Quaternary Science Reviews 181 (2018) 19e29

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Quaternary Science Reviews

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Archaeal community changes in Lateglacial lake sediments: Evidence from ancient DNA

Engy Ahmed a, b, c, 1, Laura Parducci d, 1, Per Unneberg e, Rasmus Ågren f, Frederik Schenk a, Jayne E. Rattray a, g, Lu Han d, h, Francesco Muschitiello a, i, Mikkel W. Pedersen j, * Rienk H. Smittenberg a, Kweku Afrifa Yamoah a, Tanja Slotte b, c, Barbara Wohlfarth a, a Department of Geological Sciences and Bolin Centre for Climate Research, Stockholm University, SE-10691 Stockholm, Sweden b Department of Ecology, Environment and Plant Sciences, Stockholm University, SE-10691 Stockholm, Sweden c Science for Life Laboratory, Tomtebodavagen€ 23A, SE-17165 Solna, Sweden d Department of Ecology and Genetics, Uppsala University, SE-75236 Uppsala, Sweden e Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, SE-75236 Uppsala, Sweden f Science for Life Laboratory, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Goteborg,€ Sweden g Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Canada h Ancient DNA Laboratory, College of Life Sciences, Jilin University, PR China i Lamont-Doherty Earth Observatory, Columbia University, 61 Route 9NW, Palisades, NY, USA j Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK article info abstract

Article history: The Lateglacial/early Holocene sediments from the ancient lake at Hasseldala€ Port, southern Sweden Received 1 April 2017 provide an important archive for the environmental and climatic shifts at the end of the last ice age and Received in revised form the transition into the present Interglacial. The existing multi-proxy data set highlights the complex 18 October 2017 interplay of physical and ecological changes in response to climatic shifts and lake status changes. Yet, it Accepted 30 November 2017 remains unclear how microorganisms, such as , which do not leave microscopic features in the sedimentary record, were affected by these climatic shifts. Here we present the metagenomic data set of Hasseldala€ Port with a special focus on the abundance and biodiversity of Archaea. This allows recon- Keywords: fi Ancient DNA structing for the rst time the temporal succession of major Archaea groups between 13.9 and 10.8 ka BP Shotgun sequencing by using ancient environmental DNA metagenomics and fossil archaeal cell membrane lipids. We then Archaea evaluate to which extent these findings reflect physical changes of the lake system, due to changes in Metagenomics lake-water summer temperature and seasonal lake-ice cover. We show that variations in archaeal Lipid biomarkers composition and diversity were related to a variety of factors (e.g., changes in lake water temperature, Last deglaciation duration of lake ice cover, rapid sediment infilling), which influenced bottom water conditions and the Paleoclimate sediment-water interface. Methanogenic Archaea dominated during the Allerød and Younger Dryas Paleoenvironment pollen zones, when the ancient lake was likely stratified and anoxic for large parts of the year. The in- Lake sediments crease in archaeal diversity at the Younger Dryas/Holocene transition is explained by sediment infilling Southern Sweden and formation of a mire/peatbog. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction states before Interglacial temperatures were reached in the early Holocene. These marked climatic shifts influenced past lake eco- The final stages of the Last Glacial in the Northern Hemisphere, systems directly through lake thermal and hydrological budgets, between 19 and 11.7 thousand years before present (ka BP), were which in turn affected lake biogeochemistry and aquatic organisms, punctuated by distinct and alternating warm and cold climatic and indirectly through changes in seasonality and catchment pro- cesses (e.g., erosion, run-off, vegetation development) (Birks et al., 1990; Leavitt et al., 2009; Fritz and Anderson, 2013).

* One of the best studied Lateglacial lake sediment records in Corresponding author. € E-mail address: [email protected] (B. Wohlfarth). Sweden is derived from the ancient lake of Hasseldala Port (Fig. 1A 1 These authors contributed equally to the work. and B) (Muschitiello et al., 2015b; Wohlfarth et al., 2017). Here, https://doi.org/10.1016/j.quascirev.2017.11.037 0277-3791/© 2017 Elsevier Ltd. All rights reserved. 20 E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29

Fig. 1. (A) Location of the ancient lake of Hasseldala€ Port in southern Sweden and (B) view on the peat bog, which today exists at Hasseldala€ Port. multi-proxy studies combining organic and inorganic geochem- response to past climatic and environmental shifts have rarely been istry; pollen, diatom, and chironomid assemblages; biomarkers and investigated (Jørgensen et al., 2011; Billard et al., 2015; Yang J. et al., hydrogen isotopes; and an excellent chronological framework have 2016; Vuillemin et al., 2016). Yet, Archaea are considered as active allowed reconstruction of local and regional environmental con- community members responsible for methanogenesis, methane ditions as well as summer temperatures between 14.3 and 9.5 ka BP oxidation and nitrogen transformation in the water column and in (Wohlfarth et al., 2017 and references therein). In particular, the the sediment (e.g. Liu et al., 2015; Eme and Doolittle, 2015). sequence of environmental changes at the transition into and out of Here we explore the metagenomic data set for Hasseldala€ Port the Younger Dryas cold phase (c. 12.7e11.65 ka BP) has been well using shotgun sequence analyses of sedimentary aDNA. We spe- documented (Muschitiello et al., 2015b; Wohlfarth et al., 2017 and cifically focus on the temporal succession of ancient archaeal references therein) and provides information on how the shallow communities between 13.9 and 10.8 ka BP, a time period charac- lake and its catchment responded to abrupt climatic shifts. The terised by marked shifts between warmer/colder and wetter/drier available paleoclimatic and paleoenvironmental data sets for climates. We compare the composition and diversity of the archaeal Hasseldala€ Port make the sediments of this site a perfect target for communities to sediment organic matter content, to changes in testing the application of ancient DNA (aDNA) analyses. Specifically, summer water temperatures, to lipid biomarkers (glycerol dialkyl aDNA can offer a broader dimension to conventional biological glycerol tetraethers (GDGTs; Brochier-Armanet et al., 2008; proxies and thus provide the possibility to capture a much larger Schouten et al., 2013)) (Koga and Morii, 2006) and to lake model spectrum of organisms, including non-fossilized microorganisms simulations. These comparisons allow discussing various factors (e.g. Theroux et al., 2010). that might have influenced the archaeal communities in the Ancient DNA from sedimentary records is a new emerging tool ancient lake. used to reconstruct the diversity of past biota (e.g. Willerslev et al., 2003, 2007; 2014; Pawłowski et al., 2014; Parducci et al., 2012, 2. Site description 2013; 2015, 2017; Giguet-Covex et al., 2014; Jørgensen et al., 2012). Mostly, aDNA studies have focused on flora, fauna and Hasseldala€ Port (56016 N; 15001 E; 63 m a.s.l.) in Blekinge phototrophs, while ancient archaeal communities and their province, southern Sweden is today a peat bog underlain by a E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 21 distinct Lateglacial and early Holocene sediment record (Wohlfarth sampling for aDNA (see below), we sub-sampled core #7.4 in et al., 2017)(Fig. 1A and B). Multiple sediment cores have been contiguous 1-cm increments to analyse the organic matter content obtained from this site and studied using a variety of paleo- of the sediments. Loss-on-ignition (LOI) analyses were performed environmental and paleo-climatic proxies (Davies et al., 2003, at 550 C following the procedures described in Heiri et al. (2001). 2004; Andersson, 2004; Wohlfarth et al., 2006; Watson, 2008; The LOI % curve for core #7.4 was then statistically correlated to the Kylander et al., 2013; Steinthorsdottir et al., 2013, 2014; Ampel LOI % curve of core #5 following the core-to-core alignment of et al., 2015; Muschitiello et al., 2015a, b; Karlatou- Muschitiello et al. (2015a, b). This provided a time scale for core Charalampopoulou, 2016). Correlations between the various par- #7.4 and for each of the aDNA samples. allel sediment cores and their repetitive lithology were made by statistically aligning their respective loss-on-ignition (LOI) curves 3.2. Precautions during coring, sampling and aDNA analysis (Muschitiello et al., 2015a, b). This made it possible to present the reconstructed environmental and climatic changes on a common Because aDNA is always degraded, damaged and in low con- time scale (Wohlfarth et al., 2017). centration, the potential for contamination of samples and DNA Sediment deposition in the ancient lake of Hasseldala€ Port extracts with modern DNA is high. To minimize these risks, we took started around 14.3 ka BP. During this early phase, which is specific and well established precautions during coring, sampling assigned to the regional Older Dryas pollen zone, the catchment and DNA analysis of core #7.4. Immediately after collection, the vegetation was dominated by herbs, shrubs and dwarf-shrubs core was wrapped in plastic, placed in PVC liners and quickly (Andersson, 2004). Coleoptera remains suggest that mean sum- transported to the cold room at the Department of Geological Sci- mer air temperatures were around 14 C, while chironomid-based ences (IGV) at Stockholm University, where it was stored at 5 C mean July surface water temperatures remained at around 7e8 C, until subsamples were taken for DNA analyses. The cold storage is possibly due to run-off from stagnant ice (Watson, 2008; Wohlfarth in a part of the building where no DNA analyses are being per- et al., 2017). Around 14.1 ka BP, at the start of the regional Allerød formed. Sub-sampling was conducted in September 2015 in a clean pollen zone, lake organic productivity started to increase and the laboratory at IGV using gloves and wearing lab coats and masks. ice-covered season became gradually shorter (Ampel et al., 2015; The seven DNA samples taken from core #7.4 cover the warm and Wohlfarth et al., 2017). These changes co-occur with a step-wise cold Lateglacial intervals (Allerød, Younger Dryas) and the early increase in mean July surface water temperatures to values of Holocene (Wohlfarth et al., 2017)(Supplementary Table S1). For 12e13 C between 13.3 and 13.2 ka BP. Several proxies imply a first each sample, we removed the top 1.5 cm of the outer sediment with climatic shift already around 13.2e13.1 ka BP, when mean July sterilized scalpels, sampled two times circa 5e10 g of uncontami- surface water temperatures decline and regionally drier conditions nated material from the innermost part of the core with a new set seem to have occurred (Wohlfarth et al., 2017). of sterile scalpels and placed the sample in sterile tubes that were A further decline in lake water summer surface temperatures at immediately closed. The seven samples were kept cold and frozen 12.7 ka BP, i.e. at the start of the regional Younger Dryas pollen zone, within 2 h after sub-sampling and shipped frozen to the Centre for coincides with regionally drier conditions and with an expansion of GeoGenetics, University of Copenhagen for molecular analyses. herb and grass vegetation in the catchment (Wohlfarth et al., 2017). Also, the aquatic vegetation in the lake changed as the nutrient 3.3. DNA extraction and metagenome library construction level decreased and the ice-covered season became longer (Ampel et al., 2015). Coldest lake water surface summer temperatures were DNA extractions and pre-PCR preparations of the seven samples reached at 12.4 ka BP, but started to rise again between 12.4 and were performed in the aDNA laboratory of the Centre for Geo- 11.9 ka BP from 9 to 12 C(Wohlfarth et al., 2017). Coincident with Genetics, University of Copenhagen. Here all facilities are specif- the rise in lake surface water summer temperatures, climatic con- ically dedicated to aDNA analyses from lake sediments. Total ditions became wetter, the aquatic vegetation changed and the ice- genomic DNA was extracted as described in Pedersen et al. (2016). covered season became shorter. The catchment vegetation Each sediment sample was mixed gently before DNA extraction, in responded to the change in temperature and precipitation with a order to be representative of the respective sediment layer. For each delay of a few hundred years as shrubs and trees became only of the seven samples we used 1 g of material for DNA extraction and established between 11.8 and 11 ka BP (Wohlfarth et al., 2017). Also replicated the extraction (14 extractions in total). One gram of at around 11.8e11.7 ka BP, the shallow lake started to fill in and mixed sediment was added to 3 ml of lysis buffer [68 mM N-laur- gradually transformed into a mire and later into a peat bog. oylsarcosine sodium salt, 50 mM Tris-HCl (pH 8.0)], 150 mM NaCl, and 20 mM EDTA (pH 8.0) with addition of 1.5 ml 2- 3. Materials and methods mercaptoethanol and 1.0 ml 1M DTT for each 30 ml of lysis buffer, together with 170 mg of proteinase K, and vortexed vigor- 3.1. Sediment coring, sampling and sediment description ously for 2 20 s using a FastPrep-24 at speed 4.0 ms 1. An addi- tional 170 mg of proteinase K was added to each sample and Sediment core (#5) was obtained in September 2011 and a new incubated, then gently rotated overnight at 37 C. The MOBIO C2 sediment core (#7.4) in July 2015 using a Russian corer with a and C3 buffers (MO BIO Laboratories, Carlsbad, CA) were used for chamber length of 1 m and a diameter of 10 cm. The sediment cores removal of inhibitors, following the manufacturer's protocol. The were transported to the Department of Geological Sciences, extracts were further purified using phenol:chloroform and Stockholm University, where they were stored in a cold room (5 C) concentrated using 30 kDa Amicon Ultra-4 centrifugal filters. The until sub-sampling. Sediment core #5 was sub-sampled in 2012 in phenol:chloroform step was repeated when more purification was contiguous 1-cm intervals for lipid biomarker and hydrogen needed. All DNA extracts were quantified using the Quant-iT dsDNA isotope analyses following the procedures described in HS assay kit (Invitrogen) on a Qubit 2.0 Fluorometer according to Muschitiello (2016). Core #5, which has been dated in very high the manufacturer's manual. DNA from the 14 sediment samples resolution and analysed for various proxies, is the template core for was extracted in two batches with seven samples each, together all Hasseldala€ Port sediment cores (Wohlfarth et al., 2017). The li- with two extraction control blanks per each batch (totally 18 thology of the new sediment core #7.4 was described in the labo- samples). ratory prior to sub-sampling in September 2015. After sub- The 18 samples were then prepared for shotgun metagenome 22 E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 sequencing using the NEBNext DNA Library Prep Master Mix Set for were de-multiplexed and trimmed using AdapterRemoval v. 1.5 454 (New England BioLabs) following the manufacturer's protocol (Lindgreen, 2012) with a minimum base quality of 30 and mini- with the modifications described in Pedersen et al. (2016). Meta- mum length of 30 bp. Using Holi, reads with poly-A/T tails 4bp genome libraries were amplified in two batches of nine samples were removed and low-quality reads and duplicates were removed each plus one library blank/batch, using AmpliTaq Gold (Applied using String Graph Assembler (SGA) (Simpson and Durbin, 2012) Biosystems) (14e20 cycles) and quantified using the 2100 Bio- setting the pre-processing tool dust-threshold ¼ 1, the index analyzer chip (Agilent). The 20 libraries were purified using the algorithm ¼ ‘ropebwt’ and using the SGA filter tool to remove exact MinElute PCR Purification kit (Qiagen), quantified on the 2100 and contained duplicates. Each quality-controlled (QC) read was BioAnalyzer, pooled equimolarly and sequenced using the Illumina thereafter mapped to reference sequences using Bowtie2 v. 2.2.4 HiSeq 2500 platform (100 bp single-end reads). All controls, which (end-to-end alignment and mode ek 50 for example, reads were included the four extraction blanks plus two library blanks (one allowed a total of 500 hits before being parsed). We used the full from each library batch), were also sequenced as controls for nucleotide database (nt) from GenBank (downloaded September contaminants. 2016), which due to size and downstream handling was divided into nine consecutive equal sized databases and indexed using 3.4. Bioinformatic analyses Bowtie2-build (Langmead and Salzberg, 2012). All QC checked fastq files were aligned end-to-end using Bowtie2 default settings. Each 3.4.1. DNA damage analysis alignment was merged using SAMtools (Li et al., 2009), sorted ac- To ensure that our aDNA data was authentic we examined read cording to read identifier and imported to MEGAN v. 10.5 (Huson length and DNA damage patterns due to cytosine deamination, et al., 2007). MEGAN allows visual inspection of how sequences which both are indicators of ancient origin (Ginolhac et al., 2011; are binned along the . We used a lowest common Dabney et al., 2013). We also performed k-mer analysis with the ancestor (LCA) algorithm to bin sequences along the NCBI taxon- k-mer profile analysis library kPAL (Anvar et al., 2014) and per- omy (version March 2015) with reads matching 100% in a given formed a principal component analysis (PCA) on the kmer profiles database. We extracted the number of sequences with unspecific to identify outlier samples. Reads were first trimmed of the Illu- alignments over multiple taxa (marked as ‘not assigned’), the mina 30 sequencing adapter with Cutadapt v.1.11 (Martin, 2011). We number of sequences that had no alignments to any comparative employed parameter settings -m 30 etrim-n -q 10,10 to discard sequence in the database (‘no hits’) and the number of sequences reads shorter than 30 base pair (bp), trim trailing N's, and trim low- assigned to or below taxa from the three domains of cellular life quality bases (phred score 10) from both ends of a read. Raw and (Bacteria, Archaea and Eukaryotes). trimmed reads were quality checked with FASTQC v. 0.11.5 (Andrews, 2010). K-mer analysis indicated that all extraction and 3.4.3. Diversity analysis library blanks (six in total) were distinctly different from the 14 For alpha and beta diversity measurements, we used Kraken v. samples, suggesting that they probably originated from primer- 0.10.5-beta (Wood and Salzberg, 2014) for taxonomic assignments dimers, chimera constructions, contamination from modern hu- on the same database used for the DNA damage profiling. Low- man DNA and modern laboratory contaminants (see below). Reads complexity regions were masked using dustmasker from passing quality filters were mapped against a custom database BLAST þ v. 2.6.0 (Camacho et al., 2009). The Kraken filtering using end-to-end alignment in Bowtie2 v. 2.2.8 (Langmead and parameter was set to 0.2, since it has been suggested to give the Salzberg, 2012). The custom database consisted of all bacterial, best trade-off between sensitivity and specificity on simulated data fungal, archaeal, protozoan and viral genome sequences, as well as (Wood and Salzberg, 2014). Duplicate reads were removed before Homo sapiens, available in GenBank (May 2016 version). Bam files mapping. The diversity calculations were based on binning reads were sorted with SAMtools v. 1.3.1 (Li et al., 2009), duplicate reads into operational taxonomic units (OTUs) based on their LCA. Only were removed with Picard v. 2.7.1 (http://broadinstitute.github.io/ reads mapping to Archaea were taken into account. In order to deal picard), and mapped reads with mapping quality 25 were with possible biases in the database due to over- or underrepre- filtered out with BAMtools v. 2.4.0 (Barnett et al., 2011). Filtered sentation of certain genera, we performed abundance calculations bam files were analysed with mapDamage v. 2.0.6 (Jonsson et al., on genera rather than level. The Python package scikit-bio v. 2013) and the damage pattern analysis showed that all samples 0.5.1 was used for calculating alpha and beta diversities based on consist mainly of trimmed sequences with short read lengths, and C OTU abundances. Using the Shannon index as the metric for alpha- to T and G to A nucleotide substitutions at the end of the reads are diversity, we estimated the biological diversity of a community, drastically elevated (Supplementary Figure S1A, B) compared to the taking both genera richness and variance in genera proportion into reads from blanks (Supplementary Figure S1C). In order to attest consideration. The weighted UniFrac metric for beta-diversity was that the archaeal sequences were of ancient origin, we also per- used to compare the composition of archaeal communities with formed a damage pattern analysis on those mapped sequences that consideration for occurrence and abundance of OTUs. The bioin- were classified as archaeal based on the taxonomy identifier of the formatics work was performed using the workflow manager database reference sequence. After removing duplicate sequences Snakemake v. 3.11.0 (Koster€ and Rahmann, 2012). as well as filtering on mapping quality, we used in the range 405e1828 for damage pattern modeling. Despite the low number of 3.5. Lipid biomarkers resulting reads, the ancient signal is present (Supplementary Figure S1D) in contrast to that obtained when running a damage Lipid extraction and GDGT analyses were performed on 54 pattern analysis on the blank samples (Supplementary Figure S1C). sediment samples (2e8cm3) from core #5. Samples were freeze- dried and extracted via sonication using the method described in 3.4.2. Taxonomical analysis Muschitiello et al. (2015a) and Muschitiello (2016). Three fractions For the taxonomical analysis, the data pre-treatment was per- were isolated using a silica gel column and solvent elution and the formed in a similar manner as for DNA damage analysis. Succes- third, most polar fraction was used for GDGT analysis; it was kept sively, we used the metagenomics pipeline Holi for handling read frozen prior to analysis. GDGTs were analysed using a Thermo- de-multiplexing, adaptor trimming, quality control, duplicate and Dionex system comprising a Dionex Ultimate 3000RS quaternary low-complexity read removal (Pedersen et al., 2016). Raw reads pump and an auto-sampler coupled to a Thermo Scientific TSQ E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 23 quantum access MAX triple stage quadrupole max mass spec- Port's samples (average 48%), and 80.6% of the reads for blank trometer. APCI (Atmospheric Pressure Chemical Ionization) was samples. used as the ionization source in positive mode. Prior to analysis the A general overview of the data present in Hasseldala€ Port's lipid extract was dissolved in 9:1 methanol:dichloromethane and samples is given in Supplementary Figure S2, where we show the the injection volume was 10 ml. GDGTs were detected by scanning number of sequences with unspecific alignments over multiple with selective ion monitoring over m/z 1287.2e1304.8. Chromato- taxa (not assigned, ~70%) and the number of sequences that had no graphic separation was achieved using a modified method of Zhu alignments to any comparative sequence in the database (no hits, et al. (2013). ~14%). Eukarya were divided into ten different kingdoms, which also include fungi and animals (Opisthokonta), plants and green 3.6. Numerical fresh water lake modeling algae (Viridiplantae). Whereas most of the reads (~84%) remained unidentified, the majority of the assigned reads (~16%) were To simulate the direct climatic impact on a shallow ancient lake assigned to microorganisms with more than 10% assigned to Bac- like that of Hasseldala€ Port between the warm state of the late teria, ~5% to Archaea and less than 1% to Eukaryotes. Similar results Allerød (~13 ka BP) and the mid-Younger Dryas cold period (~12.17 were recently also obtained by Slon et al. (2017), who analysed ka BP), we used the freshwater lake model FLake (Mironov, 2008)to seven cave sediment samples using shotgun sequencing; in this predict key physical lake parameters such as water temperature, study, the percentage of sequences aligned to the database varied lake ice and mixed layer depth. The lake model has undergone from 4.9% to 21%. thorough testing and is used operationally as part of Numerical Filtered reads were then mapped to a database consisting of Weather Prediction (NWP) models of national weather services, 11,635 archaeal sequences, 718,548 bacterial sequences, and e.g. by Deutscher Wetterdienst (DWD). The source code and an 298 596 eukaryote sequences. Circa 20% of the blank reads passing overview of lake studies using FLake can be found online at www. QC filters mapped, whereas only 1e3% of Hasseldala€ Port's sample flake.igb-berlin.de. Atmospheric forcing for the lake model was reads mapped. Prior to mapdamage analysis, mapping reads were taken from recent high-resolution global climate model simula- filtered on mapping quality (>¼25) and deduplicated, giving in the tions of the Allerød and Younger Dryas periods with the Commu- range 2678e29737 reads for mapDamage analysis (Supplementary nity Earth System Model (CESM1, Schenk, unpublished). The 3 Table S2, column “Singleton reads”). hourly forcing data derived from CESM1 at the closest grid point to In order to assess potential contamination effects from blank Hasseldala€ Port (56.07N; 15 E) consisted of down-welling short samples, reads were taxonomically assigned, both using Kraken wave radiation (W/m2), total cloud cover (%), air humidity at 2 m (data not shown) and based on mapping results to superkingdoms reference height (mbar), air temperature at 2 m reference height Archaea, Bacteria, and Eukaryota. Blank samples were almost (C) and near-surface wind speeds at 10 m (m/s). Based on today's devoid of archaeal sequence, suggesting that they mostly consist of topography around the ancient lake, a maximum lake depth of 5 m contaminants. In contrast, the fraction of reads assigned to Archaea was chosen. Water transparency was set to transparent water with is substantially higher for Hasseldala€ Port's samples (in the range of an extinction coefficient of 0.3 [1/m]. The lake model was initialized 405e1828 reads) (Supplementary Table S2, column “Archaea on June 1st with an initial temperature of the upper mixed layer of filtered singletons”). An overview of the Archaea taxa present in 5 C, initial bottom temperature of 4 C and an initial mixed layer Hasseldala€ Port's samples is given in Tables 1 and 2 and in thickness of 2 m. The model was insensitive to the initial conditions Supplementary Table S3, where we show the number of sequences when started during the warm season. Omitting the first half year identified at the level of phylum and species, as well as the top 20 used for spin-up, we run 20 years for both time slices, respectively. most abundant Archaea species.

4. Results 4.3. Temporal succession of major archaea groups

4.1. Lithostratigraphy and sediment organic matter content The aDNA Archaea communities observed in our 12 samples after MEGAN inspection consisted of three phyla, 12 classes, and 54 The basal sediments of core #7.4 are composed of fine sand with genera. The archaeal record was mainly affiliated with Eur- low LOI % values (14.1e13.7 ka BP) and clayey algae gyttjas with yarchaeota and , while only few reads (~1e3%) were increasing organic matter content (13.7e12.65 ka BP) (Fig. 2A, B, D). affiliated to and Candidatus Bathyarchaeota In the silty algae gyttja with fine sand and in the clayey algae gyttja (Fig. 2E; Tables 1 and 2 and Supplementary Table S3). The hydro- (12.65e11.55 ka BP), the organic matter content is low, but rises genotrophic (, M. conradii, M. again at 11.8 ka BP. The uppermost algae gyttja displays the highest arvoryzae) and Methanoregula (Methanoregula formicica) and the organic matter content of the whole sequence. acetoclastic Methanosaeta were the most abundant within Eur- yarchaeota, while uncultured crenarchaeote were most abundant 4.2. DNA sequencing within Crenarchaeota (Table 2 and Supplementary Table S3). In particular, our aDNA data suggest that Hasseldala€ Port's ancient We generated 445060968 reads in total for the 14 Hasseldala€ lake was mainly dominated by Methanocella paludicola (2593 Port samples, for which the per-sample read count lies in the range reads), uncultured archaeon (1645 reads), uncultured cren- of 29e50 million (Supplementary Table S2), except for two failed archaeote (1385 reads) and Methanocella conradii (611 reads) and € € samples (HA3.1_2 and HA5.1_2) that were almost devoid of by smaller proportions of Methanoregula formicica (240 reads), sequence. The sequence data for the 12 Hasseldala€ Port samples has uncultured euryarchaeote (233 reads), Methanocella arvoryzae (219 been submitted to the European Nucleotide Archive (ENA; https:// reads), uncultured methanogenic archaeon (191 reds), Methanor- www.ebi.ac.uk/ena/) under study accession number PRJNA378719 egula boonei (154 reads) and Methanosaeta concilii (125 reads) (https://www.ebi.ac.uk/ena/data/view/PRJNA378719). (Table 2). € € Sequencing of negative controls resulted in 187696052 reads in Sediment samples HA1.1 (14.0e13.77 ka BP), HA3.1 (13.3e13.15 € total for four extraction blanks and two library blanks, despite ka BP) and HA4.1 (12.94e12.74 ka BP), assigned to the Allerød negligible DNA concentrations as measured by Qubit. Trimming pollen zone, were dominated by (1459, 2130 and and quality filtering removed 32e71% of the reads for Hasseldala€ 1021 reads, respectively) and to a lesser extent by Crenarchaeota 24 E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29

Fig. 2. Archaeal composition and abundance in Hasseldala€ Port's ancient lake sediments compared to other proxies. (A) Simplified lithostratigraphy for core #7.4; (B) loss-on- ignition (LOI) curve for core #7.4; (C) chironomid-derived mean July surface water temperatures established on core #HP4 (Watson, 2008; Wohlfarth et al., 2017); (D) regional pollen zones (Wohlfarth et al., 2017); (E) abundance counts of archaeal phyla and sample ID for core #7.4; (F) Ratio between Euryarchaeota and Crenarchaeota in core #7.4; (H) Relative abundance of GDGT-0 and Crenarchaeol (‘Thaumarchaeol’) in core #5, both normalized against the sample with highest GDGT-0 concentration.

Table 1 Summarized counts of Archaea reads found in each sample and assigned at the phylum level.

Sample ID Candidatus Bathyarchaeota Crenarchaeota Euryarchaeota Thaumarchaeota Sum € HA1.1 6 149 1459 2 1616 € HA3.1 20 293 2130 5 2448 € HA4.1 14 244 1021 5 1284 € HA5.1 6 194 1110 5 1315 € HA6.1 13 253 961 8 1235 € HA7.1 43 377 521 10 951 € HA8.1 24 383 293 3 703

Table 2 Summarized counts of reads assigned to the top 20 Archaea species found in each sample. € € € € € € € Sample ID/species HA1.1 HA3.1 HA4.1 HA5.1 HA6.1 HA7.1 HA8.1 Sum (counts)

Methanocella paludicola 415 858 391 374 294 187 74 2593 uncultured archaeon 170 210 168 191 224 414 268 1645 uncultured crenarchaeote 102 201 175 132 184 289 302 1385 Methanocella conradii 97 198 102 85 61 55 13 611 Methanoregula formicica 48 63 14 42 49 15 9 240 uncultured euryarchaeote 51 52 23 34 32 11 30 233 Methanocella arvoryzae 56 71 31 18 17 19 7 219 uncultured methanogenic archaeon 43 49 24 29 28 9 9 191 Methanoregula boonei 26 35 13 35 23 13 9 154 Methanosaeta concilii 9 16143143102125 Methanosphaerula palustris 1522717164384 uncultured marine crenarchaeote E6-3G 2 13 7 5 4 19 16 66 Candidatus Methanomassiliicoccus intestinalis 26801255359 uncultured soil archaeon 128131414456 uncultured miscellaneous Crenarchaeota group 4370924855 Methanosaeta harundinacea 61410345244 uncultured Desulfurococcales archaeon 1 12 3 0 20018 uncultured archaeon 360500014 Methanosarcina acetivorans 434200013 Methanococcoides burtonii 442200012

Sum (counts) 1137 1864 1030 1054 1041 1112 765 8003

(149, 293 and 244, respectively), while Candidatus Bathyarchaeota respectively) and a decrease of Euryarchaeota (1110 and 961 reads, and Thaumarchaeota were only present in very low numbers respectively), while Candidatus Bathyarchaeota and Thaumarch- (Fig. 2E and Table 1). In samples assigned to the Younger Dryas aeota have similar values as before (Fig. 2E, Table 1). Methanosaeta € € pollen zone (HA5.1, 12.59e12.48 ka BP; HA6.1, 12.02e11.94 ka BP), thermophila was detected only in samples assigned to the Allerød we notice a slight increase of Crenarchaeota (194 and 253 reads, pollen zone (Supplementary Table S3). E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 25

A distinct change in archaeal composition occurs only in the two related to differential DNA degradation in the different samples, we € youngest samples in our data set assigned to the Holocene (HA7.1, compared the level of fragmentation of all our reads and found that € 11.75e11.54 ka BP and HA8.1, 11.10e10.80 ka BP), with Cren- there was no substantial change in fragmentation between samples archaeota increasing (377 and 383 reads, respectively) and Eur- of different ages (Supplementary Figure S3A-D). It should be noted, yarchaeota decreasing (521 and 293 reads, respectively), while however that this does not entirely rule out differential fragmen- Candidatus Bathyarchaeota and Thaumarchaeota show a slight in- tation, nor that there is no effect of differential fragmentation on crease, but overall remain relatively low in abundance (Fig. 2E, species composition. Table 1). This change is also seen in the Euryarchaeota: Cren- archaeota ratio, which displays a gradual increase in Crenarchaeota 4.5. Lipid biomarkers in the lower five samples and a marked increase in the uppermost two samples (Fig. 2F). The abundance of the generic archaeal membrane lipid GDGT- 0(Schouten et al., 2013) shows an increase during the later part 4.4. Diversity changes of the Allerød pollen zone, reaches highest concentrations during the Younger Dryas pollen zone (Fig. 2H) and abruptly decreases at e In addition to community changes in Archaea composition be- the Younger Dryas/Holocene transition around 11.8 11.7 ka BP. fi tween 13.9 and 10.8 ka BP, we also detected alpha and beta di- Crenarchaeol, which is speci c for Thaumarchaeota (formerly versity changes (Fig. 3A). Lowest levels of alpha diversity were group Ia Crenarchaeota) (Schouten et al., 2013), is detected in € € found in samples HA1.1 and HA3.1 from the Allerød pollen zone. relatively low concentrations in samples assigned to the Allerød € Archaeal diversity gradual increased in sample HA4.1 assigned to and Younger Dryas pollen zones, but increases in Holocene the later part of the Allerød pollen zone and in Younger Dryas samples. € € samples (HA5.1, HA6.1) and reached high levels in the two youngest € € samples (early Holocene, HA7.1 and HA8.1) (Fig. 3A). Dissimilarity 5. Temporal changes in archaeal community abundance and values between sample pairs calculated using the weighted UniFrac diversity metric for beta diversity, shows distinct temporal clusters (Fig. 3B). € € Early Holocene samples (HA7.1 and HA8.1) form one major cluster, Earlier analysed proxies in Hasseldala€ Port's sediment cores while the rest of the samples cluster together and can be sub- show that the lake basin registered the marked climatic shifts that € € € € divided into two main sub-clusters: HA1.1/HA3.1 and HA4.1/HA5.1/ characterised the last glacial/interglacial transition, both in terms of € HA6.1. Fig. 3B clearly highlights that the two youngest Holocene summer temperatures and evaporation (Watson, 2008; € € samples (HA7.1 and HA8.1) are markedly different from all other Muschitiello et al., 2015b), and that it started to fill in around samples. In order to ascertain that the changes in diversity were not 11.8e11.7 ka BP and gradually transformed into a mire and later

AB Dissimilarity

HÄ 8.1 HÄ 6.1_2 HÄ 8.1_2 5.0 HÄ 5.1

HÄ 6.1 Similarity

4.5 HÄ 4.1_2 HÄ 7.1 HÄ 7.1_2 HÄ 6.1 HÄ 4.1 HÄ 5.1 HÄ 6.1_2 HÄ 1.1_2 4.0 HÄ 4.1

HÄ 1.1 HÄ 1.1

Shannon's diversity index Shannon's diversity HÄ 3.1 3.5 HÄ 4.1_2

HÄ 1.1_2 HÄ 7.1_2

HÄ 3.1 HÄ 7.1

3.0 Allerød Younger Dryas Holocene HÄ 8.1_2 Open lake system Mire/peat bog

HÄ 8.1

HÄ 8.1 HÄ 8.1_2 HÄ 7.1 HÄ 7.1_2 HÄ 3.1 HÄ 1.1 HÄ 1.1_2 HÄ 4.1 HÄ 4.1_2 HÄ 6.1 HÄ 5.1 HÄ 6.1_2 13.3 − 13.15 11.1 − 10.8 14.0 − 13.77 12.94 − 12.7412.59 − 12.48 11.75 − 11.54 Time (ka BP)

Fig. 3. Archaeal diversity in Hasseldala€ Port's ancient lake sediments. (A) Alpha diversity using Shannon's diversity index shows a gradual increase in archaeal diversity through time. The regional pollen zones as well as lake status changes are shown (Wohlfarth et al., 2017). (B) Clustered heatmap for Beta diversity using a weighted unifrac distance metric. Red indicates a large dissimilarity between samples and blue indicates greater similarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 26 E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 into a peat bog (Wohlfarth et al., 2017). The sediment composition dominate in the water column and in the sediments of eutrophic of core #7.4 mirrors these changes with rising organic matter and mesotrophic lakes (Yang et al., 2016). Higher relative abun- content during the (warmer) Allerød pollen zone, lower organic dances for Crenarchaeaota were observed in other freshwater lakes, matter content during the (cold and dry) Younger Dryas pollen but also large variations in abundances for Crenarchaeaota and zone and high organic matter content during the (warm and Euryarchaeota between different lakes and between water and wetter) early Holocene. As shown by several studies (e.g., Bjorck€ sediment samples (Zhang et al., 2015). Temporal changes in and Moller,€ 1987; Wohlfarth et al., 2017), changes in organic mat- archaeal community composition have been indirectly related to ter content in late-glacial lake sediments in southern Sweden can past climate conditions as these would have changed the inflow of be used as an indirect proxy of climatic conditions as lake aquatic organic and inorganic material, in-lake processes (e.g., water productivity increased and detrital input into a lake decreased chemistry, aquatic productivity and sedimentation of organic during warmer climate states and vice versa. However, sediment matter) and sediment properties (Yang. et al., 2015; Vuillemin et al., organic matter content is not only coupled to climatic conditions, 2016). Below we evaluate how temporal changes in Hasseldala€ but also to lake status changes and to natural sediment infilling. Port's ancient archaeal communities compare to changes in sedi- Our aDNA analyses show that archaeal community composition ment organic matter, lake status, and surface water temperatures to and diversity changed through time in Hasseldala€ Port's sediments discuss the influence of climate versus natural lake infilling. and that the most distinct shift occurred at the Younger Dryas/ Archaeal abundance is highest in Allerød and Younger Dryas Holocene transition, i.e. when air and lake water surface summer samples and decreases in early Holocene samples (Fig. 2E). Lipid temperatures rose distinctly (Wohlfarth et al., 2017) and sediment biomarker analysis supports this and shows high relative abun- organic matter content started to increase markedly. Beta diversity dances of GDGT-0 (Schouten et al., 2013) during the later part of the analysis moreover indicates two sub-clusters within the Allerød- Allerød pollen zone and during the Younger Dryas pollen zone and € € Younger Dryas pollen zones, comprising samples HA1.1/HA3.1 and an abrupt decrease at the Younger Dryas/Holocene transition € € € HA4.1/HA5.1/HA6.1 (Figs. 2E and 3B). These observations lead to the (Fig. 2H). Euryarchaeota, which dominate in Allerød and Younger question whether changes in archaeal community composition and Dryas samples, decrease in abundance in early Holocene samples diversity were linked to (external) climatic changes, which influ- where Crenarchaeota and partly also Candidatus Bathyarchaeota enced lake status, sediment lithology and lake physical properties increase (Fig. 2E and F and Table 1). Thaumarchaeota, on the other € € or whether these communities only responded to (internal) hand, have slightly higher abundances in samples HA6.1 and HA7.1 changes, such as lake infilling and corresponding changes in sedi- only (Table 1), i.e. below and above the Younger Dryas/Holocene ment lithology. transition. These observations generally agree with the lipid DNA analyses of recent and sub-recent Archaea communities in biomarker analyses, which detect an increase in Crenarchaeol various lake systems have shown that highly organic sediments (‘Thaumarchaeota’; formerly group Ia Crenarchaeota; Schouten hold high abundances of anaerobic Euryarchaeota (Fan and Xing, et al., 2013) concentrations around 11.8 ka BP (Fig. 2H). This in- 2016; Ma et al., 2016; Mandic-Mulec et al., 2012) and that these crease suggests that nitrogen-based redox processes, including

Table 3 Correlation between sediment organic matter content (LOI%) and major Archaea groups found in Hasseldala€ Port's sediments.

Sample ID Depth (cm) LOI (%) averaged Candidatus Bathyarchaeota Crenarchaeota Euryarchaeota Thaumarchaeota € HA 1.1 433e438 8.61277078 6 149 1459 2 € HA 3.1 414e418 23.10613688 20 293 2130 5 € HA 4.1 401e406 29.15005124 14 244 1021 5 € HA 5.1 391e395 17.78441084 6 194 1110 5 € HA 6.1 374e378.5 18.83143639 13 253 961 8 € HA 7.1 363e368 25.74090522 43 377 521 10 € HA 8.1 350e354 55.73213664 24 383 293 3

r2 0.187639085 0.597138964 0.363559039 0.013722118 P-value 0.331627205 0.04165777 0.151816372 0.802496123

Table 4 Comparison between inferred lake water July surface water temperatures (Fig. 2C) and the most abundant Euryarchaeota in Hasseldala€ Port's sediments. € € € € € € € HA1.1 HA3.1 HA4.1 HA5.1 HA6.1 HA7.1 HA8.1

Age interval (ka BP) 14.0e13.8 13.3e13.1 12.9e12.7 12.6e12.5 12.0e11.9 11.5e10.7 11.1e10.8 Averaged July surface water temperature (C) 9.3 12.4 10.9 9 9 NA NA

Methanocella paludicola 415 858 391 374 294 187 187 Methanocella conradii 97 198 102 85 61 55 13 Methanoregula formicica 48 63 14 42 49 15 9 uncultured euryarchaeote 51 52 23 34 32 11 30 Methanocella arvoryzae 56 71 31 18 17 19 7 uncultured methanogenic archaeon 43 49 24 29 28 9 9 Methanoregula boonei 26 35 13 35 23 13 9 Methanosaeta concilii 916143143102 Methanosphaerula palustris 15 22 7 17 16 4 3 Candidatus Methanomassiliicoccus intestinalis 26 8 0 12 5 5 3 uncultured soil archaeon 1 2 8 13 14 14 4 Methanosaeta harundinacea 614103452 uncultured Desulfurococcales archaeon 1 12 30200 E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 27 archaeal ammonium oxidation, became more important in the mire weeks (126.1 ± 1.75 days) is significantly reduced by around two and peat bog as compared to the methane cycle, which appeared weeks (14.7 ± 2.24 days, p ¼ 2$10 7) in YD. As a result, the lake predominant during the lake stage. starts to warm up later leading to a significantly shorter growing Correlations between the sediment organic matter content (LOI season length (N days > 5.5 C per year) of around one week %) and the four archaeal groups show that Crenarchaeota correlate (7.25 ± 2.91 days, p ¼ 0.0211). The onset of the growing season positively with LOI% (r2 ¼ 0.597; P-value 0.041), while Candidatus does not occur before mid (~16th of June, AL) to late (~23th of June, Bathyatchaeota, Euryarchaeota and Thaumarchaeota display no YD) June and ends in late September (~24th of September). The significant correlations (Table 3). Higher organic matter content in simulated lower YD mean summer (June, July and August) tem- € € the uppermost analysed samples (HA7.1, HA8.1) can therefore peratures of ~12.75 C can mainly be attributed to a roughly 3 K explain the increase in abundance of Crenarchaeota. The increase in colder June, while July temperatures remain on average warm with organic matter content in Hasseldala€ Port occurs around 11.8e11.7 17.56 C (17.46 C in AL) (Fig. 4A). These very warm conditions last ka BP and coincides with infilling of the ancient lake and the however for a very short period only during YD. transition into a mire and later into a peat bog (Wohlfarth et al., Extrapolating these results to the diversity differences observed 2017). This transition also concurs with an important climatic among the late-glacial archaeal assemblages (Fig. 3B), we suggest transition, which led to a rapid rise in summer water temperatures that the length of the growing season and of the lake-ice season (Fig. 2C) and ambient air temperatures and to marked changes in were more important factors than warm peak July temperatures catchment vegetation (Wohlfarth et al., 2017). Although the coin- only. Higher abundances of certain methanogenic Euryarchaeota in € cidence of a climatic shift and natural lake infilling make it difficult sample HA3.1 on the other hand, may be related to a combination of to disentangle the importance of these factors on past archaeal abundances, we hypothesize that lake infilling and the accompa- nying changes in sediment organic matter content were the un- A derlying causes for the observed increase in Crenarchaeota. Alpha diversity generally increases from the oldest to the youngest samples and displays highest diversity in the two Holo- € € cene samples HA7.1 and HA8.1 (Fig. 3A). The increase in archaeal diversity during the early Holocene was accompanied by a decrease in archaeal abundance, except for Candidatus Bathyatchaeota and partly also Thaumarchaeota (Table 1). The two Holocene samples also stand out in the similarity/dissimilarity analysis as clearly different from all other samples (Fig. 3B) and imply a distinct change in ecosystem functioning and restructuring of the archaeal communities. Higher diversity is readily explained by the formation of many micro-environments within a mire or a peat bog and the much larger temporal and spatial physicochemical gradients and variations compared to a relatively simple lake environment. Beta diversity analysis moreover suggests two sub-clusters within the Allerød-Younger Dryas pollen zones, comprising sam- € € € € € ples HA1.1/HA3.1 (Allerød) and HA4.1/HA5.1/HA6.1 (late Allerød and Younger Dryas) (Fig. 3B). These two sub-clusters correlate to some extent with reconstructed July lake surface water tempera- B tures derived from chironomids (Fig. 2C), which could indicate that summer lake water temperatures might have been important for archaeal communities during Hasseldala€ Port's lake phase. Specif- € € ically, samples HA1.1 and HA6.1 compare in time to low lake water € C July surface temperatures, while sample HA3.1 temporally corre- € sponds to higher lake water temperatures and samples HA4.1 and € HA5.1 concur with a decrease in temperatures. To test this further we compare the most abundant Euryachaeota genera with Allerød and Younger Dryas summer lake surface water temperatures inferred from chironomid assemblages (Fig. 2C; Table 4). We observe that Methanocella paludicola, Methanocella conradii, Meth- anoregula formicica and Methanocella arvoryzae have highest € abundances in sample HA3.1 (highest summer lake surface water temperatures), while Methanosaeta concilii increases in abundance € € in samples HA5.1 and HA6.1 (lower summer lake surface water temperatures) (Table 4). To test if lake physical properties had an influence on the di- versity of methanogenic Archaea we use a lake model simulation Fig. 4. Comparison of simulated lake water temperatures and lake ice cover for fi Younger Dryas (YD; 12.17 ka BP) and Allerød (AL; 13 ka BP). (A) Simulated 20 year- and focus on a speci c warmer and colder time interval (13 ka BP - mean annual cycle for lake water temperatures [C] for AL (red) and YD (blue). (B) AL and 12.17 ka BP e YD, respectively). Compared to AL, simulated Mean lake ice thickness [m] during AL (light blue) and YD (purple). (C) Temperature lake water temperatures for YD are dominated by severe winter difference of the annual cycle for YD minus AL [K] (left axis) and change in ice cooling and cold spring temperatures lasting throughout May thickness [cm] (right axis) for YD minus AL. The dominant change of YD relative to AL is a ~3 K colder June, which is caused by thicker lake ice and a longer ice-covered (Fig. 4A). Consequently, the lake model simulates thicker lake ice season during YD. The small warming in late summer may not compensate for the fi cover (Fig. 4B) and a signi cantly longer ice-covered season for YD overall colder and shorter warm season of the YD. (For interpretation of the references as compared to AL (Fig. 4C). On average, the ice-free season of ~18 to colour in this figure legend, the reader is referred to the Web version of this article.) 28 E. Ahmed et al. / Quaternary Science Reviews 181 (2018) 19e29 a longer growing season, a shorter lake ice cover season and higher Port, southeastern Sweden. Boreas 44, 94e102. Andersson, M., 2004. Pollen Stratigraphic Response for Tephra Horizons during the July lake water temperatures. Taken all proxies together, it appears e € Last Glacial Interglacial Transition (LGIT) in Southernmost Sweden (M.Sc. that the conditions in Hasseldala Port's ancient lake were most thesis 4108). Stockholm University, pp. 1e65. favourable for methanogenic Archaea during the Allerød and Andrews, S., 2010. 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Comm. 5, 4211. https://doi.org/10.1038/ncomms4211. aDNA, can thus be an indirect proxy for changes in lake status. Their Ginolhac, A., Rasmussen, M., Gilbert, M.T., Willerslev, E., Orlando, L., 2011. map- paleoecological importance however needs to be further studied in Damage: testing for damage patterns in ancient DNA sequences. Bioinformatics concert with other paleoenvironmental indicators. 27, 2153e2155. Heiri, O., Lotter, A.F., Lemcke, G., 2001. Loss on ignition as a method for estimating organic and carbonate content in sediments: reproducibility and comparability Acknowledgement of results. J. Paleolimnol. 25, 101e110. Huson, D.H., Auch, A.F., Qi, J., Schuster, S.C., 2007. MEGAN analysis of metagenomic data. Genome Res. 17, 377e386. We would like to thank Eske Willerslev (University of Copen- Jonsson, H., Ginolhac, A., Schubert, M., Johnson, P.L.F., Orlando, L., 2013. Map- hagen, Denmark) for using the facilities of the Centre of Geo- Damage2.0: fast approximate Bayesian estimates of ancient DNA damage pa- Genetics, Denmark during DNA extraction and library preparation. rameters. Bioinformatics 29, 1682e1684. https://doi.org/10.1093/bioinformati We also thank Love Dalen (Swedish Museum of Natural History, cs/btt193. Jørgensen, S.L., Hannisdal, B., Lanzen, A., Baumberger, T., Flesland, K., Fonseca, R., Sweden) and Anders Hansen (University of Copenhagen, Denmark) Øvreås, L., Steen, I.H., Thorseth, I.H., Pedersen, R.B., Schleper, C., 2012. Corre- for valuable discussions and Sarah Mak (University of Copenhagen, lating microbial community profiles with geochemical data in highly stratified e Denmark) for guidance during the lab work. The present study was sediments from the Arctic Mid-Ocean Ridge. PNAS 109, 2846 2855. 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