmentsbeingcharacteristicofsulfidethatisconfinedtotheporewaters(Scott&Lyons,2012).Molybdenumenrichmentcanalsotracktheavailabilityoforganiccarbon ineuxinic systems (Algeo&Lyons,2006),butweobservethesamestratigraphictrendforMo/TOC(Figure2)—suggesting thatwe are notmissing a euxinic intervalbecauseoflowTOC.UraniumandVontheotherhandrequirelessreducingconditionsandcanbeenrichedinsedimentsmarkedbyanoxicbutnotnecessarilyeuxinicconditions(Tribovillard,Algeo,Lyons,&Riboulleau,2006; seeSupporting Information for a re-lateddiscussionofVgeochemistryandPartinetal.,2013,forad-ditionalbackgroundonU).Pronouncedenrichmentsforallthreemetals, combinedwith the Fe proxy data, point convincingly todominantlyeuxinicconditionsinthemiddleVelkerri.Thehigh-endconcentrationsforMoandU inthis intervalareamongthe larg-estknownforthemid-Proterozoic.MagnitudesoflocalMoandUenrichmentineuxinicsedimentsarecontrolledinpartbythespa-tialextentofeuxiniaandanoxiaintheglobalocean(Partinetal.,2013;Reinhardetal.,2013;Scottetal.,2008).Assuch,theserela-tivelyhighMoandUvaluesmaymarkatransientoxidationeventin the ocean–atmosphere system during the mid-Proterozoic.Importantly, other recent, independent studies of Velkerri tracemetal and U isotope data have suggested transient, perhapsglobal-scalemarineoxygenation for this timeperiod (Mukherjee&Large,2016;Yangetal.,2017).

ConcentrationsforMo,U,andVaboveandbelowthemiddlein-tervalareatnear-crustallevels.Despiteevidenceforintermittently

ferruginouswatersintheupperpart,FeHR/FeT ratios are generally lowinbothintervals.Collectively,thetrace-metalandFespeciationresults imply at least moderate levels of marine oxygenation lo-cally,andperhapsglobally,throughouttheVelkerritimeofthemid-Proterozoic.Consequently,thesesamples,forbothlocalandglobalreasons, are particularly appropriate for our organic geochemicalsearchforeukaryotes.

Consistent with our integrated, conceptual paleoredox modelfortheVelkerriFormation,manganese(Mn)concentrationbehavesantithetically toMo,U,andV,exhibiting lowvalues in theeuxinicintervals and high values when the prevailing redox was domi-nantlyoxic (lowerVelkerri)orvacillatingbetween ferruginousandoxic conditions (upper Velkerri). These Mn enrichments suggestgreaterMn-oxideburial,implyingappreciableoxygenperiodicallyinthewatercolumn(Calvert&Pedersen,1993;Froelichetal.,1979).Furthermore,MnappearstobetrackingTIC,whichweassumetore-flectauthigenicMn-richcarbonate.Inthiscase,theprerequisiteforMn-carbonateprecipitationisoxicbottomwaterswhereMnoxidesare trapped in the sediments and redox cycled repeatedly, result-ingindissolvedMnbuildupinthesubsurfaceanoxicportionsoftheporewaterprofileandsubsequentcarbonprecipitation (Calvert&Pedersen,1993,1996).

3.4 | Lipid biomarkers

Our inorganicgeochemical frameworkcanyieldnuanced informa-tion that can resolve longstanding questions about the extent ofmid-Proterozoiceukaryoticdevelopment, theenvironmentalback-ground,andthenutrientconditionsoftheirearlyevolution.Inthiscontext,welookforpossiblebiomarkersbytargetingstratathathadappreciablyhightotalorganiccarbon(TOC)content(>1wt.%);high

F IGURE  2 ChemostratigraphicrecordsforAltree-2drillcorefortheVelkerriFormation,RoperGroupwithstratigraphicvariationsof:Mn;Mo;Mo/TOC;U;V;andtheprevalentmarineredoxconditionsforeachinterval.Mo/TOCpanelhasbeenculled(TOCwithvalues

8  |     NGUYEN Et al.

and lowMoconcentrations;andfaciesoverall that reflectvaryingdepositional redox environments, including independent sugges-tionsofatleastintermittentlyhospitableoxicconditionsinthewa-tersbelowthesurfacemixedlayer.Consequently,selectedsamplesfromAltree-2 core covermost of the Velkerri Formation, rangingfrom452to1,205mdepth(seeTable1).

Althoughlipidbiomarkerscancontributetoabetterunderstand-ingofmid-Proterozoicoceanchemistryandbiology,importantana-lyticalself-consistencychecksarerequiredtoverifysyngenicityofthecompoundsdetected.Contaminationisaprimaryconcern—frommigrationofhydrocarbonsacross abillionormoreyearsofburialhistoryorfromoil-basedadditivesusedtodrillthecores(Grosjean&Logan,2007).Fortunately,wecanscreenforthispossibilitythroughmeticulousmethodologydesignedtoidentifyandpreventcontami-nation(Brocks,2011;Brocksetal.,2017;Flannery&George,2014;Frenchetal.,2015;Loveetal.,2009;Luo,Hallmann,Xie,Ruan,&Summons,2015;Pawlowska,Butterfield,&Brocks,2013;Zumbergeetal.,2018).

Saturated and aromatic hydrocarbon fractions, obtained fromrockbitumensextractedfrominnercoreportions,wereanalyzedforthis study to generate various parameters such asmethylphenan-threneindex(MPI-1;Radke&Welte,1983),aswellasvarioushopaneandsteranemolecular ratios (Peters,Walters,&Moldowan,2005;Summons,Powell,&Boreham,1988).Theseconstraintswereusedtocheckforsystematicchangesinmaturityratioswithburialdepthtoensurethatthebitumenrecordshavenotbeencompromisedbyhigh thermal overprints or obvious exogenous hydrocarbon/othercontaminant inputs (Table1). Methylphenanthrene and phenan-threneratios, in thiscaseusing theMPI-1 index, (Table1)showedsystematic increases with increasing burial depth—values beganincreasing from0.49at725m (middleVelkerri) to ashighas1.48at1205m (lowerVelkerri).Thispattern isverysimilar toprevious

reports of aromatic hydrocarbons from the same core (Summonsetal.,1994). Inaddition,data for%Rc (vitrinite reflectancecalcu-latedfromMPI-1)pointtoathermalmaturityrangeforthesesam-ples spanning fromearly oilwindow towithin themiddle intervaloftheoilwindow(Figure1,Table1),asindependentlyverifiedfromRock-Evaldata(hydrogenindex)andfromthealkanebiomarkerma-turityparameters.Cross-checkingwithratiosofC27hopanes18α(H)-22,29,30-trisnorneohopane(Ts)and17α(H)-22,29,30-trisnorhopane(Tm)—whereTsisthemoststablethermodynamicconfiguration,andthushighervaluesofTs/(Ts+Tm)representanincreaseinthermalmaturity (Seifert &Moldowan, 1978)—shows that the alkane andaromaticmaturityratiosareself-consistent.ATs/(Ts+Tm)ratioof0.59isobservedat452m(upperVelkerri);theseratiosincreasetoashighas0.78at852m.Highervaluesaregenerallyfoundindeepersamples, although mineralogy can exert a secondary control onabsolutevalues.Moreover,moretanes (βα–isomers)are thermody-namicallylessstablethantheαβ-hopanes,andthelowratiosofC30 βα–isomers/αβ–isomersobserved(0.06–0.07,Table1)fortheupperandmiddleVelkerristrataarealsoconsistentlyuncontaminatedho-panesignaturesfromrockswithintheoilwindow.

Acrossthisfullgradientinoceanchemistry,asdistinguishedbyinorganic geochemical characteristics, we found no evidence forany C27–C30 sterane biomarkers in any samples at levels detect-able above our lowdetection limit usingMRM-GC-MS (estimatedat

     |  9NGUYEN Et al.

orregularsteranespresentmustbelowerthan0.1ppmofthetotalalkane fractions in concentration. Overall, three important self-consistencycheckssupportourfindingthateukaryoticsteranebio-markers in these rocksareeither absentorpresent invanishinglysmall amounts below detection limits: (a) C27–30 regular steranes were also below detection limits in the hydropyrolysates derivedfrom the corresponding kerogen phases for seven pre-extractedrock powders, even though kerogen-bound hopaneswere detect-able;(b)anydetectableamountsofextractabletracesteraneswereonly found in outer exposed core portions, which are more sus-ceptibletocontaminant inputs,butwerecompletelyabsent inthepristine“inner”coreportionsanalyzedseparately(Figure3);and(c)short-chain sterane compounds (in our case,wemonitoredC21+22 steranesusingtheappropriateMRM-GC-MStransitions),whicharecommonlyfoundtogetherwiththeusualC27–C30parentsteranesinoilwindow-maturerocks,werealsoabsentintheinnercoreextracts

andkerogenhydropyrolysates.Additionally,gammacerane,whichisthefossilalkanebiomarkerderivedfromtetrahymanolwhichcanbesynthesizedbyciliatedprotozoans,some low-oxygen-adapteduni-cellulareukaryotesaswellassomebacteria(Takashitaetal.,2017),isonlyfound intraceamountsrelativetohopanes intwosamplesandisbelowdetectionlimitsinalltheothers(Table1).

Inadditiontothelackofdiscerniblesteranes,patternsoflinearandbranchedalkanes(mostlyintheC13–C24rangeforn-alkanesandmethylalkanesintotalionchromatograms),aswellasidentificationofrobusthopanesignalsusingMRM-GC-MSdetectionforboththebitumenandkerogenproducts(Figures4and5),suggestabacteri-allydominatedecologythatdroveprimaryproductivityasfoundpre-viously fromanalysesofextractablebitumenfrommiddleVelkerriFormationblackshalesfromtheWalton-2core(Flannery&George,2014).Analysesofoil inclusions fromtheBessieCreekSandstoneunderlying theVelkerriFormation (which is themost likely source

F IGURE  4 ComparisonofC27–C33 hopanedistributionsfromMRM-GCMSofsaturatedhydrocarbonfractionsfor(a)Phanerozoicoilstandardand(b)Altree-2rockbitumenextract(ca.452mdepth)fromtheuppermemberoftheVelkerriFm.SeeTable1forpeakassignments

Ts TmBNH

29H

29Tso

30H

30-nor30M

31HS+R 32H

S+R 33HS+Rγ

(a)

(b)

TsTm

29H

29Ts

30H

SR S

RS R

Oil standard

Velkerri Fm. (452 m)

60:00 66:00 RT (min)62:00 64:00 68:0058:00

F IGURE  5 Comparisonoffree(extractable)andkerogen-boundC27–C31 hopanesfromMRM-GCMSofsaturatedhydrocarbonfractionsfor(a)solvent-extractablebitumenand(b)kerogenhydropyrolysateofAltree-2samplefromca.452mdepth.SeeTable1forpeakassignments.MRMparent/daughtertransitions and relative abundances arealsoshown.Thekerogen-boundhopanesexhibitaslightlylessmaturediasteroisomericdistributionthanthefreehopanesduetostericprotectionthroughbindinginthekerogenmatrix(Loveetal.,1995),whichistheclassicancientbiomarkerpatternforrocksofallagesandapositivetestforsyngenicityofthehopaneseriesintheserocks 54:00 60:00 66:00

C31H426→191

45 | 18

C30H412→191

97 | 42

C29H398→191100 | 100

C27H370→191

72 | 91

TsTm

αβ

Tsβαdia

dia

αβ

βα

diaS+R

R

Tm

Ts

(a) (b)

βα

αβ

βα

αβ

βα

βα

Sαβ

RSαβ

27β

RT (min) 54:00 60:00 66:00 RT (min)

10  |     NGUYEN Et al.

rock) revealedabroadrangebiomarkersderivedmainly frombac-teria but either lacking or having only trace quantities of eukary-oticsteranebiomarkers(Dutkiewicz,Volk,Ridley,&George,2003).Overall,thegeochemistryoftheoilinclusions,intermsofthemajorhydrocarbonconstituents,isverysimilartothebitumenandsourcerocksobserved from theRoperGroup (Flannery&George, 2014;Volk,Dutkiewicz,George,&Ridley,2003;Volk,George,Dutkiewicz,&Ridley, 2005). Further, since other polycyclic biomarker alkaneswere preserved in these rocks (such as tricyclic terpanes and ho-panes),theabsenceofsteranesignal isnotduetopoorbiomarkerpreservationarisingfromstructuralmodificationordestructionduetothermalcrackingduringburialmaturation,consistentwiththelowthermalgradeofthestrataasalreadydemonstrated.

Notonlywereregularsteranesabsentintheeuxinicinterval,buttheywerealsonotdetectedintherelativelymoreoxicfacies,sug-gestingthat(steroid-synthesizing)eukaryoteswerenotecologicallywidespreadandabundantinsuchdistalshelfmarinesettingsduringthismid-Proterozoic interval—in contrast to the later Proterozoic.Previousmodelshaveassertedthatlownutrientavailabilityandas-sociateddeficiencies in oxygenmayhave throttled early diversityandabundancesofeukaryotes(Anbar&Knoll,2002;Javaux,Knoll,&Walter,2001;Lyonsetal.,2014;Reinhardetal.,2013).Alternatively,a taphonomicbiaseffecthasbeenproposed for lowpreservationpotential of steroids in mid-Proterozoic rocks (Pawlowska etal.,2013), a hypothetical mechanism termed the “mat-seal effect” inwhichenhancedmicrobialreworkingofsteroidlipidsdepositedonthesurfaceofbenthicmicrobialmatswasproposedtopreferentiallydepletesedimentarysteroidcompoundsintheextractablebitumenphase.Inourstudy,wefindnoevidencefor(a)shortsteranecom-pounds (C21+22steranes)withpartiallydegradedside-chains intherockbitumens,or (b)oranyboundsteranecompoundscovalentlylinkedwithinthe(insoluble)kerogenphasewhichwouldhavebeensequesteredearlyinthedepositionalhistory.Ithasbeenshownpre-viouslyfromarangeofmodernaquaticenvironmentsthatkerogenformationcommencesveryearlyduringdiagenesis,beginninginthewatercolumnandresultinginappreciablesequestrationofbiolipidsintoan insolublemacromolecularorganicphase that isnot readilybiodegradable (Farrimond etal., 2003). Since the macromolecularhostmatrixprotectstheboundmolecularconstituentsfrommicro-bial degradation, the complete lack of any free or kerogen-boundsterane biomarkers detected in Velkerri Fm. rocks shown heresuggeststhatthedearthofregularsteranesisunlikelytobeacon-sequenceofa“mat-seal”taphonomicbiasoperatingagainstpreser-vationofancientsteroidbiomarkers.

FullscanGC-MSanalysisofaromaticfractionsfromtherockbi-tumensalsorevealednodiscerniblearomaticcarotenoidbiomarkersderivedfromanoxygenicphotosyntheticbacteria,suchasChlorobi,in 133 and 134Da mass chromatograms. Despite other series ofpolyaromatic hydrocarbons being abundant, both the 2,3,6- and2,3,4-trimethyl-substitutedarylisoprenoidalserieswerebelowourdetection limit estimated at 1ppm of the total mass of aromatichydrocarbonsfor individualcompounds.Abundantaromaticcarot-enoid biomarkers for both green and purple sulfur bacteria were

detectedpreviouslyinrocksofthe1.64GaBarneyCreekFormation,alsofromtheMcArthurBasin(Brocksetal.,2005).Incontrast,theVelkerri Formation shows no detectable aryl isoprenoids and C40 parent carotenoid markers for anoxygenic phototrophic bacteria,which suggests that photic zone euxinia was not commonly sus-tainedinthedepositionalenvironment.Further,sincethewatercol-umndepthforVelkerriFm.strata,depositedindeepmarinesettingsontheouterslopeandmoredistal,islikelytohavebeensignificantlydeeperthanphoticzonewaterdepths(typicallydowntoca.100m),then proliferation of photosynthetic benthicmats on the seafloorwouldbelikelyunsustainableduetolowlightlevelrestrictions.

With our new results,we can comfortably assert that the ab-sence of diagnostic biomarkers for eukaryotic organisms is nota product of locally inhospitable conditions due to a continuousabsence of oxygen in the bottom waters and a correspondingpersistenceof anoxiaor euxinia. The suggestion, then, is that theenvironmentoftheVelkerriFormationmayhavebeen,ifanything,morefavorablethanmanymid-Proterozoicsettings.Specifically,theVelkerriwaslikelymarkedbyatleastepisodicallyhospitablewatersduringanintervalinEarthhistorymoregenerallycharacterizedbydelayed eukaryotic proliferation inmore prominently inhospitableoceans.Inotherwords,theVelkerriFormationprovidesaspecialifnotuniqueenvironmentalwindowtosampletheglobalevolutionarystateofcomplexlife—freeoflocalredoxbias.

Molecular clock studies (Gold, Caron, Fournier, & Summons,2017)predictthatsterolsynthesisevolvedearlyineukaryoticevo-lution significantly prior to the diversification of crown group eu-karyotesandthat the lasteukaryoticcommonancestorLECAwasprobablycapableofmakingsterols(Desmond&Gribaldo,2009).Insupport of this, bacterially derived steroidswithmethylation pre-servedatC-4(Wei,Yin,&Welander,2016)havebeenreportedinimmaturerocksfromca.1.64GaBarneyCreekFormation (Brocksetal., 2005) showing that steroid synthesis significantly predatesthe1.3–1.4GaRoperGroup.Afundamentalconclusionofourstudythen is that eukaryotes did not make significant contributions tothe sedimentaryorganicmatter deposited in theMcArthurBasin,asgaugedfrombiomarkerabundancepatterns,suggestingthateu-karyoteswerenotabundantmarineprimaryproducersduringthisintervalof themid-Proterozoic.One importantcaveat is that low-oxygen-adaptedeukaryoteswhichdidnotpossesssterolsmayhavebeenpartofthecommunitystructure,sinceafewanaerobicprotistshaverecentlybeenshowntolackeithersterolsortetrahymanolintheircellmembranes(Takashitaetal.,2017).Bothalowabundanceof microbial eukaryotes relative to bacteria and the possibility ofadaptedeukaryoticprotists thatdidnotmakesteroidsor tetrahy-manolcanpotentiallyreconcilethefindingofeukaryoticmicrofossilsinRoperGroupstrata(Javaux,Knoll,&Walter,2004)butnodetect-ablerecordofestablishedeukaryoticbiomarkers.

Arecentconsensushasemergedfromcarefulanalysisoflowma-turityancientsedimentaryrocksthatC27–C30steranes,biomarkerssourcedfromeukaryotes,arebelowdetectioninrockbitumensformid-Proterozoic strata andmore generally in rocks older than ca.800Ma (Blumenberg, Thiel, Riegel, Kah, & Reitner, 2012; Brocks

     |  11NGUYEN Et al.

etal., 2005, 2015, 2017; Flannery & George, 2014; French etal.,2015; Isson etal., 2018; Luo etal., 2015; Pawlowska etal., 2013).Indeed, theoldest robust reports of ancient steranes derive fromrocksoftheChuarGroupandVisingsöGroup(700–800Ma)usingthehighsensitivityofMRM-GC-MSforbiomarkerdetection(Brocksetal.,2015,2017).Incontrast,abundanthopanesderivedfromdi-versebacterialsourceareubiquitousinimmaturerocksasfarbackas1.64Ga(Brocksetal.,2005).Themostparsimoniousinterpretationofthelackofdetectablesteranesinourviewisthataerobiceukary-otesdidnotbecomeecologicallywidespreadandabundant in theglobaloceansuntilwell into theNeoproterozoicera (Brocksetal.,2017) and that source biota in mid-Proterozoic and older oceansweredominatedbybacteria(Brocksetal.,2005,2017;Flannery&George,2014;Issonetal.,2018;Luoetal.,2015).Arecentmolecu-larclockanalysis(Gibsonetal.,2017)predictsthatphotosynthesisdidnotactuallyemergeinEukaryauntilca.1.25Ga,andthiscouldhelpexplainthebacterialecologicaldominanceofRoperGroupbio-markerassemblages,althoughthedivergencetimeuncertaintiesforsuchestimatescanbe largeand this innovationdoesnotcoincidewiththeubiquityofeukaryoticsteranesinancientrocksbeingde-layeduntilca.800Maandyounger,asdescribedabove.

Previous microfossil studies indicate only a modestMesoproterozoic eukaryote diversity as gauged from acritarchs(Beghin etal., 2017; Javaux etal., 2004), even from settings in-terpreted as oxic in the Mesoproterozoic ocean (Sergeev, Knoll,Vorob’eva,&Sergeeva,2016;Sperlingetal.,2014).Furthermore,theapparentdisparitybetweensomeoccurrencesofMesoproterozoiceukaryotic acritarchs yet no detectable record of regular ster-anesappearstocontinuetill1.0GaintheMalginFormationoftheSiberianPlatform(Suslova,Parfenova,Saraev,&Nagovitsyn,2017),while thermally immature rocks from the1.1GaAtarGroupwerealso shown tobedevoidof steroidbiomarkers (Blumenbergetal.,2012).Whilemicrofossil analysis is themost sensitive and appro-priate approach for assessing the first appearance of eukaryoticcellsinancientsedimentaryrocks,discernibleacritarchmicrofossilstypically represent only a smallmass fraction of the total ancientsedimentaryorganicmatter (mainly amorphous kerogen) and thusit isdifficult toquantify theeukaryoticcontributionswithout lipidbiomarkersourceinputconstraints.InsightsfromacombinationofmicrofossilandbiomarkerapproachesinthestudiesofProterozoicgeobiologyarehenceusefulandcomplementary.

4  | CONCLUSIONS

Ironandtrace-metaldatafromtheVelkerriFormationrevealthattherewasmoreapparenttemporalvariabilityinthemarineredoxconditions, at least locally and perhaps globally, during themid-Proterozoic than was previously recognized through analysis ofthesameinterval (Kendalletal.,2009;Mukherjee&Large,2016;Shen,Canfield,&Knoll,2002;Shenetal.,2003;Yangetal.,2017).Overall, the system appears to have evolved from one that wasdominantlyoxicduringdepositionofthelowerVelkerriFormation

toeuxinicinthemiddleVelkerri.Morespecifically,themiddleunitismarkedbyevidenceforatransientperiodofeuxiniabracketedbyevidenceformorepersistentwatercolumnsulfidepriortothedep-ositionoftheupperVelkerri,assupportedbythecombinedFeandModata.TheupperVelkerriFormationshowsanintriguingcombi-nationoflowtrace-metalabundancesbutnumerousFespeciationdatasuggestingfrequentferruginousbutnoteuxinicdeposition—withotherFespeciationdatathataremorereasonablyattributedtooxicconditions.ThelackofUandVenrichmentplacespossibleconstraintson the limiteddurationof theanoxicconditions—andtherelativeratesofUandVversusFeenrichment—andthepos-sibilityofVandUremobilizationunderconditionsofrapidredoxvariability(andsubsequentoxicoverprintsspecifically).TheFesig-naturesof ferruginous conditions, by contrast, couldbe retainedevenifreducedFephaseswereoxidized.Themostlikelyscenariofortheupperintervalisoneofoscillatoryredoxbetweenoxicandanoxicmarinedeposition.

Arecentviewofthemid-ProterozoicoceanredoxstructurewaspresentedinPlanavskyetal.(2011)andPoultonandCanfield(2011),whichsuggestsadominantly ferruginousdeepoceanwitheuxinialimitedtoproductivemargins(seealsoReinhardetal.,2013;asre-viewed in Lyons etal., 2014).Ourdata—specifically comparativelyhigh Mo contents in the middle euxinic interval and suggestionsfrom the Fe speciation data for at least intermittently oxic condi-tions above and below, perhaps at some appreciablewater depthgiventhefine-grainednatureofthesediments—suggestredoxvari-ability and an interval of relatively oxic marine conditions in themid-Proterozoic. The depth, lateral extent, and concentrations ofmarineoxygenationaredifficulttoconstrainandinnowayunambig-uouslydemandwidespreadoxygenationduringthisinterval.Theseinferred conditions for the Velkerri Formation do, however,makeit a particularly attractive target in our search for eukaryotic bio-signatures—withinaframeworkofboth localandglobalredoxandimpliednutrientcontrols.Moreover,weshouldbeabletoinferqual-itativelyvariationsinproductivityandorganicmatterpreservationacross local redoxgradients, if those localcontrolsdominated theeukaryoticsignalstrength.Inotherwords,welookedforeukaryoticsignalsinfaciesexpectedtofavortheirproduction(oxic)andpres-ervation(anoxic)—recognizingthatanoxicbottomwaterscanrecord(withstrongfidelity)eukaryoticproductioninshallow,overlyingoxicwaters.Atthesametime,proximaldeeperanoxicwaterscanchal-lengeshalloweukaryoticlifethroughupwardmixingofthosewaters(Hardistyetal.,2017).

Despiteourcareful, independentconsiderationofprimaryredoxcontrols, biomarker data across the three intervals of the VelkerriFormation show no clear evidence for the presence of the regularsteranecompoundsthatareconsidereddiagnosticofeukaryoticlife,eveninstratawithappreciableTOCandsuggestionsofoxicdeposi-tion.Inaddition,therearenosignificantdetectablelevelsofaromaticcarotenoidbiomarkersforgreenandpurplesulfurbacteria;thus,sub-stantial anoxygenic photosynthesis could not have been supportedasamajormodeofprimaryproductionatleastinthissettingatthistime (Johnston etal., 2009). The lack of aryl isoprenoids and C40

12  |     NGUYEN Et al.

parentcarotenoidmarkersforanoxygenicphototrophicbacteriaintheVelkerriFormationcontrastsbiomarkerevidence forgreenandpur-plesulfurbacteriafoundintheolderBarneyCreekFormation(Brocksetal., 2005).Weare leftwith the conclusion that photic zoneeux-inia,ifpresent,wasperhapsofrelativelylimitedextentduringdeposi-tionoftheVelkerriFormationeventhoughdeepereuxinicconditionswererelativelywidespreadcomparedtothemodernrecord(Reinhardetal.,2013).Whileweareprovidingonlyonesnapshotof themid-Proterozoicworld,italsoraisesdoubtsastowhethereuxiniawasshal-lowandabundantenoughtosupportappreciableanoxygenicprimaryproductionviasulfideoxidation—particularlyinopenmarinesettings.Overall, the absence of eukaryotic biomarker signals in our sam-ples—despiteourcarefulsearchwithinapaleoenvironmentalcontextinformedby ironmineraland traceelementgeochemistry—suggeststhatothercontrolssuchassynergisticlong-termnutrientandecolog-icalfactorsmayhaveheldcomplexeukaryoticlifeatbaypendingtheprofoundenvironmentalchangesthatmarkedthelaterProterozoic.

ACKNOWLEDG MENTS

Funding was provided by the Earth-Life Transitions and FESDprograms of the U.S. National Science Foundation and theNASA Astrobiology Institute under Cooperative Agreement No.NNA15BB03AissuedthroughtheScienceMissionDirectorate.GDLthankstheAgouronInstitutefortheAutospecGC-MSinstrumentatUCR.Manythankstothepeoplewhohelpedwithlaboratorywork,analysis,anddiscussion:DaltonHardisty,EmilyHaddad,RosemarieBisquera, Konstantin Choumiline, Petra Schoon, Alexandria Ruiz,LauraWehrmann,NataschaRiedinger,andMichaelDahl.

CONFLIC T OF INTERE S T

Theauthorsdeclarenoconflictinginterests,financialorotherwise.

ORCID

Gordon D. Love https://orcid.org/0000-0002-6516-014X

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How to cite this article:NguyenK,LoveGD,ZumbergeJA,etal.AbsenceofbiomarkerevidenceforearlyeukaryoticlifefromtheMesoproterozoicRoperGroup:Searchingacrossamarineredoxgradientinmid-Proterozoichabitability.Geobiology. 2019;00:1–14. https://doi.org/10.1111/gbi.12329

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