Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , and the PASADO 3 , L. Bunting 3,4 18345 18346 , P. R. Leavitt 2 5,6 , D. Ariztegui 1 This discussion paper is/has beenPlease under refer review to for the the corresponding journal final Biogeosciences paper (BG). in BG if available. GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Sect.Department 4.5 of Earth Sciences, University of Geneva, rue desLimnology Maraîchers Laboratory, 13, Department 1205 of Geneva, Biology, University of Regina, Regina, Saskatchewan, Institute of Environmental Change and Society, University of Regina, Regina,GEOPOLAR, Saskatchewan, Institute of Geography, University of Bremen, Celsiusstr. FVG-M, 28359 GEOTOP Research Centre, Montréal, Canada 1 Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany 2 Switzerland 3 Canada 4 Canada 5 Bremen, Germany 6 Science Team Received: 12 October 2015 – Accepted: 2Correspondence November to: 2015 A. – Vuillemin Published: ([email protected]) 16 November 2015 Published by Copernicus Publications on behalf of the European Geosciences Union. Recording of climate and diagenesis through fossil pigments and sedimentary DNA at Laguna Potrok Aike, Argentina A. Vuillemin Biogeosciences Discuss., 12, 18345–18388, 2015 www.biogeosciences-discuss.net/12/18345/2015/ doi:10.5194/bgd-12-18345-2015 © Author(s) 2015. CC Attribution 3.0 License. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | were Archaea 18348 18347 erent sedimentary microbial assemblages derive from spe- ff Ancient DNA has already been successfully employed to study the succession of These patterns reveal that microbial assemblages identified from environmental DNA Here geomicrobiological and phylogenetic analyses of a Patagonian maar lake were and diversity of microbes (Dong etin al., terrestrial 2010; Vuillemin plant et cover al., 2013b). along Similarly, climate-related changes environmental gradients influence sedi- species as a resultson, of 2009). environmental changes For in example,and lacustrine wet diversity settings and in (Coolen the warm and sediment, climates whereas Gib- result cold in and dry high climates bacterial favour abundance lower abundance blages underwent structural changesesis and to selective result preservation during inenvironmental early the DNA diagen- obtained final from composition lacustrineformation entombed to sediments complement in provides paleoenvironmental essential the indicatorspost-depositional genetic sediments. and diagenetic in- trace We processes climate conclude over change tens that and of millennia. 1 Introduction Lacustrine sediments represent(Meyers excellent and Lallier-Vergès, archives 1999), while of providing asedimentary past wide microbes. range environmental of Despite ecological conditions the nichesganisms for fact shows that the a compositionat strong of the correspondence sedimentary time to microor- ofabout geological deposition the in and relative marine geochemical influence environmentssedimentary conditions (Inagaki of processes et extant as al., environmental controls 2003),custrine conditions of little sedimentary and is microbial settings post-depositional (Vuillemin known assemblage etshould composition al., influence in 2013a). In the deep principle, flux la- rial climatic deposited and conditions at geochemical the make2001), lake while up microbial bottom of activity (Meyers in organic andtion the (Freudenthal Ishiwatari, water and et column 1993; inorganic al., (Chen Meyers et 2001; mate- sediments al., and Lehmann and 2008) et Teranes, and associated al., after 2002) microbial deposi- will biota.during further Finally, early evolution influence diagenesis of the is sediment nature expected environments crobial of to consortia influence (Nelson the et final al., composition 2007; of Zhao entombed et mi- al., 2008). stemmed from a variety oftors sedimentary (catchment niches inflows, associated with water climate-dependent column fac- conditions, productivity), but that initial assem- used to indicate that the di predominant in sediments dated from thealternated Last with Glacial fine Maximum, volcanic in material whichwater pelagic characteristic conditions, clays of but a reduced lake water level column highstand productivity. and fresh- Abstract Aquatic sediments recordecological past niches for climatic microorganisms. conditions Althoughblages marine are while sedimentary often providing microbial defined assem- byenvironmental a their features surrounding wide upon geochemical microbial range conditions, developmentmains the and of largely influence post-depositional unknown of survival in re- thecomposition lacustrine of environmental realm. DNA Due can to beover long-term expected time to microbial and evolve activity, with the therefore sedimentbut should depth this and reflect hypothesis has both rarely ancient been and tested using extant a microbial multiproxy populations, approach. cific lacustrine regimes duringtervals whose defined sediments climatic harboured periods. activewere microbial sampled Two for populations well a and comparative defined environmental measurable climatic studyrRNA ATP based gene in- on sequences. fossil pigments Bacterial and andfrom 16S archaeal the 16S Holocene rRNA record geneditions revealed sequences actively a recovered producing microbial methane communityteristics during adapted were organic to associated matter subsaline with degradation. con- sedimentshigh These resulting charac- evaporative from stress endorheic lake andclone conditions concomitant libraries with high established algalstratification throughout of productivity. the these Moreover, populations, archaeal Holocene consistentafter record deposition. with In a indicate contrast, gradual sulphate-reducing an use bacteria of age-related and organic lithotrophic substrates 5 5 25 10 15 20 25 10 15 20 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | C. Dissolved oxygen nor- ◦ 18350 18349 erences in the age and composition (lability) of ff erentiation between extant and ancient populations. ff In this contribution, we take advantage of previous paleoclimatic reconstructions This paper tests the hypothesis that climate, in-lake processes, sedimentary envi- Potrok Aike is a maar lakePali located Aike in volcanic southern field Patagonia, (Coronato Argentina eterly (Fig. al., winds 1a) 2013). on within Due the the to areacurrently the (Mayr persistent unstratified et influence throughout al., of the 2007), West- (Fig. the year. 1b), while lake The mean is basin annual polymictic temperatures has range and from a the 4 to water maximum 10 column depth of 100 m 2 Material and methods 2.1 Study site In the framework ofpiston the core (Ohlendorf ICDP-PASADO et project, al., a 2011)biological was 100 study m collected of and long the sampled by lacustrine for a subsurface 7 detailed cm biosphere geomicro- (Vuillemin wide et hydraulic al., 2010). Laguna mally shifts from oxic toet dysoxic al., conditions at 2006) the andet water-sediment oxygen interface al., (Zolitschka penetration 2013b). within Thisclimatic regime hydrologically-closed surface in basin sediment southernmost South contains is Americaand a restricted in ice which sedimentary cap (Vuillemin changes distribution in record the in ofditions Westerly the winds the Andes and regulate in-lake variations conditions in (Fig. regional environmental 2) con- such as mixing and hydrological balance (Mayr (Gebhardt et al., 2012;data Kliem reflecting et planktonic production al.,complement by 2013) geomicrobiological algae investigations and (Vuillemin and et blend phototrophic al.,lected their 2013a, bacteria. phylogenetic 2014a) results data As with with using well, se- 16Sin new we LGM rRNA pigment and gene Holocene. libraries Thislast to approach 25 focus allows 000 us on years to discrete in compareevolution horizons response variations of to in the both DNA over sediments. past the intervals environmental Finally, throughout we conditions the established and microbially-active sediments archaeal geochemical ofstructural clone the criteria Holocene libraries of period at di to regular provide mentary microbes via variations in(OM) erosion to and export lakes oftions (Clark organic of and and the inorganic water Hirsch, column matter (Coolen regime, 2008). further et Shifts induce al., large in changes 2006, in lake 2008), bacterial salinity, while populations as di well as modifica- ronments and post-depositional alterations shouldcomposition all of be reflected environmental in DNA thecrobial final by assemblages microbial in comparing an thethose unproductive phylogenetic characteristic glacial of signatures environment the (ca. productive of Holocene 25tection mi- (ca. 000 of years 5000 in years ago) ago). situ to Moreover, microbial theMaximum activity de- (LGM) within sediments periods from provided theronmental the Holocene opportunity DNA and over Last to Glacial time assessassemblages and the at preservation discriminate of the nucleic envi- acid timeet sequences of al., of 2012) deposition from initial (Anderson-Carpenter those microbial sediments arising et (Freudenthal from et al., diagenetic al., processes 2011; 2001). followingwas Jørgensen entombment Finally, characterized evaluation in by of the high the cell Holocene densitiesinary record, and evaluation which homogeneous of sediments, how allowed the prelim- and over structure time. of active microbial populations varied with depth sedimentary OM can also createthe distinct long-term bacterial niches persistence (Nelson andfurther et al., modify activity 2007). geochemical of Finally, conditions microbes viaselective diagenesis preservation in (Inagaki and sediments et modification following al.,Boere of burial 2006), bacterial et leading can assemblages al., to (Miskin 2011a,nities et b). deep al., Therefore, 1998; sedimentary in environmentscatchment principle arises conditions at the from the composition a timements, of combination of diagenetic deposition, microbial of modifications the following the commu- nature burial, and climaticand and diversity provenance and the of of metabolic microbial the populations activity, sedi- distribution date, (Ariztegui these et al., hypotheses have 2015; not Kallmeyer et been al., clearly 2015). tested To using multiple proxies. 5 5 15 10 25 20 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | a -fixing 2 ,6-diamidino-2- gr total organic cient conditions 0 ffi × -triphosphate (ATP) assays. 0 18351 18352 atmosphere, filtered through 0.45 µm pore mem- 2 , pigments normally indicative of total algal abundance (Leavitt et al., a ) were calculated for pigments usually characteristic of green sulphur bacteria C years (Kliem et al., 2013). Sediments for pigment analysis were frozen im- 1 14 − methanol) and water under an inertbrane N filters, and injected intophoto-diode HPLC array system detector, fitted and with fluorescenceand a detector. HPLC reversed-phase peaks C18 systems identified column, were calibrated tion using Agency authentic and DHI pigment Labof standards Products, sedimentary Denmark), (US unialgal pigments. Environmental cultures, Biomarker and Protec- concentrations reference stocks (nmol pigment mediately after coreisolation collection and (Ohlendorf quantification of et carotenoid,ing chlorophyll al., (Chl) a 2011) and Hewlett derivative and Packard pigmentssystem. us- model stored All 1100 in extraction, high the isolation performancedetailed elsewhere dark liquid and (Leavitt chromatographic until and quantification (HPLC) Hodgson, followedfrom 2002). In the freeze-dried all sediments standard cases, into pigments procedures degassed were mixtures extracted of organic solvents (i.e. acetone, 2.3 Pigment analysis Historical changes in algaltified abundance by and gross analyzing community fossil50 composition 000 algal were pigment quan- concentrations in 2500 samples spanning ca. (isorenieratene), total cyanobacteria (echinenone), Nostocales and potentially N carbon cyanobacteria (canthaxanthin, aphanizophyll), purplediatoms (diatoxanthin). bacteria Preservation index (okenone) was calculated and from the mainly ratio of chlorophyll Pore water was retrievedsoil moisture from samplers small (Rhizon Eijkelkamp). holesgeochemical All drilled protocols analyses for in related lithostratigraphic and theistry, to bio- cell liners count bulk using and sediment denaturing 0.15been µm composition, gradient published gel pores pore elsewhere electrophoresis (Vuillemin (DGGE) waterable procedures et at geochem- http://doi.pangaea.de have al.,under 2013a, accession811524. numbers b). 10.1594/PANGAEA.811521 to Complete datasets are avail- to pheophytin elsewhere (Vuillemin et al., 2013b,tablish 2014a). clone The libraries. same PCR DNA products extracts were were purified used using to the es- High Pure PCR Product 1997). Shifts in productivity associatedthe with ratio lacustrine of conditions total eukaryotic were pigments estimated to from total prokaryotic2.4 pigments. Clone library and phylogenetic analysis Detailed procedures for DNA extraction, PCR amplification and DGGE are described phenylindole (DAPI) cell counts, and on-site adenosine-5 Sediment sampling protocols werecontamination optimized (Kallmeyer to et avoid al.,ration potential 2006; of sources the Vuillemin et drilling of platform al., microbial prevented 2010). an The on-site size laboratory and with su configu- et al., 2007, 2013; Ohlendorfflux et enhances al., lake primary 2013). During productivitycolonization wetter in of periods, the the elevated lake nutrient sediments (Recasensthese in- et by insights al., microbes with 2012), a (Vuillemin astracted new et well from 16S as al., the rRNA whole 2013b). gene Holoceneas We record analysis well supplement and of as one the deep aand total ancient full sedimentary prokaryotic LGM sequence DNA horizon phototrophs. (Fig. ex- analysis Fossilthe 2b), of pigment two key and climatic sedimentary sedimentary intervalsgenetic carotenoids records also DNA in from allows extractions the eukaryotic for from frame of a well-established unique paleo-environmental reconstructions. comparison2.2 between climatic On-site and sampling and procedures of asepsis, therefore retrieved coresback to were the transported field laboratory everyunder where 90 min a the detailed from most protocol sterile the was conditions platform a applied possible. to quick The retrieve retrieval sediments aperture and of sampling conditioning windows of allowed sediments for DNA extraction, 4 5 5 15 10 20 25 20 25 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Es- NaCl, ampi- 1 1 − − and vector Bacteria C; Pollock and Bush, ◦ . Sequencing was per- 3 − Archaea Bactoagar and 2 mLL 1 − . 2 µL PCR product were ligated to 1 − 18354 18353 C for 20 h on a LB medium containing 1 gL ◦ value (Supplement). All our sequences have been de- Bactoyeast, 1.5 gL ff v.1.32.1 software (Schloss et al., 2009) and number of 1 − ® C; Pollock and Bush, 2013). Sedimentary features of horizon A consist ◦ 1 cells. Cloning procedure was performed using the TOPO TA Cloning Kit − . Chimeras were detected using the online program Bellerophon (Huber ff Bactotryptone, 0.5 gL 1 − Previous sedimentary studies (Kliem et al., 2013; Gebhardt et al., 2012; Ohlendorf volcanic detritus to the centercalibration of of the Laguna basin Potrok (Zolitschka Aikecorrespond et stratigraphy to al., showed specific that 2013). Furthermore, these climaticand five time periods, A1, lithological namely LGM, units the Youngeret Dryas Last (YD) al., Glacial, and 2013). Antarctic Holocene An events times A2 important (Buylaert sedimentary et transition al., was 2013; identified Kliem between lithological et al., 2013) defined fiveAike. main These lithological five units units are throughout based theof on record stratigraphic gravity of features inflows Laguna associated with Potrok in theations frequency response promoted to climatic important lake reworking level fluctuations of (Fig. the 2c). catchment Such with fluctu- influx of terrestrial and ing a continuous pelagicof to the hemipelagic LGM regime horizon (Fig. Bhighstand, (Fig. 2a). 2b) In and corresponded contrast, colder with paleoconditions a annual2013). freshwater mean water Sedimentary column surface features lake temperatures level ofwith ( horizon numerous intercalations B of mainly mafic sands consist associated of with terrestrial compacted events (Fig. greyish 2b). clays of fine intercalations of laminated silts with soft methane-saturated black clays, reflect- posited in the GenBankJX472282 database to under JX472399 accession and numbers KT381303 JX272064 to KT381433. to JX272122, 3 Results 3.1 Holocene and LGM environmental features 3.1.1 Sedimentary features Lake basin conditions at thesubsaline time (1.2 of % the NaCl HoloceneAnnual eq.) horizon mean during A surface a (Fig. atmospheric 2a) water-column temperaturespresent were were day lowstand defined ( slightly (Ohlendorf as colder et than al., those of 2013). the OTUs, rarefaction curves, Chao, Shannonat 97 and % Dominance-D sequence identity indices cut-o were calculated cherichia coli 1 gL cillin. To constitute libraries,spectively selected 83 from bacterial samplesat clones at 0.25, and 4.97 0.55, 228 anding 1.90, 29.77 archaeal m cycles 2.51, clones sediment were 4.97, were depth performed(Applied and 7.81, re- using BioSystems) samples the with 9.37 universal BigDye andprimers primers Terminator D4 29.77 v.3.1 27F and m R5 Cycle and sediment fromformed Sequencing 1492R the depth. on Kit for BigDye an Sequenc- sequencing ABIPRISM kitquences 3130xl for were Genetic assembled Analyzer with (Applied CodonCode BioSystems,aligned Hitachi). Aligner on Se- v.3.7.1 Seaview (CodonCode v.4.3.0 Corporation), tively (Gouy cut et o al.,et 2010) al., with 2004). ClustalW2. 16S PrimersBLAST rRNA gene) were (http://www.megx.net sequences selec- and were SILVA(Pruesse identified comprehensive using et ribosomal the al., megx RNA 2007).align, Geographic- databases The search SINA and classify onlineSILVA sequences v.1.2.11 database and (Pruesse as their et taxonomic closest al., references.platform matches All 2012)) (http://www.arb-home.de/ downloaded sequences from was and were the used phylogeneticimum uploaded trees to Likelihood on established method the with using ARB thement the Max- of RAxML bootstrap algorithm tree withwere advanced using exported bootstrap 100 to refine- replicates the (Ludwig Mothur et al., 2004). Distance matrices (Invitrogen by life technologies) followingformed the cells manufacturer’s were recommendations. incubated Trans- at 37 Purification Kit (Roche Diagnosticstrophotometer SA), (Witec AG), measured and with dilutedthe a to pCR4-TOPO 10ngµL vector Nanodrop (Invitrogen ND-1000 by life Spec- technologies) and cloned into competent 5 5 25 20 15 10 10 15 20 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ; Fig. 3c) suggested that a in Holocene sediments and nitrate concentrations (Sup- 1 ) suggest that bacteria related − 1 + − ) was often below detection limit + 2 grTOC pheophytin × gr TOC a/ × 18356 18355 erence between the Holocene and LGM times (i.e. ff -fixing cyanobacteria contributed substantially to the labile OM during the YD and The pigment preservation index (chlorophyll 2 plement) were alwaysin very between low 0.2 throughout andHolocene 0.6 the sediments ppm. sedimentary and Phosphate most concentrations sequence,of (Fig. often with the close 3d) sedimentary values to were(3.7 sequence. ca. detection ppm), 10 limit Dissolved but ppm (0.4 iron was in ppm) quantifiable (Fe tions within from between the 5 55 rest and to 15quent 15 ppm. m variations The sediment with sulphate baseline depth, concentration values reaching profile oscillating (Fig. concentra- between 3d) 5 displays and fre- 70 ppm. Extraordinary 37 m) likely promoted preservation3b). of bacterial OM during lake lowstand (Figs. 2b3.1.4 and Pore water chemistry Chloride concentrations (Supplement) indicatedsubsaline a (600 ppm) shift conditions from during freshwater (200 the ppm) YD. to Nitrite the main patterns of pastdiagenesis production (Leavitt, were 1993). robust to However,sizes changes peaks associated in of with lake the increased morphometry sedimentation index and ing rates also that (Fig. there correlated 3, may top coarse have andSuch grain been bottom), a intervals suggest- of phenomenon poor may preservationimportant be related common lake to during level low fluctuations OM theof content. LGM (Kliem the and et ratio YD al., of eukaryotic transition, 2013;portance to a Zolitschka prokaryotic of period et pigments eukaryotic of al., (Fig. algae 3c)early 2013). increased revealed Holocene; Analysis that during Fig. the climatic 3, relative bottom), transitions im- published and diatom (late that counts LGM, this and YD pattern theiret and link exhibited al., to important 2015). warming similarities Otherwise, events to and baselineotic tephra values biomass inputs oscillated (Recasens is around 2.0, considerably indicatingperiod. less that However, abundant the prokary- water than depth the di eukaryotic one during the glacial diatoxanthin showed that diatoms (Fig. 3b)Holocene were period abundant (Recasens during et the al., late YD 2015). and early close to detection limitnenone, in canthaxanthin the and glacial aphanizophyllN (Fig. record. 3b) Taken together, revealedHolocene concentrations that times, of cyanobacteria but echi- and are present only sporadiccally within the glacial interval. Finally, to sulphur metabolism were annity important during component the of late the YDconcentrations primary and were producer early also commu- Holocene observed (Fig. intrations 3b). the (not Sporadic shown) glacial peaks were record. always in In below isorenieratene contrast, 20nmol okenone concen- units 5 and 4ment (Fig. deposits, 2c) some that events(Kliem corresponded exceeding et with 1 m increased al., percentage ina 2013). desiccation of thickness These stage mass and of horizons move- the containing were maar fine dated interpreted around gravels 533.1.2 as kaBP paleo-shorelines (Gebhardt et following Organic al., matter 2012). Total organic carbon (TOC), total nitrogen (TN)very and similar organic stratigraphic variations, phosphorus with (OP) all displayed profilescurrence covarying of with gravity grain size events and (Fig. thegrain 3, oc- sizes top). and Low gravity events OM as contentsIn they were contrast, regularly four associated occurred sediment with during intervals coarse the displayeddepth Last increased and Glacial OM period. values uppermost around sediments 70, (Fig. 40,bottom), 3a). 10 these m In intervals context correspond of to theHolocene the overall times, stratigraphy Antarctic respectively. (Fig. event 3, A2, early LGM, YD and3.1.3 late Pigment concentrations Analyses of bacterial and algalgal pigment abundance concentrations related provided clear to indicationduring biomass for the (i.e. al- LGM assessed and productivity) Holocenesil being periods, concentrations respectively lower of (Fig.3b). and isorenieratene Specifically, (100nmol higher elevated fos- 5 5 25 20 10 15 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Ar- Aci- liated ffi Atribac- liated to ffi were maximal at 5 100) were recorded in > sequences were a 50) were observed in LGM > Bacteria signal disappeared below 60 m that were partly related to syntrophic (Fig. 4) belonging to the SVA0485 can- Bacteria Proteobacteria δ 18357 18358 liated with known iron reducers (Liesack et al., Clostridia ffi Proteobacteria δ , respectively former candidate divisions OP9 and OP8 (Rinke identified from Holocene deposits were mainly a sequence was a Bacteria (Barns et al., 1999) and Aminicenantes profile displayed a reduced but stable number of DGGE bands along the entire and Acidobacteria O ranged between 25 and 30 RLU. Thus, a value of 30 was systematically subtracted Analyses of DGGE gel features were used to assess structural changes in micro- In contrast, the microbial assemblage from the ca. 25 ka old LGM interval revealed 2 sedimentary record, with maximal values located around 8 and 35 m depth (Fig. 3f). chaea sediment depth in horizons potentially correspondingearly with reflooding increased of gravity the events maar and (Gebhardt et al., 2012; Kliem et al., 2013). Similarly, the sediments (ca. 40 toactivity 20 m in discrete depth), horizons. pointing Analysisbial to of populations a DAPI sustained were cell but counts densesttotal lower (Fig. cell in 3e) level abundance Holocene of showed decreased that sediments microbial graduallywith micro- from (ca. minimal values the 5 in m YD the down core deepest through depth), glacial LGM record but sediments, and that onebial minor communities. peak Number at 34 ofand m 30 depth. DGGE m depth, bands which (Fig.appeared corresponds 3f) with active the for based two intervals on where ATP microbial levels. populations The teria 3.2.3 Bacterial clone libraries 16S rRNA gene sequences from ca. 5 ka old Holocene sediments showed that 3.2.2 Sedimentary DNA from LGM andTogether DAPI Holocene ATP, and horizons DGGE profiles (Fig.crobial 3e assemblages and in f) the provided Holocene evidenceingly for and reflected viable LGM mi- specific sediments. habitats Theselected two which to horizons promoted establish seem- microbial comparative assemblages cloneedge and that libraries DAPI were (Fig. staining does se- 2a not and distinguishthe between b). present active, DGGE That inert bands said, or represent dead we shortextracellular cells acknowl- and sequences DNA that (150 preserved base under pairs) anoxic thattheless, conditions may the (Corinaldesi include detection et of al., small 2011).in amounts Never- LGM of sediments. ATP Thus, (Fig. thewere 3e) obtained considered suggested 16S active to rRNA metabolism gene reflectcroorganism sequences the assemblages (1400 (Figs. major and 4 900 componentsarchaeal bp) and clone of libraries 5). former established In throughout andof the addition, currently-viable discrimination Holocene we between mi- record ancient established to5 provide and six and criteria recent additional 6). 16S rRNA sequences (see below; Figs. et al., 2014), dominatedrepresentative the sedimentary microbial assemblage (Fig. 4). Additionally, dobacteria species (Liu et al., 2011). Similarly, some the Holocene record in betweenmicrobial processes. 8 In and contrast, only 4 m small peaks burial of depth, ATP ( indicating significant ongoing peaks were located at 49,1590, 38 1270 and and 25 980 m ppm, sediment respectively, depth, in reaching concomitance with concentrations tephra of3.2 layers ca. (Fig. 3, top). Microbial characteristics 3.2.1 Microbial activity, density and diversity Rapid ATP detections (Fig. 3e) weresediments used (Nakamura as and an Takaya, 2003). index Background ofH values in measured situ on microbial micropure activity within from the readings for background correction. Maximal values ( didate division likely involved inone sulphate reduction (Bar-Or et al., 2015). Remarkably, the significant presence of with syntrophic species (Jackson et al., 1999; Liu et al., 1999). 5 5 10 15 20 10 15 20 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , , for- Chloroflexi , Spirochaetes Methanomicrobi- Bathyarchaeota, and Planctomycetes and uncultured along with some sequences related to the Methanomicrobiales sequences were present throughout Holocene 18360 18359 , former candidate division WS3 (Rinke et al., Methanoregula could not be uniquely associated with either the , receded and gave way to the phylum South African Methanoregula Bathyarchaeota Latescibacteria Actinobacteria Methanosarcina , and 4-29 and Methanomicrobiales (Fig. 6). Finally, usion and cell migration can occur in soft methane-saturated clays and may re- Methanolinea In general, the LGM horizon (Fig. 2b) coincided with a period of active hydrology ff within the lake basin,et with al., both overflow 2007),Throughout and Patagonia, glaciers but active started waning inflows reduced fromtion maximum into extent vegetation phase to the (ca. initial in lake deglacia- 25–22 ka),2010). (Haberzettl the promoting Overall, periglacial catchment the and sedimentary wind-relatedpeared (Haberzettl erosion record reliant (Hein et of on et Laguna mass al., al.,shifts Potrok movements in 2009). and Aike productivity tephra during layers (Hahn thiscorresponded (Fig. to period et 2c) a ap- al., period triggering of 2013). small-scale lake In level rise contrast, after the an extreme Holocene lowstand (Anselmetti horizon et (Fig. al., 2a) 4 Discussion 4.1 Paleoclimatic and geochemical conditions revealedThe by sediments sedimentation regime ofpendent Laguna on Potrok climatic variations Aike and overerosion river the and inflows last as reworking water 51 of kaing level the fluctuations was glacial led catchment times mainly to gave (Coronato de- shore moister way et to conditions al., regression promoted 2013). phases transgression and phaseset Dry and multiple conditions al., pelagic gravity dur- events, 2007; conditions whereas istry (Haberzettl Gebhardt reflected such et historical al., changesand and 2012; sedimentary was processes. interpreted Ohlendorf Lithological in(Wastegård et terms horizons et of concealing al., al., paleoconditions 2013) rhyolitic 2013). and tephrasulphate mafic material Pore concentrations sands (Fig. contributed water 3d). to Water geochem- punctual columnto increases salinity subsaline in rapidly iron conditions shifted and from during freshwater site the (Killian and YD Lamy, 2012; as Pollock the andration Bush, and position 2013), lake of resulting level in the decline. elevatedet At wind Westerlies al., this evapo- moved 2013), time, to resulting the in the basincentrations nitrogen-limiting also conditions (Fig. became (Zhu endorheic et 3d) (Ohlendorf al., withintrophic 2013). Phosphate level Holocene con- of sediments, the basin, althoughOM were degradation potentially instead by microorganisms reflecting considered (Vuillemin representative past et of al., post-depositional 2013a, 2014a). 1994). Other sequences specific to the LGM horizon clustered with Nitrospirales 2014; Youssef et al., 2015). Finally, sequences related to Marine Benthic Group D, whereassequences. the LGM assemblage was dominated by SAGMEG Goldmine Group (SAGMEG). In detail,to methanogen sequences correspond with depth sediments and, while uniformly presentters in associated every with sample the below main 2 samplingand m intervals depth, Holocene (Fig. formed 5). horizons clus- Direct (Figs. comparisonblage of 5 mainly the LGM and consisted 6) of revealed that the Holocene archaeal assem- Bacteroidetes Holocene or LGM horizonformed separate (Figs. clusters 2 (Figs. 4 and and 4), 6). although3.2.4 their respective sequences Archaeal clone still libraries Di work layered microbial communities. Nevertheless, archaealthe sequences Holocene obtained from recordwith provided evidence depth for (Figs. gradualpresence 5 evolution of and of phyla 6). these Marinemeters The Group1 assemblages are dominated uppermost and by Marine meter sequencesmerly Benthic of of Miscellaneous Group Crenarchaeotal sediment B. Group mainlysemblage The (Evans plus revealed underlying et the the two al., phylum Marine5 2015), m Benthic while depth, Group the D same constituted as- the next meter. Below aceace 5 5 25 20 10 15 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , Syn- , and 2 Chloroflexi Methanomi- methyl com- Methanoreg- and CO 2 and (Jiang et al., 2008) may reflect a dynamic Archaea Bathyarchaeota (Bräuer et al., 2011), a taxon Archaea (Jackson et al., 1999) and have been described recently as Methanosarcina equally present (Pachiadaki et al., to (Moe et al., 2009) and widely abundant erent geochemical surroundings (Fig. 3d). ff , although present in every sample, formed Syntrophus Atribacteria Methanoregula 18361 18362 sequences (Rinke et al., 2013) dominated the Bacteroidetes Methanolinea and Dehalogenimonas Bathyarchaeota Aminicenantes (Liu et al., 2011). Genes from alkalotolerant species, such as and Actinobacteria (Nakagawa et al., 2006) and Marine Benthic . Such substrate evolution during prolonged OM diagenesis could promote Atribacteria (Vuillemin et al., 2014b). Marine-related sequences also shifted from Group 1 to In the organic-rich Holocene clays buried at 5 m depth, 16S rRNA gene sequences Overall, microbial populations were defined according to an apparently depth- Taken together, these assemblages suggest that sedimentary microorganisms first transition in the subsurface biosphere during early diagenesis ofdependent OM. trend reflecting(Vuillemin the et receding al., activity 2014a).tervals and However, (Fig. within slow 2) this deathand appear trend, f) of to the for microorganisms mid-Holocene which be genetic(Figs. and preferentially 4, analyses 5 LGM colonized showed and in- distinct 6) sedimentary corresponding assemblages with horizons of their microbial di (Fig. consortia 3e provided evidence for the presence of pounds (Evans et al., 2015). Thus, the present series of First, at 5 m depthactive (ca. microbial 5 kaBP), assemblage capable 16S of rRNAdation on-site gene (Wüst methane sequences et production revealed during al., aproductive OM predominantly 2009; degra- lake Vuillemin conditions et (Fig. al., 2).sequences 2014b) In exhibited during contrast, an microbial at intervallithotrophy 30 populations m of (Figs. depth characteristic subsaline 4 (ca. and of and 25clayey kaBP), sediments 5) sulphate overlain retrieved when by reduction fine an and basaltic unproductive material freshwater (Fig. 2). lake episode deposited which was identified introphomonadaceae association with Clostridia were also detectedment (Figs. labile 4, organic compounds 5degradable (Wüst and by et 6). al., 2009), These whereas organisms, cellulose when and lignin active, are mainly fer- unique clusters according tochaeal sampling assemblages depths have also (Figs. been 5below identified and the in 6). seafloor marine Similar (Vigneron environmentspected from changes et to 1 in al., degrade to ar- 2014). 10 complex m compounds In organic (Lloyd this matter, et latter such al., case, as 2013; marine cellulose, Meng groups proteins et are and al., ex- aromatic 2013), and 2011). energy-conservative heterotrophic anaerobes whichof act sugars either as or primarysyntrophic secondary fermenters propionate fermenters catabolism of (Sieber organic et al., acids 2012). (Nobu Additionally, et GIF9 al., 2015) capable of Benthic Group B and then BenthicMEG Group D sequences before disappearing were below only 5semblage m first depth. at identified SAG- 29.8 m at depth. 7.8 m depth, but then dominated the as- ula Holocene horizon (Figs. 4springs and (Hugenholtz 6). et These al.,ments (Inagaki microbes 1998), et were but al., are initially 2003). often described In addition, abundant from in hot anaerobic marine sedi- 2009) due to strong Westerlies(Waldmann and et positive al., temperature excursion 2010; in Kilianwith South and important Patagonia Lamy, nutrient 2012). fluxes Periods intocasens of the lake et closed-basin, level al., elevated rise primary 2015),conditions correspond productivity (Vuillemin and et (Re- al., high 2014a). microbial colonization of4.2 the sediment Preservation under and pelagic interpretation of fossilAnalysis DNA of ATP detectionulations and were sedimentary abundant cell and densitiesriod metabolically showed (Fig. active that in 3e). microbial the Archaeal pop- assemblages sediment phylotypes within of (Figs. the the 5 uppermost Holocenemethanogen and 10 varied pe- m 6) with depth of indicated from sediment. gradual evolution For of example, the the predominant which are closely related to in organic-rich anoxicet sediments, al., are 2014). presumably homoacetogenic fermenters (Hug degraded the labile OMthat from in algae turn, before these generatingcrobiales gases served fermentative as H substrates for methane production by 5 5 25 20 10 20 15 15 10 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | erent ff Bacteria (Takai et al., (Hoover et al., Aminicenantes and (Fig. 6). This pattern Archaea (900 bp) and Spirochaeta Archaea Bacteroidetes Bathyarchaeota and SAGMEG erent intervals. In addition, the presence ff , which is consistent with the degradation of 18364 18363 (Liesack et al., 1994), Proteobacteria δ Chloroflexi and 4-29 have been shown to be abundant in temporary streams (Pachiadaki et al., 2011) and S production during sulphate reduction was thought to promote Acidobacteria 2 have been recently presented as anaerobes mediating the turnover of Proteobacteria Nitrospirales δ Actinobacteria Several lines of evidence suggested that patterns of microbial activity and compo- Some sedimentary nucleic acids could not be unambiguously associated with a spe- Microbial communities recovered from ca. 2 ka old LGM sediments also showed sition did not ariseFirstly, phylogenetic from results contamination of fromsingle OTU ancient Holocene in sediments common and (Fig. with LGMtwo 4). modern Secondly, sediments hours sedimentary microbes. ATP displayed after activity recorded onlythat core less one measured than recovery substantially showed later,ratory the analyses and same (Supplement). was pattern Thirdly, alsolithological deep of characteristics coherent sediments ATP of lacked with concentration the any more youngersulphides, than of lower sediments extensive the salinity, (Fig. labo- pigment chemical 3), composition, or including(Supplement). color framboidal of Finally, iron clays 16S and rRNA absence gene of gas sequences vugs of kinds of environmental2015). change Global with patterns highthe of functional main bacterial drivers redundancy distributiontion of (Sunagawa in in community et the the composition environment al., tary oceans were have environments (Raes temperature shown (Lozupone et and that andanaerobic al., primary Knight, metabolisms 2011) produc- 2007). appeared and Inlink as salinity to deep pore-water and sedimentary the sulphate substrate settings,(Kallmeyer concentrations dominant et (Orsi OM type al., activities, et 2012), in al., with consistentlythe sedimen- 2013) with methanogenesis cell the and zone present sedimentation densities overlies microbial rates the assemblages in sulphate although reduction zone. (1400 bp) analyzed in ourtact phylogenies were bacterial considered cells long rather enough thancould to arising have derive also from from been extracellular in- extracted DNA.2011), from However, we because dead DNA urge and dormant cautionrial intact to growth cells avoid and (Corinaldesi interpreting active et sediment metabolisms al., in populations the in di terms of bacte- sequences were not specificallyof associated either with the environmental Holocene or metabolic and features LGM horizons. Indeed, microbes easily tolerate di cific sedimentary interval.studies For example, (Kubo consistent et with al., their 2012; ubiquity Farag noted et in al., other 2014), presence of sulphate-reducing 2001). Substantial H sustained metabolic activity,sequences albeit from at this lower LGM population assemblage densities (Figs. (Fig. 3e). 4, Genetic 5 and 6) recorded the important the recycling of end productsserved with and autotrophic syntrophic methanogenesis hydrogen and consumption,Wüst homoacetogenesis as et (Inagaki presently et al., ob- al.,assemblage 2009). 2003; arose Such from a metabolicgeochemical complementarities pattern cycling involved of also in OM. Thus, the suggests microbialification degradation populations that within displayed and an the the age-related strat- sedimentary finalproductive regime environment Holocene (Fig. made microbial 6). possible by a steady pelagic and the alteration of maficlithotrophic minerals species and (Johnson, providetion molecular 1998; hydrogen, of Blanco iron authigenic et and sulphidesconditions al., sulphur such at 2014) to as and such framboidsproduction eventually sedimentary (Vuillemin (Schubert the interfaces et et forma- (Fig. al., al.,sulphate 2) 2013a). and 2011) iron In would and reduction instead. general, select greatly However,among syntroph for limit sequences a were any also microbial identified methane secondary assemblage metabolites capable such of asmada propionate et (Liu al., et al.,were 2007). represented 1999; Otherwise, by De organotrophs Bok capable et al., of 2001; refractory Ya- OM degradation 2003), under dry conditions (Febria et al., 2015). multiple complex algal polymers inwhereas deep anoxic aquatic habitats (Youssef et al., 2015), of sequences was interpretedwith as volcanic material arising that fromLatescibacteria could the act intercalation as of sources organic-poor of clays iron and sulphate (Fig. 3). Finally, 5 5 15 20 10 25 10 15 20 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Acti- , Planctomycetes 18366 18365 erent types of bacteria (Sinninghe Damsté and ff S as an electron donor for photosynthesis, and so 2 were among the heterotrophs (Fig. 4) which can produce Bacteroidetes and ka et al., 2013). Moreover, alteration of these compounds can form isorenieratane č Comparison of fossil pigment and sedimentary DNA assemblages suggested that the initial nucleic acidtogeny composition following deposition. of For sedimentsatene example, could from although be brown high modifiedwere varieties concentrations by recorded of of in microbial green isorenier- the on- sulfurevant sediments carotenoid-producing throughout bacteria phototrophic the (Glaeser taxa Holocene, weresubject and genetic rare to Overmann, markers in of the 2001) DNA the mid-Holoceneto analysis. rel- intervals anaerobic Early taxa research which suggested used that H isorenieratene was unique the pigment preservation index suggests thatduring pelagic intervals production of is elevated accurately sedimentation recorded (Fig. 3c). could be used as(e.g., an Leavitt index et of al.,like the 1989). compounds can penetration However, be more of produced recent by light di work to suggests anoxic that waters isorenieratene- or sediments Hopmans, 2008; Krügel etinvolved al., in 1999; isorenieratene Krubasik biosynthesis and maywal, Sandmann, be 2005). 2000) Therefore found environmental in and 16S cyanobacteria thatmeans rRNA as genes to sequences well evaluate may (Phad- the provide an compositionmann alternative et of al., past 1999; bacterial Coolen phototrophic and communities Overmann; (Over- 2007). In this paper, nobacteria Jehli in sedimentary environments (Brockstion and that Pearson, these 2005). heterotrophic taxa Of arepersisting interest common in long is ancient after the algal associated mats observa- phototrophic assemblages,et often bacterial al., species 2012; have Cole been lost ettaxonomic (Antibus al., blasts (Fig. 2014; 4) Lage confirmed andof that Bondoso, anoxic these 2011 aquatic heterotrophic and and species sedimentary 2015).et are lacustrine Present characteristic habitats al., results (De of 2007; Wever et Songwould al., result 2005; et Schwarz in al., selective 2012). recycling Such of bacteria colonization (Antibus of et algal al., material 2012), after and suggest deposition that carotenoids pigments (Hahn et al., 2003; Warnecke et al., 2005; Fukunaga et al., 2009; of extracellular DNA amidst DGGErelative band bacterial sequences features (150 at bp)rate 30 may m of have depth the increased related (Fig. the compounds 3f),in depends with ancient on sediments the the (Corinaldesi restriction maintenance et that of al., an the 2007 activity turnover and by 2008). 4.3 microbes Preservation and interpretation of fossilAnalyses pigments of bacterial and algaltivity pigment during concentrations indicated the high Holoceneperiod primary (Fig. while produc- 3b). oligotrophic During conditions theshort characterized LGM, intervals pigment the of concentrations elevated last were productivityflows. glacial variable Profiles related and of to exhibited preservation mass index movement2013) (Fig. suggest and 3c) the tephra presence and layer of organic in- primarysediments, macro respectively and remains (Fig. reworked 3b (Hahn OM and in et c). pelagicfraction al., Still, and of bacterial gravity-related sources the constituted organic anearly important sedimentary YD, strengthened record winds (Fig. andprimary 3a nitrogen producers and limitation (Zhu b). appeared et Duringof to al., the be cyanobacteria 2013). late unfavourable clearly However, for LGM concentrations increasedtance of and at of pigments this nitrogen indicative time fixationYD, (Fig. deglacial processes warming 3b), in and revealing nutrients the awith inflows basin associated potential a (Mayr with lake impor- vegetation et lowstand recoveryindicated al., along improved conditions 2009). by for During elevated planktonic the concentrationsote production : late of prokaryote by pigment diatom eukaryotes, ratios as pigmentsprimary (Fig. (diatoxanthin) productivity 3b and and appeared eukary- c).sity restrained During of (Fig. the the 3b), early WesterliesChanges possibly Holocene, and in planktonic lasting due basin nitrogen-limited to morphometry, conditionstentially increased light (Zhu bias inten- penetration, the et correlation bottom al., between1993; 2013, water fossil Leavitt 2014). stratification and and algal can Hodgson, abundance po- factors through 2002). vary time Primarily as (Leavitt, because a of function of the lake maar level morphology, and these gravity events. Fortunately, interpretation of 5 5 10 15 25 20 10 15 20 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | and and char- 500bp). ∼ in fine Atribacteria Actinobacteria , 4-29 (Febria et al., 2015) as the dominant taxa (Figs. 4, Planctomycetes Nitrospirales and the SAGMEG group. 18367 18368 Methanomicrobiales and Proteobacteria δ (Youssef et al., 2015). Surrounding geochemical conditions asso- Atribacter liated to ) may have also emerged from the early degradation of algae and mi- ffi Latescibacteria Further research using a combination of DNA and other proxies will advance our un- During the Holocene interval, elevated rates of OM deposition under pelagic regime During the LGM period, limited nutrient inputs to the water column and volcanic in- acteristic of conditions associated with past environmental factors, with and ciated with the formation ofa OM-poor subsurface but biosphere iron- capable of andsequences sulphate sulphur-rich a reduction sediments and selected lithotrophy, mainly for including ognize that our analytical platformLaguna represents Potrok Aike only sediments an and insightlikely that into prevented the genetic the length variations of detection of the of targeted partially sequence (1400 preserved bp) phototrophic bacteria ( derstanding of the mechanisms formingat fossil present, nucleic it acid is assemblages. unclear For whetherimentary example, microorganisms environments actively or grew for whether centuries theirstudy in sequences past period sed- were (Hugenholtz merely et entombed al., during 1998; the Nakagawa et al., 2006). Similarly, we also rec- methanogens, sulphate reducers and SAGMEGand as LGM dominant sediment, species respectively. in the Holocene However, the rapid development ofscriptomic single analysis cell sequencing will technologies enable andsive a parallel metatran- refined sequencing view will of provide deep extensive phylogeny biosphere of activities, environmental while DNA. mas- 5 Conclusions Climatic and lacustrinethe conditions main factors at defining sediment the geochemistrypreservation time of and microbial microbial of sources substrates. occurred Preferential sediment duringburial, synsedimentary deposition leading processes appear to and after changes tositions. in Sedimentary be the niches DNA atdevelopment assemblages the of with time the of conserved deposition subsurface pigmentated exerted biosphere. compo- with initial Changing sustained constraints geochemical metabolic on activityover conditions the time performed associ- and a defined selection the final of microbial viable assemblages. microorganisms Identified taxa were Bacteroidetes crobial biofilms (see above).the Seemingly, expense these of heterotrophic phototrophic speciesintact species actively only their (Antibus grew respective et at pigments. Phylogenetic al.,face sequences 2012; representing biosphere the Cole were subsur- et characteristicmetabolism, al., of with 2014), those leaving exhibiting solely anaerobic heterotrophic from ancient assemblages seemedtions to refer (Cole to et complex autotroph-heterotroph al., interac- 2014) and likely included led to increased pigment concentrationspotentially in derived the from sediment ancient (Figs. assemblages 2a (i.e. and 3b). Sequences flows engendered low primary production mainlymicrobial by mats bacteria, reworked to presumably the in basin the during form gravity of events. Similarly, sequences issued sedimentary genetic assemblages maybetter be conserved. altered while fossil pigment composition is 4.4 A model for ancient andTogether, extant our microbial results assemblages suggest thattrol prevailing climatic on conditions microbial exerted substrates thefined at initial the the con- time subsurface ofdepositional biosphere deposition alterations. (Fig. assemblages In 2), and this whichorganic set subsequently model, material de- the climate to regulates pathway thealgal the lake for productivity influx basin, further and of which sedimentation post- in organic(e.g., of turn salinity) and particulate and determines in- material. sediment waterthe Water lithology column sediment column then chemistry, (Figs. conditions interact 2 and to 3) determine and final dominant geochemistry subsurface assemblage. of 5 and 6). Theyindicative reflected of the advanced OM sediment degradation surrounding during geochemical early conditions diagenesis. and were 5 5 20 15 25 10 15 25 10 20 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 18370 18369 in the environment, Appl. Environ. Microb., 65, 1731–1737, 1999. A. Vuillemin carried out field sampling, 16S fingerprinting techniques and We thank funding support from International Continental Scientific Drilling , L. G., Hall, B. L., Baeseman, J. L., and Blackwood, C. B.: Cultivable bacteria ff Acidobacterium er among major historical climate zones and that some fossil elements even sus- The help during cloning procedures of J. Pawlowski, M. Holzmann, F. Lejzerowicz, L. This study provides new evidence for mechanism underlying the preservation of sed- Poinar, H. N.: Ancientand DNA genetics, BMC from Evol. lake Biol., 11, sediments: 1–15, bridging 2011. thefrom gap ancient algal between mats paleoecology from1014, the 2012. McMurdo Dry Valleys, Antarctica, Extremophiles, 16, 105– dorf, C., and Zolitschka,seismic B.: stratigraphy of Environmental Laguna history Potrok of Aike, Sedimentology, southern 56, Patagonia 873–892, 2009. unraveledtrine by sediments the through scientific drilling, Int. J. 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S.: 5 25 30 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | of (b) depth Water [m] -20 -40 -60 -80 Site Site -100 5022-2 5022-1 Depth below lake bottom [m] from the volcanic 0 10 20 30 40 50 60 70 80 90 93 ff

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A1 A2 H YD LGM

0 5

50 10 20 30 40 15 25 35 45 Age [ka BP] [ka Age and bathymetric map During Holocene times with C) PelagicPelagic GravityGravity Tephra Tephra (a) (a) B A 1 km snowmelt B) events gravity stratification subsaline tephra Site 1 Site 1 tephra 66° 18384 18383 freshwater volcanic inflows 68° low productivity Westerlies Rio Gallegos high productivity Site 2 Site 2 mixing evaporation 70° Aike Potrok 72° Laguna During LGM times with lake highstand and active overflow, freshwater -15 m asl +22 m asl (b) 74° D D Tierra del Fuego 76° ...... HORIZON B Surface T° -3 °C Age 25 ka BP depth 138 m Water Sed. depth 29.8 m NaCl eq. 0.4 % 0.2 % TOC HORIZON A Surface T° -1 °C Age 5 ka BP depth 101 m Water Sed. depth 4.9 m NaCl eq. 1.2 % 1.5 % TOC Lithology log in age scale displaying the 5 units defined in Kliem et al. (2013), A) HOLOCENE B) LGM (c) Map of Southern Argentina displaying the location Sketches depicting paleoenvironmental conditions at Laguna Potrok Aike. From left A) 50° 52° 54° catchment. and stratigraphic sequence in meter(black) scale layers. displaying Time pelagic abbreviations (white),Maximum stand gravity (LGM), (grey) Antarctic for and events Holocene 1 tephra (H), (A1) and Younger 2 Dryas (A2). (YD), Last Glacial active Westerly winds, lake lowstand,basin subsaline and conditions catchment. and highconditions, primary low productivity primary in productivity in the the basin and inflows restricted to runo Figure 2. to right: Climatic and lacustrinelocation parameters; of sagittal view the of 16S the rRNA basin; core library; picture and with stratigraphic sample sequence. Laguna Potrok Aike showinggeomicrobiological samples the were two retrievedpigment drilling from analysis sites cores were obtained at (Zolitschka from site et cores 5022-1, at al., whereas site 2006). 5022-2. sediments Pore for water and Figure 1. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | (e) Number of (f) , with peaks indica- - + m a m 93 10 age age age depth depth depth 4-29 OP8) (OP9) ( (WS 3) Atribacteria Microbial activity Microbial structure Bacteroidetes Clostridia Actinobacteria Aminicenantes Chloroflexi Planctomycetes Microbial cell density Nitrospirales Spirochaetes Acidobacteria Dominant Species Proteobacteria 45 Latescibacteria d Dominant Species 40 50 A1 A2 DGGE 40 50 60 70 80 90 Age [ka BP] to pheophytin 2+ 30 35 Fe a are the main taxa encountered in the 30 B ATP 2- Depth below lake bottom [m] Depth below ,6-diamidino-2-phenylindole (DAPI) cell 4 Pore water geochemistry DAPI 0 Bacteria Archaea 3- ATP and cell count detection SO 4 (AB517712) 15 25 PO sp. (NR028963) 10 20 1 x 4.97 m - Holocene 10 20 1 x 4.97 m - Holocene ) H YD LGM 5 1 x 29.77 m - LGM A

0 0 (DQ317601)

1 x 4.97 m - Holocene (EU679418) 1 x 29.77 m - LGM

15 10 5 0 0 0 1000 500 020406080 ·71·E8 5·E7 0 wetland 0 200 100 MSBL9 Pacific abyssal sediment (FJ746095) OTU 13 ( ) 44

OTU 12 ( ) Dongping

cl × [cell e ] sed [RLU] [ppm] [ppm] [nb of bands] of [nb 2 x 29.77 m - LGM Anaerophaga gr MSBL9 SO -CH transition sediment (FJ455909) OTU 15 ( ) MSBL9 Japan Holocene mud (AB300077) 1 x 4.97 m - Holocene D) E) F) log unit size OTU 11 (OTU 11 ) MSBL9 de Daimiel (FJ517127) Tabla OTU 14 ( ) (AY254883) 2 x 29.77 m - LGM grain MSBL9 tar oil contaminated sediment (EU266844) Cytophaga fermentans 1 x 29.77 m - LGM 1 x 29.77 m - LGM (AB243672) 93 ) OTU 16 ( ) (AF373921) 1 x 4.97 m - Holocene BD2−2 deep−sea sediments (AB015532) 1 x 4.97 m - Holocene OTU 24 ( ) age age age TN OP 1 x 4.97 m - Holocene depth depth depth 1 x 29.77 m - LGM TOC Pir4 lineage Florida sea grass sediment (EU488313) vadinHA17 LakeOTU 27 ( sediment (GQ472362) ) 18386 18385 vadinHA17 Lake Kinneret sediment (AM086122) OTU 26 ( ) 2 x 29.77 m - LGM 1 x 4.97 m - Holocene 1 x 29.77 m - LGM 1 x 29.77 m - LGM 1 x 4.97 m - Holocene OTU 34 ( ) Diatoms Japan vadinHA17 Lake Anderson sediment (EU283477) OTU 25 ( ) OTU 9 ( aa OTU 28 ( ) Scalindua brodae AKAU3564 South China Sea (EU048688) seagrass bed Aminicenantes 1 x 4.97 m - Holocene OTU 35 ( ) OTU 31 ( ) GIF9 methane hydrate (AY093456) Spirochaeta OTU 30 ( ) OTU 33 ( ) GIF9 Peru methane hydrate (AB177135) OTU 10 ( ) Anammoxoglobus propionicus GIF9 in situ reactor system (AF407200) Cyanobacteria 2 x 4.97 m - Holocene 1 x 4.97 m - Holocene 1 x 29.77 m - LGM 1 x 4.97 m - Holocene GIF9 sediment (EU488317) 1 x 4.97 m - Holocene 4 x 29.77 m - LGM 1 x 29.77 m - LGM 1 x 29.77 m - LGM 1 x 29.77 m - LGM Bellilinea caldifistulae GIF9 biomat cenote La Palita (FJ901649) (GU993263) 1 x 4.97 m - Holocene Yellowstone Spirochaeta americana OTU 32 ( ) 4−29 Peru methane hydrate (AB177154) leachate sediment (HQ183815) OPB41 active tundra soil (EU644120) OPB41 Taiwan mud volcano (JQ245645) OPB41 Taiwan OPB41 biomat cenote El Zacaton (FJ485014) 7 x 4.97 m - Holocene OPB41 Amsterdam mud volcano (HQ588500) OPB41 Peru gas hydrate (AB177140) Holophaga OTU 8 ( ) Dehalogenimonas lykanthroporepellens OPB41 Lake Tanganyika anoxic waters (DQ463730) OPB41 Lake Tanganyika Green sulfur bacteria OTU 44 ( / ) Uncultured biomat Le Zacaton (FJ485365) OTU 41 ( ) OTU 7 ( ) OTU 40 ( ) OPB41 hydrothermal oxides (JN860326) 4 x 4.97 m - Holocene OTU 42 ( OTU 43 ( ) 2 x 29.77 m - LGM OTU 23 ( ) OTU 29 ( ) 2 x 29.77 m - LGM MSB 5B2 Japan Holocene mud (AB300063) 1 x 29.77 m - LGM (AF126282) and 45 Amsterdam OTU 22 ( ) 2 x 29.77 m - LGM sp. (HQ397226) 1 x 4.97 m - Holocene OPB95 Nankai trough sediments (AB013270) ake Taihu s ake Taihu OPB95 methanogenic lotus pond (AB426208) D8A−2 black rust formation (AB260059) WS3 Peru methane hydrate (AB177136) OTU 21 ( ) Eukaryotes / Prokaryotes 40 50 WS3 NorthSea (GQ246366) Yellow Preservation Chl / Pheo OTU 6 ( ) OPB95 anaerobic UASB reactor (FJ535533) OTU 18 ( ) 4 x 29.77 m - LGM AKIW659 Mexico cenote biomat (FJ484803) AKIW659 Japan Holocene mud (AB300082) D8A−2 microbial mat (FJ206828) OPB95 biomat cenote La Palita (FJ901520) OTU 20 ( ) Nitrospirales 12 x 4.97 m - Holocene OTU 17 ( ) OPB95 deep−sea mud volcano (HQ588598) A1 A2 KF−JG30−18 uranium tailing (AJ519381) D8A−2 gold mine deep groundwater (DQ088758) 1 x 4.97 m - Holocene OTU 19 ( ) KF−JG30−18 biogenic Fe−nodules (AB254785) (AF002671) OTU 38 ( ) 1 x 4.97 m - Holocene OPB95 L ediment (FJ755774) 1 x 29.77 m - LGM 1 x 4.97 m - Holocene OTU 36 ( ) 40 50 60 70 80 90 OP9 petroleum reservoir (EU721783) Desulfovirga 1 x 4.97 m - Holocene OP9 Japan Trench sediment (AB015269) OP9 Japan Trench OTU 37 ( ) Age [ka BP] OP9 Kazan mud volcano (FJ712605) OP9 marine sediment (FN429787) Syntrophomonadaceae OTU 39 ( ) 30 35 Syntrophus aciditrophicus 7 x 29.77 m - LGM OTU 2 ( ) () Lake Kinneret sediment (AM086113) Smithella propionica OTU 3 ( ) Pigment ratios Diatoxanthin OTU 1 ( ) Aphanizophyll Isorenieratene Organic content SVA0485 mine shaft (GQ921432) SVA0485 OTU 4 ( ) 30 Specific pigments SVA0485 MoSVA0485 mine (EF562249) Pore water concentrations for phosphate, iron and sulphate. B 0.10 Biomat cenote El Zacaton (FJ484529) SVA0485 Lake Hongku sediment (HM243935) SVA0485 SVA0485 cenote El Zacaton (FJ484529) cenote El Zacaton SVA0485 OTU 5 Depth below lake bottom [m] Depth below Echinenone Canthaxanthin scale: 15 25 (d) Desulfobacca acetoxidans 10 20 Atribacteria 10 20 H YD LGM 5

A

0 0

2.0 0 1.0 . 0.2 0.1 0100 50 2 2 4 4 0 80 6 6 0 200 0 0 100 100 100

- +

T C] OC [nmole × gr × [nmole [%] [%] [ppm] -1 A) B) C) Specific pigments usually accounting for green sulphur bacteria (isorenier- (b) Maximum likelihood phylogenetic tree of bacterial 16S rRNA gene sequences Paleoclimatic and geomicrobiological multiproxy. Top: Stratigraphic sequence of La- Total organic carbon (TOC), total nitrogen (TN) and organic phosphorus (OP) from (a) Preservation index based on the ratio of chlorophyll (1400 bp) recovered at(blue types) 4.97 sediments. and 29.77 m depth from Holocene (orange types) and LGM Figure 4. Holocene organic-rich pelagic sediments, whereashorizon sulphate reducers composed are of dominant intercalated intypes the volcanic signify LGM mafic database sands references with and sequence hemipelagic accession sediments. numbers Boldface in parentheses. Figure 3. guna Potrok Aike,grey). followed by grainbulk size sediment. with clay (black), silt (dark grey) and sand (light counts respectively used asbands indices from of microbial DGGE activity gelscommunities. and is Bottom: population used Lithology density. as logand relative displaying their the index corresponding five of climatic units structural intervals. established shifts by in Kliem bacterial et and al. archaeal (2013) atene), cyanobacteria (echinenone, canthaxanthin, aphanizophyll)(c) and diatoms (diatoxanthin). tive of increased preservation associatedto with high prokaryotic sedimentation pigments. rates, andOn-site ratio adenosine of triphosphate eukaryotic (ATP) detections and 4 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , ) as they , whereas (MCG) Methanolinea Bathyarchaeota Marine Benthic Gp D Marine Benthic Gp B Marine Gp 1 SAGMEG Bathyarchaeota Methanomicrobiaceae Methanoregula Methanosarcina Methanolinea Bacteroidetes , Nb of OTUs Sediment depth [m] Nb of sequences Bathyarchaeota Gp 1 24 10 (MCG) Marine and Gp B Gp D 6 11 Bathyarchaeota Goldmine Gp (AB517987) South African Thermoprotei Marine Benthic Marine Benthic Dominant Species Methanomicrobiales (EU887810) (HI592326) OTU 55 (1 x 7.81 m) 21 (AY196681) 13 Dominant Species (AB479390) (EU156000) acidic bog lake (AF481343) 4 x 4.97 m- Holocene (AY260430) (AF028693) OTU 59 (1 x 0.25 m) (AB162774) alpine lake (AM851080) 10 27 acidic fen (EU155976) permafrost (FJ982666) OTU 58 (1 x 2.51 m) 11 x 4.97 m11 - Holocene Lake Hongku (HM244180) OTU 47 (1 x 0.25 m) OTU 46 (1 x 0.25 m) OTU 54 (1 x 7.81 m) OTU 57 (1 x 0.25 m) Methanospirillum hungatei OTU 60 (1 x 0.25 m) Methanoregula boonei OTU 51 (1 x 2.51 m) OTU 48 (1 x 0.25 m) 11 x 29.77 m - LGM 11 Planctomycetes 12 27 OTU 53 ( / 2 x 7.81 m / 1 x 9.37 m) Archaea OTU 50 (1 x 1.90 m / 1 x 2.51 m) Methanoregula sp. Alpine lake sediment (AM851082) Rich minerotrophic fen (EU155985) OTU 45 (2 x 0.25 m / ) Methanosarcina acetivorans OTU 49 (1 x 0.25 m / 17 x 1.90 m) Sulfur-rich sinkhole sediment (EU910622) Salar de Huasco (EU481619) Methanoplanus petrolearius 1 x 4.97 m- Holocene , OTU 56 (4 x 0.25 m / 4 x 0.55 m / 12 x 2.51 m) Methanoregula formicicum Methanolinea 2 x 4.97 m- Holocene Methanolinea tarda Methanosphaerula palustris Methanosarcinales Eel River methanic sediment (GQ356893) x 7.81 Methanosarcina lacustris Methanoregula sp. x 7.81 m Methanosarcinales 1 x 4.97 m- Holocene 8 Methanoculleus marisnigri 42 OTU 52 (3 x 9.37 m) x 7.81 OTU 26 ( ) x 7.81 1 x 29.77 m - LGM 1 x 29.77 - LGM Lake Hongku upper sediment (HM244093) Lake Wivenhoe sediment (HQ330676) Lake Wivenhoe OTU 39 (OTU 41 (1 x 0.55 m) ) OTU 61 (2 x 7.81 m) x 7.81 OTU 40 (4 x 0.55 m) OTU 15 (1 ) 1 x 29.77 m - LGM OTU 66 (1 ) x 7.81 1 x 4.97 m- Holocene OTU 25 (1 x 0.55 m) 1 x 29.77 m - LGM 3 x 29.77 m - LGM Okhotsk seep (FJ404091) methane 7 OTU 16 (1 ) 41 OTU 43 (1 x 0.25 m) 0.6 1.9 2.5 4.9 7.8 9.4 29.8 OTU 65 ( ) 1 x 4.97 m- Holocene OTU 14 (1 ) OTU 44 (1 x 0.25 m) 1 x 29.77 m - LGM OTU 12 (1 x 9.37 m) OTU 35 (1 x 1.90 m) OTU 63 ( ) 1 x 29.77 m - LGM OTU 17 (1 ) OTU 27 ( ) OTU 64 ( ) 1 x 4.97 m- Holocene OTU 18 (1 ) Hong Kong harbour sediment (EF203585) 1 x 29.77 m - LGM 1 x 4.97 m- Holocene Spain freshwater sulfurous lake (EU683399) OTU 62 (4 x 7.81 m / 2 x 9.37 m / ) Obsidian pool prime community (AY861953) South China subseafloor sediment (EU385847) 3 x 29.77 m - LGM Ohio-Mississipi confluence (GQ906595) OTU 37 ( ) OTU 19 (2 x 9.37 / ) Lake Hongku middle sediment (HM244135) OTU 24 ( ) Nirano mud volcano (HQ654860) Oyster shell (EU370093) 17 35 0.3 Holocene mud sediment (AB300143) OTU 42 (14 x 0.25 m / 31 x 0.55 m) OTU 13 (4 x 7.81 m) Germany marsh sulfidic spring (AM055707) OTU 23 ( ) OTU 38 (1 x 1.90 m) OTU 36 (1 x 1.90 m) Finland boreal forest lake (AJ131315) x 2.51 m Pearl River estuary sediment (JX870390) South Africa deep gold mine water (AB050223) OTU 34 ( ) Petroleum contaminated soil (AB161328) OTU 22 ( ) Lake Baikal methane seep (KJ922116) Methanomicrobiales 18388 18387 Russian caldera spring (GQ328203) Waikato coalfields sediment (AM883005) Waikato OTU 8 (1 x 0.25 m) OTU 20 ( ) OTU 33 (1 x 0.55 m) Spain freshwater sulfurous lake (AM076830) Submarine permafrost (FJ982763) OTU 21 (2 x 9.37 m / ) Serpentinization alkaline groundwater (FN690972) OTU 31 (1 x 2.51 m) OTU 9 (1 x 2.51 m) OTU 32 (1 x 2.51 m) Mt Kunlun permafrost soil (JQ684417) OTU 11 (1 x 2.51 m) OTU 11 0 OTU 10 (1 x 2.51 m) Tibet hypersaline lake (EF201709) Tibet OTU 7 (3 ) Chloroflexi 90 80 70 60 50 40 30 20 10

OTU 30 (2 x 1.90 m)

100 OTU 29 (3 x 1.90 m / )

OTU 28 (1 x 0.55 m / 15 x 1.90 m / 3 x 2.51 m / )

OTU 6 (1 x 2.51 m) Cascadia margin methane hydrate (AB177287) Japan Holocene mud (AB300142) filiation [%] filiation Mongolia groundwater (JX196210) af Sequence 1 x 4.97 m - Holocene OTU 4 (1 x 7.81 m) OTU 5 ( ) OTU 2 (1 x 0.25 m) ; marine-related sequences to Group 1, Benthic Group B OTU 3 (1 x 0.25 m) OTU 1 (1 x 0.25 m / 1 x 7.81 m) OTU 1 (1 x 0.25 m / 1 x 7.81 Salt marsh sediment (EU284658) China estuarine sediment (JQ245927) China estuarine sediment 0.10 (WS 3) (OP8) 4-29 Iceland giant submarine smectite (AF411230) Iceland giant submarine smectite (OP9) scale: Proteobacteria Aminicenantes Atribacteria Actinobacteria Acidobacteria d Latescibacteria Chloroflexi Planctomycetes Nitrospirales Clostridia Spirochaetes Bacteroidetes Methanoregula Nb of sequences Nb of OTUs Sediment depth [m] 40 and 43 4.9 29.8 23 22 Bacteria 0

Maximum likelihood phylogenetic tree of archaeal 16S rRNA gene sequences 90 80 70 60 50 40 30 20 10

100

Histograms of identified phylotypes displayed in relative %, with OTU and sequence filiation [%] filiation Sequence af Sequence are known ubiquists inevolution with aquatic depth environments. of Right:Methanosarcina the Archaeal assemblages. phylotypes Methanogens indicate correspond a in gradual turn to and Benthic Group D andfrom disappear 7.8 m below 5 depth, m but depth; dominate SAGMEG the sequences assemblages are at only 29.8 identified m depth. Figure 6. numbers at theby top the and Holocene bottom, and LGM respectively. Left: horizons (i.e. Several bacterial phylotypes are shared Figure 5. (900 bp) recovered atClone 0.25, series 0.55, established 1.90, throughoutrelated the 2.51, evolution of Holocene 4.97, the record assemblages, 7.81, withending (dark a with 9.37 grey SAGMEG general sequences. and trend types) Comparatively, the from 29.77 indicate Holocene marinedepth m archaeal (orange a groups assemblage types) sediment to at depth- is methanogens 4.97 mainly depth. m composedthe of LGM archaeal assemblageand at SAGMEG 29.77 division. m Boldface depthnumbers types in (blue signify parentheses. types) database is references restricted with to sequence accession