Martina I Gocke
email: mgocke@uni-bonn.de

Arnaud Huguet
email: arnaud.huguet@upmc.fr

Sylvie Derenne
email: sylvie.derenne@upmc.fr

Steffen Kolb
email: steffen.kolb@zalf.de

Michaela A Dippold
email: dippold@gwdg.de

Guido L B Wiesenberg
email: guido.wiesenberg@geo.uzh.ch

Disentangling interactions between microbial communities and roots in deep subsoil

Keywords: biopore, microbial hotspot, molecular marker, paleoenvironmental archive, rhizosphere, terrestrial sediment

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The existence of microbial life in deeper parts of the unsaturated subsurface like e.g. in terrestrial sediments is well known [START_REF] Holden | Microbial Processes in the Vadose Zone[END_REF]. Hotspots in the deep subsoil >> 1 m including soil parent material stimulating microbial thriving are e.g. paleosols [START_REF] Brockman | Microbiology of Vadose Zone Paleosols in South-Central Washington State[END_REF] or places of preferential flow with accumulated dissolved organic carbon (C org ; [START_REF] Bundt | Preferential flow paths: biological 'hot spots' in soils[END_REF]. In addition, deep roots entering the sediment during and after its deposition may provide such places of preferential water flow as well as C org and nutrient accumulation [START_REF] Kautz | Nutrient acquisition from arable subsoils in temperate climates: A review[END_REF], as demonstrated by the positive correlation of C org contents and root biomass distribution [START_REF] Wang | Profile storage of organic/inorganic carbon in soil: From forest to desert[END_REF]. The rhizosphere as potential microbial hotspot, however, has been largely ignored below 0.5 m depth, although the ability of roots to penetrate subsoil and underlying soil parent material to several meter depth is known for various plant groups [START_REF] Canadell | Maximum rooting depth of vegetation types at the global scale[END_REF]. Deep roots have been studied in various soils and unconsolidated terrestrial sediments including loess-(paleo)soil and sand-(paleo)soil sequences in Central and Southeast Europe (Gocke et al., 2014a(Gocke et al., , 2014b[START_REF] Gocke | Paleosols can promote root growth of the recent vegetationa case study from the sandy soil-sediment sequence Rakt, the Netherlands[END_REF], where they were observed to occur with considerable abundances >> 2 m below the surface. The incorporation of root-and microorganism-derived organic matter (OM) in deep sedimentary subsoil does not only potentially cause an overprint of the paleoenvironmental signals recorded in such archives, followed by insecurities for the interpretation of biomarkers like e.g. n-alkanes or glycerol dialkyl glycerol tetraethers (GDGTs; Huguet et al., 2013a;Gocke et al., 2014c). It might also affect terrestrial carbon (C) stocks, of which considerable amounts are stored in paleosols [START_REF] Marin-Spiotta | Long-term stabilization of deep soil carbon by fire and burial during early Holocene climate change[END_REF].

So far, it is unknown whether deep roots influence the microbial community in unconsolidated deep subsoil > 1 m. Especially their influence on long-term maintenance of microbial life after the lifetime of the root is difficult to study, but might be important in the context of C sequestration in buried soils and sediments [START_REF] Fisher | Carbon storage by introduced deep-rooted grasses in the South American savannas[END_REF][START_REF] Johnson | Carbon cycle: Sequestration in buried soils[END_REF]. Ancient lithified rootsrhizolithswhich often form through calcification of the root during or shortly after its lifetime [START_REF] Klappa | Rhizoliths in terrestrial carbonates: classification, recognition, genesis and significance[END_REF][START_REF] Gocke | Carbonate rhizoliths in loess and their implications for paleoenvironmental reconstruction revealed by isotopic composition: δ 13 C, 14[END_REF] give a time-integrated insight into root-related processes over the lifespan of the root and thereafter.

Here we present a multi-proxy approach to characterize both the living and fossil microbial community in deep sedimentary subsoil based on abundance and composition of five biomarker classes. The molecular approach includes two compound classes with high potential for preservation in soils and sediments: Free-extractable fatty acids (FAs) give a broad overview over plant and microbial remains and degradation products [START_REF] Harwood | Lipids in plants and microbes[END_REF][START_REF] Lichtfouse | Stable carbon isotope evidence for the microbial origin of C 14 -C 18 n-alkanoic acids in soils[END_REF], whereas core lipids of GDGTs (CL-GDGTs), presumed to be of fossil origin, are solely produced by some microorganisms (Suppl. Fig. 1). Thus, GDGTs with isoprenoid alkyl chains (iGDGTs) are attributed to archaea, whereas those with branched alkyl chains (brGDGTs) are biosynthesized by not yet identified bacteria [START_REF] Schouten | The organic geochemistry of glycerol dialkyl glycerol tetraether lipids: A review[END_REF] potentially feeding on root remains [START_REF] Huguet | Branched tetraether membrane lipids associated with rhizoliths in loess: Rhizomicrobial overprinting of initial biomarker record[END_REF]. Some of these bacteria might belong to the phylum Acidobacteria [START_REF] Sinninghe Damsté | 13,16-Dimethyl Octacosanedioic Acid (iso-Diabolic Acid), a Common Membrane-Spanning Lipid of Acidobacteria Subdivisions 1 and 3[END_REF][START_REF] Johnson | Carbon cycle: Sequestration in buried soils[END_REF]. Further, we included the respective intact polar counterparts of these compound classes, the phospholipid FAs (PLFAs) and intact polar lipid GDGTs (IPL-GDGTs), which are attributed to living or recently deceased microorganisms due to low stability of their polar headgroups [START_REF] Kindler | Fate of bacterial biomass derived fatty acids in soil and their contribution to soil organic matter[END_REF][START_REF] Schouten | Fossilization and degradation of intact polar lipids in deep subsurface sediments: a theoretical approach[END_REF][START_REF] Lengger | Differential degradation of intact polar and core glycerol dialkyl glycerol tetraether lipids upon post-depositional oxidation[END_REF]Suppl. Fig. 1). As third compound class, 16S ribosomal ribonucleic acid (rRNA) genes from bacterial deoxyribonucleic acid (DNA) provided an overview of the bacterial community. Depth distribution of these biomarkers was compared with that of several parameters of pedogenic, weathering and rooting processes, including C contents, alkane contents and composition, abundances of living roots and root remains, density, clay contents, color and magnetic properties of a Central European sediment-paleosol sequence.

We aimed to elucidate i) which physical, chemical and biological factors influence microbial life in sedimentary deep subsoil, ii) if microbial community composition in deep subsoil is notably affected by root distribution, iii) if root effects on microbial community can be traced after lifetime of the root, and iv) whether roots maintain microbial life in terrestrial sediments for centuries or millenia.

The multi-proxy approach was applied to the Late Pleistocene loess-paleosol sequence at Nussloch, Southwest Germany, which has been investigated intensely for paleoclimate and paleovegetation (e.g. [START_REF] Antoine | Rapid and cyclic aeolian deposition during the Last Glacial in European loess: a high-resolution record from Nussloch, Germany[END_REF]. The current study was performed on the uppermost 9.5 m of the profile P 2011 (Gocke et al., 2014b;Fig. 1a), which is defined here as associated pair of Holocene soil (0-1.1/1.5 m; Fig. 1b) and Holocene deep subsoil (1.1/1.5-9.5 m) because rhizoliths (Fig. 1b-f) of Holocene ages occur continuously between 1.5 and 9 m depth [START_REF] Gocke | Carbonate rhizoliths in loess and their implications for paleoenvironmental reconstruction revealed by isotopic composition: δ 13 C, 14[END_REF]Gocke et al., 2014b). Time lags between sedimentation, lasting from 35-17 ky on the one hand, and root growth on the other hand cover a wide range of 10 2 -10 3 y. Abundant recent roots, rhizoliths between 3 and 10 ky in age, and root-derived biopores were observed along the profile, with ages of the latter likely between sedimentation and today (Gocke et al., 2014a). This provides the unique opportunity to assess shortterm and long-term effects of postsedimentary rooting on microbial community composition in deep subsoil. Detailed description of the loess-paleosol sequence at Nussloch, Southwest Germany, and its paleoenvironmental record was provided by e.g. [START_REF] Antoine | Rapid and cyclic aeolian deposition during the Last Glacial in European loess: a high-resolution record from Nussloch, Germany[END_REF] and Gocke et al. (2014b). The 13 m thick, 17-35 ky old sequence includes 16 weakly developed and three well developed paleosols, as well as the 1.1-1.5 m thick Holocene Calcic Luvisol Siltic (IUSS, 2014). The recent vegetation, which relates to living roots and likely formed an unknown portion of root-related biopores throughout the profile, comprises natural broad-leaf forest and non-native robinia, as well as smaller shrubs and herbaceous plants [START_REF] Gocke | Differentiation of plant derived organic matter in soil, loess and rhizoliths based on n-alkane molecular proxies[END_REF]. Rhizoliths at Nussloch and nearby sites are of Holocene age between 3 and 10 ky. They occur with diameters of up to 5 cm but mostly 1-2 cm (reviewed by Gocke et al., 2014a), and were formed by roots of unknown trees or shrubs, likely including hazel, oak, beech and alder [START_REF] Gocke | Differentiation of plant derived organic matter in soil, loess and rhizoliths based on n-alkane molecular proxies[END_REF] , respectively (Suppl. Fig. 2; Gocke et al., 2014b).

Materials and methods

Study site

Profile preparation, field methods and sampling

The profile was prepared by removing ≥ 1 m of material from the top and the side, respectively, followed by counting of roots, root-related biopores and rhizoliths on horizontal levels (Gocke et al., 2014b(Gocke et al., , 2014c)). Afterwards, 200 g of soil, sediment or paleosol material were collected distant from visible root remains, from topsoil down to 9.5 m depth in 0.5-1.0 m increments. After sample splitting into two aliquots, one aliquot was dried at 40 °C and used for free FA and DNA analyses, whereas the other half was immediately cooled and kept frozen at -18 °C until PLFA and GDGT analyses. The sample set for DNA analyses did not include the topsoil, because microbial diversity in topsoils is very high, which would produce a bias for the other parameters. It was further reduced to seven depth intervals between 2 m and 9.5 m due to sample loss.

Analytical methods

Free-extractable fatty acids

Using an aliquot of dried material, free-extractable lipids were extracted and free FAs separated, derivatized and subsequently measured by GC-FID and GC-MS according to the protocol by [START_REF] Wiesenberg | Analysis of lipids and polycyclic aromatic hydrocarbons as indicators of past and present (micro)biological activity[END_REF]. Free FAs with a chain length of 12 to 32 carbons were quantified because they include microbial and plant tissues, as well as degradation products [START_REF] Harwood | Lipids in plants and microbes[END_REF]. Dicarboxylic as well as unsaturated and branched free FAs were kept as separate groups of compounds, but not further assigned to specific sources, which was done for PLFAs, as multiple sources exist for most of these compounds.

Phospholipid fatty acids and glycerol dialkyl glycerol tetraethers

An aliquot of each frozen sample was subjected to lipid extraction following a modified protocol of the Bligh-Dyer method [START_REF] Bligh | A rapid method of total lipid extration and purification[END_REF][START_REF] Apostel | Biochemical pathways of amino acids in soil: Assessment by position-specific labeling and 13 C-PLFA analysis[END_REF] with 0.15 M citric acid, chloroform and methanol 0.8:1:2 (v/v/v). Using one half of the splitted extract, the PLFA fraction was purified over an activated silica column by elution with acetone, and derivatized for GC-MS measurement as previously described [START_REF] Apostel | Biochemical pathways of amino acids in soil: Assessment by position-specific labeling and 13 C-PLFA analysis[END_REF]. Assignment of PLFAs with chain length of 14 to 20 carbons to the respective microbial groups was performed according to literature [START_REF] Zelles | Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review[END_REF][START_REF] Fernandes | Comparison of Whole-Cell Fatty Acid (MIDI) or Phospholipid Fatty Acid (PLFA) Extractants as Biomarkers to Profile Soil Microbial Communities[END_REF].

The second part of the extract was separated over an activated silica column into three fractions as described by Huguet et al. (2013b), with fraction F1 containing apolar lipids (eluted with 30 ml dichloromethane), fraction F2 containing CL-GDGTs [eluted with 30 ml dichloromethane/acetone (2:1, v/v) followed by 30 ml dichloromethane/acetone (1:1, v/v)], and fraction F3 containing IPL-GDGTs

[eluted with 30 ml dichloromethane/methanol (1:1, v/v) followed by 30 ml methanol]. A small aliquot of the IPL fraction (F3) was analyzed directly using high performance liquid chromatographyatmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) to determine any carryover of CLs into the IPL fraction. The analysis showed nearly complete separation of the CL-and IPL-GDGTs. The rest of the fraction F3 was subjected to acid methanolysis (24 h at 100 °C in 1 M HCl/methanol) to cleave off the polar head groups of IPL-GDGTs. CL-and IPL-derived GDGTs were analysed by HPLC-MS using a procedure described by Huguet et al. (2013b).

Bacterial 16S rRNA genes

Prior to analysis of the bacterial genetic fingerprint, a sterility test was successfully performed.

Metagenomic DNA was extracted in replicates from 0.5 g aliquots of dry sample material using the FastDNA ® SPIN Kit for Soil (MP Biomedicals). Bacterial 16S rRNA genes were amplified with primers 27f/907mr [START_REF] Schellenberger | Metabolic responses of novel cellulolytic and saccharolytic agricultural soil Bacteria to oxygen[END_REF]. Primer 27f was labeled with infrared dye 700 for t-RFLP (terminal restriction fragment length polymorphism) analyses. PCR (polymerase chain reaction)

products were purified with a DNA Gel Extraction Kit (Millipore, MA) and single-stranded extensions at terminal ends were removed with mung bean endonuclease digest [START_REF] Egert | Formation of pseudoterminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure[END_REF].

Subsequent restriction digestion of PCR products was performed with MspI [START_REF] Degelmann | Enterobacteriaceae facilitate the anaerobic degradation of glucose by a forest soil[END_REF].

DNA concentrations were determined with a DNA Quantification Kit (Invitrogen, Germany) and adjusted to 2 ng µl -1

. T-RFLP analyses were performed as previously described [START_REF] Hamberger | Anaerobic consumers of monosaccharides in a moderately acidic fen[END_REF]. PCR products from all samples were pooled and used to construct one 16S rRNA gene library.

Amplicons were cloned into Escherichia coli JM 109 competent cells using the pGEM-T Vector System II (Promega, WI). Inserted 16S rRNA gene fragments were re-amplified and sequenced [START_REF] Messing | New M13 vectors for cloning[END_REF]MacroGen, South Korea). In total, 270 different inserts were sequenced. Identification of phylotypes was done by RDP classifier. Based on in silico digestion, terminal restriction fragments detected by software GelQuest (version 3.1.7, SequentiX GmbH, Germany) were manually affiliated to sequenced genotypes. Ribonucleic acid (RNA), representing viable microorganisms due to its presumably fast degradation, was not analysed, because i) very low DNA contents in Quaternary sediments like loess [START_REF] Liu | Contribution of inherent organic carbon to the bulk δ 13 C signal in loess deposits from the arid western Chinese Loess Plateau[END_REF] current study) imply that RNA is likely too low for amplification and further analysis, and ii) rarefaction analysis (Suppl. Fig. 3) revealed that even DNA results are reliable solely at the phylum level.

Statistic evaluation

Variables within the individual datasets (free FAs, PLFAs, core GDGTs, intact polar GDGTs and DNA)

were reduced by factor analysis. Factor values of most significant factors were compared between the datasets by canonical correlation analysis, and redundancy values for the datasets were computed.

Further, environmental and profile parameters (EPP) significantly affecting the depth distribution of the biomarker groups were identified by regression analysis. The bulk elemental composition, measured via X-ray fluorescence analysis (Gocke et al., 2014b), did not reveal significant correlations with any of the biomarker sets and is therefore not shown. A significance level = 0.1 was chosen, because of i)

small size of the sample set, ii) investigation of natural samples in contrast to samples from laboratory experiments performed under controlled conditions, and iii) heterogeneous nature of the sample set in terms of age and material. All statistical analyses were performed with Statistica 6.0 (StatSoft, Tulsa, USA).

Results and Discussion

Lipid distribution

Lipids were more abundant in the topsoil than in subsoil collected at 1 m depth (Fig. 2a-d), and concentrations in deep subsoil reached max. 28% of the respective topsoil value.

Distribution patterns of free-extractable and phospholipid fatty acids

Free FA distribution patterns were mostly dominated by short-chain homologues (Gocke et al., 2014c;C 12-19 ; Fig. 2a). The ratio of short-vs. long-chain (C 20-32 ) free FAs (R S:L ) was mostly > 1.2 (Suppl. Tab.

1), indicating the dominance of belowground OM including microbial remains, rhizodeposits and degradation products over higher plant aboveground biomass inputs [START_REF] Harwood | Lipids in plants and microbes[END_REF].

Contents of unsaturated free FAs (C 16,18,19 ) were low due to their high degradability. Branched free FAs (brC 12-18 ), most likely originating from gram positive (Gram + ) bacteria, and dicarboxylic acids (C 9- 11,18,20 ), deriving mainly from plant above-and belowground tissues, contributed only to a minor extent to free FAs. -11,18,20) deriving from cutin and suberin biopolymers [START_REF] Kolattukudy | Biochemistry and function of cutin and suberin[END_REF], whereas the right diagram displays contents of mainly microorganism-derived saturated short-chain free FAs (C12:0-19:0), plant-derived saturated long-chain (C20:0-26:0) and very long-chain free FAs (C27:0-32:0; [START_REF] Kolattukudy | Biochemistry of plant waxes[END_REF], branched free FAs (brC12:0-18:0) from total Gram + bacteria [START_REF] Zelles | Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review[END_REF][START_REF] Fernandes | Comparison of Whole-Cell Fatty Acid (MIDI) or Phospholipid Fatty Acid (PLFA) Extractants as Biomarkers to Profile Soil Microbial Communities[END_REF], as well as microorganism-and plant-derived unsaturated free FAs (C16: Specific PLFAs suggested a dominance of bacteria (methylated, cyclo-and most mono-unsaturated FAs) over fungi (C 16:1ω5,18:2 : Fig. 2b) in all depth intervals except for 9 m (Suppl. Tab. 1). At four sampling depths between 5.0 and 8.5 m, ratio of bacterial to fungal PLFAs (R B:F ) exceeded the value of the topsoil by a factor ranging between 2 and 4. Higher relative portions of fungi occurred solely in the lowermost part of the profile (8.0, 9.0 and 9.5 m) and might be attributed either to pedogenic conditions in well-developed paleosols or to postsedimentary penetration by fossil and/or living roots, both of which coincide in these depths (Gocke et al., 2014b). The ratio of Gram Thaumarchaeota, in surface soils (uppermost 0.2 m; [START_REF] Harvey | The effect of organic matter and oxygen on the degradation of bacterial membrane lipids in marine sediments[END_REF][START_REF] Buckley | Phylogenetic analysis of nonthermophilic members of the kingdom Crenarchaeota and their diversity and abundance in soils[END_REF]. The current work thus provides extended knowledge on the occurrence of archaea in deep subsoil. The relative amounts of iGDGTs vs. brGDGTs decreased for both core and intact polar GDGTs from 0 to 1 m and from 0 to 2 m, respectively (Suppl. Tab. 1). Below these depths, R i:b was mostly higher for intact polar (25-65%) than for core GDGTs (3-33%; Suppl. Tab. 1), which might be due to the more labile nature of the predominantly phospho-headgroup of brGDGTs compared to the usually glycoheadgroup of iGDGTs [START_REF] Harvey | The effect of organic matter and oxygen on the degradation of bacterial membrane lipids in marine sediments[END_REF].

Bacterial genetic fingerprint

Phylotypic composition of bacterial DNA in deep subsoil was dominated by Actinobacteria, Firmicutes, Chloroflexi and Proteobacteria, besides minor abundance of Acidobacteria, Bacteroidetes and candidate division OP11 (Fig. 2e). Throughout identified phylogenetic groups with abundances > 3%, the predominance of Gram + bacterial phyla (Actinobacteria and Firmicutes) at 2, 3, 4 and 6.5 m matched well the absence of Gram -bacterial PLFAs at 3 and 6 m, but not at 5 m (Suppl. Tab. 1).

Further, the much lower R P:N in lower parts of the loess-paleosol sequence, excluding 9 m, agreed with higher portions of Gram -bacteria (Chloroflexi and Proteobacteria).

Fossil and living microbial communities in Holocene soil and deep subsoil

The multi-proxy approach indicated the dominance of bacterial signatures over fungal, archaeal and microeukaryotic ones throughout the Holocene soil and deep subsoil, suggesting a microbial community typical for the vadose subsurface [START_REF] Fierer | Variations in microbial community composition through two soil depth profiles[END_REF]. Also, bacterial DNA composition resembled that in modern soils [START_REF] Janssen | Identifying the Dominant Soil Bacterial Taxa in Libraries of 16S rRNA and 16S rRNA Genes[END_REF]. However, as bacterial genes were already close to detection limit, the presumably less abundant archaeal or fungal genes were not investigated. The deep subsoil properties of the loess-paleosol sequence were strengthened by its increased ratio of bacterial to fungal PLFAs compared to topsoil (Suppl. Tab. 1), which matches previous studies showing an increase of the latter with soil depth [START_REF] Holden | Microbial Processes in the Vadose Zone[END_REF][START_REF] Stone | Changes in extracellular enzyme activity and microbial community structure with soil depth at the Luquillo Critical Zone Observatory[END_REF]. Further, the high ratio of Gram + to Gram -PFLAs below the topsoil (Suppl. Tab. 1) was already described for the uppermost 2 m of agricultural and grassland soils [START_REF] Blume | Surface and subsurface microbial biomass, community structure and metabolic activity as a function of soil depth and season[END_REF][START_REF] Fierer | Variations in microbial community composition through two soil depth profiles[END_REF].

Presence of PLFAs and intact polar GDGTs, though in amounts up to one order of magnitude lower compared to their free/core counterparts, revealed the presence of living microorganisms at any depth throughout the profile, including the deep subsoil of Late Pleistocene age. This contradicts the traditional assumption of organic compounds being incorporated solely during deposition of terrestrial sediments [START_REF] Conte | Molecular and carbon isotopic composition of leaf wax in vegetation and aerosols in a northern prairie ecosystem[END_REF]. Intact polar GDGTs contributed less than 15% to total GDGTs below 2 m (Suppl. Fig. 4b), similarly to brGDGT data from topsoils [START_REF] Peterse | Influence of soil pH on the abundance and distribution of core and intact polar lipid-derived branched GDGTs in soil[END_REF], whereas the percentages of PLFAs from total FAs scattered between 24% and 88% (Suppl. Fig. 4a).

Figure 3.

Statistical factors (SF) of the biomarker groups obtained by factor analysis. Compounds with strong positive and negative loadings (min. > 0.6 or < -0.6, but mostly > 0.9 or < -0.9) on these factors are listed for free FAs (SF 1-4), PLFAs (SF 5), CL-GDGTs (SF 6-8), IPL-GDGTs (SF 9, 10) and DNA (SF 11, 12), respectively.

FAs, GDGTs and bacterial DNA are produced independently from each other and partially by different source organisms. To elucidate the degree of overlap between individual biomarker data sets, and consequently disentangle the time range represented by free/core lipids, redundancy values between the five biomarker classes (Tab. 1) were calculated after reduction of the respective data sets by factor analysis. Free/core lipids of FAs and GDGTs could be well explained by the respective other free/core lipid group with 65% and 52% conformity, whereas they were less well predictable by their intact polar counterpart and the respective intact polar lipid group of the other lipid category. Best predictor of free FAs and core GDGTs was DNA with 88% and 79% redundancy. Similarly, DNA was better explained by free FAs and core GDGTs than by intact polar lipids. The high conformity of free FAs and core GDGTs with DNA, together with the potential of DNA to be preserved outside a cell in soils for centuries to millenia [START_REF] Agnelli | Purification and isotopic signatures (δ 13 C, δ 15 N, Δ 14 C) of soil extracellular DNA[END_REF], support the assumption that these lipids represent a timeintegrated signal of microorganism remains accumulated since sedimentation until today. We aimed to determine the linkage of living and fossil microorganisms to biological, physical or chemical conditions that prevailed during phases of sedimentation, pedogenesis, rooting or inbetween.

Therefore, the twelve statistical factors (SF; Fig. 3), obtained after reduction of the individual compounds of each biomarker class by factor analysis, were tested for correlation with the following 13 environmental and profile parameters (EPP; Tab. 2, Suppl.

Free-extractable fatty acids

Positive correlation of long-chain (C 22-32 ; SF 1) and dicarboxylic free FAs (C 9-11 ; SF 3) with alkane contents (Fig. 2, Tab. 2) confirmed the higher plant origin of the former and suggested the same for the latter, because average chain length of long-chain alkanes (C 25-37 ), which was mostly > 29 at the Nussloch deep subsoil [START_REF] Gocke | Differentiation of plant derived organic matter in soil, loess and rhizoliths based on n-alkane molecular proxies[END_REF], indicated major contribution of higher plant leaf waxes [START_REF] Eglinton | Hydrocarbon constituents of the wax coatings of plant leaves: a taxonomic survey[END_REF]. Gram + bacteria-derived branched free FAs (C 14,17 ; SF 2; C 12,15,16 ; SF 3; C 13,16 ;

SF 4) positively correlated with indicators of advanced pedogenesis (high C org and clay contents) and weathering (high a* and κ), low density, abundant living roots and high alkane contents. They negatively correlated with rhizolith abundances (SF 3, 4), suggesting that Gram + bacteria in subsoil feed mainly on old bulk OM and not younger plant-derived OM [START_REF] Kramer | Variable use of plant-and soil-derived carbon by microorganisms in agricultural soils[END_REF].

Composition of higher plant-derived long-chain free FAs was probably hardly affected by microbial overprint, as implied by the absence of correlation between long-chain and dicarboxylic free FAs, and recent roots. Plant-derived free FAs can thus be used as (paleo)environmental tracer [START_REF] Reiffarth | Sources of variability in fatty acid (FA) biomarkers in the application of compound-specific stable isotopes (CSSIs) to soil and sediment fingerprinting and tracing: A review[END_REF], as long as material free of root remains is collected.

Table 2.

Significance levels of the correlations between twelve statistical factors (SF) derived from the five investigated biomarker groups, and 13 environmental and profile parameters (EPP). All EPP were adapted from Gocke et al.

(2014b) except for alkane contents and CPIalk. For depth diagrams see Suppl. C org [mg g -1 ]

- + + --- + + C carb [mg g -1 ] -- + - alkanes [µg g -1 ] a + + + CPI alk [ ] b - + roots [m -2 ] c -- +++ --- - ++ - rhizoliths [m -2 ] c - + + + biopores [m -2 ] c + + - dry bulk density [g cm -3 ] ++ clay [wt-%] - - +++ - - + color a* [ ] d - ++ - ++ + color L* [ ] e -- + - κ [m 3 kg -1 ] f - -- ++ - - S-ratio [ ] g - -
+/-significant positive/negative correlation (.01 < p < .1); ++/--very significant positive/negative correlation (.001 < p < .01); +++/---highly significant positive/negative correlation (p < .001).

a Alkanes in terrestrial sediments like loess are traditionally assumed to originate from aboveground biomass of synsedimentary vegetation, incorporated via litterfall and abraded particulate organic matter [START_REF] Conte | Molecular and carbon isotopic composition of leaf wax in vegetation and aerosols in a northern prairie ecosystem[END_REF].

b CPIalk = [(Σn-C17-35 odd/Σn-C16-34 even)+(Σn-C17-35 odd/Σn-C18-36 even)]/2

Carbon preference index of odd over even n-alkane homologues. High CPIalk indicates dominance of fresh plant aboveground biomass, whereas low values suggest predominantly microorganism-derived and degraded organic matter [START_REF] Cranwell | Diagenesis of free and bound lipids in terrestrial detritus deposited in a lacustrine sediment[END_REF].

c Frequencies of roots, rhizoliths and biopores were determined on horizontal areas using a grid with dimension 0.5x0.5 m (Gocke et al., 2014b).

d High a* values indicate reddish color and low values green color (CIE, 1931). Throughout the investigated profile, highest a* in the Holocene soil indicates pedogenic processes like formation of iron oxides, whereas in sedimentary deep subsoil, high a* (mostly loess > paleosol) likely result from hydromorphic bleaching.

e High L* values characterize light color and low values dark color (CIE, 1931). Although organic matter content can strongly influence L*, Corg and L* do not correlate with each other at Nussloch. Rather, high L* may result from hydromorphic bleaching of paleosols (Gocke et al., 2014b).

f Measure for concentration and magnetic grain size variation of ferrimagnetic minerals [START_REF] Maher | The magnetic properties of Quaternary aeolian dusts and sediments, and their palaeoclimatic significance[END_REF]. Strong pedogenesis is represented by very high κ in the Holocene soil, but contrary to Eurasian loess deposits, high κ of the periglacial Nussloch loess-paleosol sequence represents seasonal waterlogging and subsequent in situ weathering rather than pedogenesis (Gocke et al., 2014b).

g Parameter for the relative portions of ferrimagnetic vs. antiferromagnetic minerals [START_REF] Wang | Magnetic properties of loess deposits on the northeastern Qinghai-Tibetan Plateau: palaeoclimatic implications for the Late Pleistocene[END_REF]. More intense pedogenesis caused lower S-ratio and weak pedogenesis higher S-ratio in the loess-paleosol sequence, whereas the opposite holds true for the Holocene soil.

Phospholipid fatty acids

The SF created from PLFA composition (SF 5) yielded factor loadings similarly high for all of the compounds with positive loading on this SF, i.e. Gram + bacteria, Actinomycetaceae, Gram -bacteria and fungi, and showed significant to highly significant correlations with nine EPP. Living bacteria and fungi were associated with depths characterized by strong pedogenesis and weathering. For eight out of nine EPP, the chronological context is ambiguous, as they might be of synsedimentary / synpedogenic nature or could partially be related to postsedimentary waterlogging effects (high a* and κ) or later pedogenic (high C org and low L* due to OM accumulation, low C carb due to carbonate dissolution, low CPI alk from degradation, high clay) and rooting phases (abundant biopores from recent/former roots). Root abundances, showing one of the highest significances throughout the sample set (p = .00001) with SF 5, emphasized the role of the recent vegetation for living microbial community in the deep subsoil. Large similarities of short-chain free FAs and PLFAs were found: Both showed that Gram + bacteria and Actinomycetaceae prefer depths characterized by stronger pedogenesis and weathering as well as recent rooting (SF 2-5). This implies that notable parts of short-chain free FAs in terrestrial sediments might result from rhizodeposits and microorganisms stimulated by plant roots, and thus represent a mixed paleoenvironmental signal from a broad timespan.

Core lipid glycerol dialkyl glycerol tetraethers

In terms of core GDGTs, archaeal GDGT 1292,1298,1300 (SF 6) were more abundant in depths that are influenced by both ancient/long-term pedogenic processes (high C org and clay contents, low L*), and recent rooting. The correlation of archaeal iGDGTs and recent roots points to a similar direction as previous studies, which demonstrated the increased occurrence of bacterial brGDGTs in vicinity of roots (Huguet et al., 2013a). Analogously to brGDGT source organisms [START_REF] Weijers | Carbon isotopic composition of branched tetraether membrane lipids in soils suggest a rapid turnover and a heterotrophic life style of their source organism(s)[END_REF][START_REF] Ayari | Distribution of archaeal and bacterial tetraether membrane lipids in rhizosphere-root systems in soils and their implication for paleoclimate assessment[END_REF], our findings thus suggest that iGDGT source organisms might have a heterotrophic metabolism as well. However, the high significance of this relation as well as the correlation between core GDGTs, considered as fossil markers, and recent roots was unexpected. The latter might indicate low stability of archaeal intact polar GDGTs in soil, leading to a fast release of the respective core GDGTs. The assumed link of archaea mainly to recent and less to ancient rooting is enforced by the absence of correlations between any archaeal core and intact polar GDGTs (SF 6, 9, 10) and rhizoliths or biopores. Short residence times of archaeal intact polar GDGTs of few days to weeks were shown for marine sediments [START_REF] Ingalls | Distribution of Intact and Core Membrane Lipids of Archaeal Glycerol Dialkyl Glycerol Tetraethers among Size-Fractionated Particulate Organic Matter in Hood Canal, Puget Sound[END_REF], and might be similar in soils. Among bacterial compounds, homologues with two cyclopentyl moieties (GDGT C org as main driving factor. This supports the assumption that C org is crucial for microbial abundance [START_REF] Helgason | Microbial distribution in an eroded landscape: Buried A horizons support abundant and unique communities[END_REF], community [START_REF] Hansel | Changes in Bacterial and Archaeal Community Structure and Functional Diversity along a Geochemically Variable Soil Profile[END_REF] or long-term survival of microorganisms in terrestrial sediments [START_REF] Breuker | The deep biosphere in terrestrial sediments in the Chesapeake Bay area, Virginia, USA[END_REF], presumably due to close spatial association of microorganisms and C org [START_REF] Holden | Microbial Processes in the Vadose Zone[END_REF]. As roots represent an important source of available C org in C org -poor eolian sediments, root distribution considerably affected microbial communities. At Nussloch, the main root effect was that intact polar lipids representing living microorganisms were affected solely by recent root features, which includes partially also biopores. In contrast, microbial remains reflecting an ancient or time-integrated signal, i.e. free/core lipids and DNA, correlated predominantly with ancient root features including rhizoliths and biopores but simultaneously also with living roots.

These findings strongly suggest that microbial life in sedimentary deep subsoil is not only altered by postsedimentary rooting, but may thrive more or less continuously long after the lifetime of the root in remaining hotspots. This is potentially stimulated by successive generations of roots partially reutilizing existing root-derived biopores (Fig. 4), a mechanism well known for crops in shallow subsoil [START_REF] Kautz | Nutrient acquisition from arable subsoils in temperate climates: A review[END_REF]. Analogously to deep sea sediments [START_REF] Takano | Sedimentary membrane lipids recycled by deep-see benthic archaea[END_REF] or topsoils [START_REF] Dippold | Direct incorporation of fatty acids into microbial phospholipids in soils: Position-specific labeling tells the story[END_REF], in ancient terrestrial sediments and root systems like at the Nussloch loess-paleosol sequence, microorganisms may thus feed on microbial degradation products and, in case of terrestrial settings, rhizodeposits even millenia after decay of the initial microorganisms and/or root. Likely, Conceptual figure of root-related microorganism distribution and associated processes over time for vegetation growing on terrestrial sediments like e.g. loess or dune sands. 1) Synsedimentary situation with scarce vegetation cover consisting of grasses and herbaceous plants, with shallow roots. 2) Roots of tree vegetation A enter the deep subsoil, stimulating microbial life in the rhizosphere adjecent to roots. 3) After death of the root, the ratio living : dead microorganisms and thus the ratio intact polar : free/core lipids decreases. 4) Millenia after root death, biopores and/or rhizoliths remain. Quantities of signature molecules of dead microorganisms are reduced by degradation. Process rates decrease with time, but proceed continuously over long time spans. Living microorganisms feed on dead microbial remains as well as on plant remains and sedimentary organic matter. In the figure, roots of the next tree vegetation (B) approach the ancient root systems, because the latter provide plant-available nutrients and old microorganism hotspots can be re-activated subsequently. New root effects may overprint old ones, e.g. if subsequent roots use old biopores. After phase 3, the cycle may thus continue with phase 2 and tree species B.

Our results disprove the traditional assumption of organic remains being solely incorporated during sediment deposition [START_REF] Conte | Molecular and carbon isotopic composition of leaf wax in vegetation and aerosols in a northern prairie ecosystem[END_REF]. This implies uncertainties for paleoenvironmental records in Quaternary terrestrial archives with allegedly high chronological resolution like loess-paleosol sequences. Horizontal transects from rhizoliths via rhizosphere towards root-free sediment at various study sites revealed the postsedimentary overprint of free FAs and core GDGTs [START_REF] Huguet | Branched tetraether membrane lipids associated with rhizoliths in loess: Rhizomicrobial overprinting of initial biomarker record[END_REF](Huguet et al., , 2013a;;Gocke et al., 2014c), and similarly, PLFAs and intact polar GDGTs from microorganisms associated with living roots might have been incorporated (Fig. 4). The overprint of synsedimentary records and time-integrated character of free/core lipids are further enforced by the suspiciously similar depth diagrams of paleotemperature, reconstructed based on core and intact polar brGDGTs at Nussloch, which strongly disagree with Glacial conditions (Suppl. Fig. 5). This urges us to recommend the following cautions prior to sampling, i) at least 0.5 m of sediment should be removed from each side of the profile to avoid recent root effects, ii) the archive should be carefully regarded with respect to deep roots and iii) samples should be collected distant from visible root remains.

Further, ongoing OM incorporation and turnover by root-related microorganisms in deep subsoil will not necessarily entail the sequestration of additional C as recently postulated [START_REF] Kell | Large-scale sequestration of atmospheric carbon via plant roots in natural and agricultural ecosystems: why and how[END_REF], but might rather cause net loss of considerable amounts of C stored in paleosols [START_REF] Marin-Spiotta | Long-term stabilization of deep soil carbon by fire and burial during early Holocene climate change[END_REF] due to priming effects. Thus, the biogeochemical and ecological disequilibrium induced by fresh younger root and microbial biomass might even stimulate degradation of stabilized sedimentary OM [START_REF] Fontaine | Stability of organic carbon in deep soil layers controlled by fresh carbon supply[END_REF]. To assess this question, carbon stocks in the deep rhizosphere and in respective bulk soil/sediment have to be determined and opposed to each other. Generally, studies on the terrestrial C cycle as well as soil microbial studies should be extended to greater depths, including the soil parent material which has been largely neglected so far [START_REF] Richter | How deep is soil?[END_REF][START_REF] Harper | The hidden organic carbon in deep mineral soils[END_REF].

Conclusions

Signature molecules for both living and fossil microorganisms were found throughout a Central European 9.5 m thick soil-sediment profile, i.e. in the Holocene soil and in associated sedimentary deep subsoil of Pleistocene age. For the first time, we demonstrated significant correlations of microbial communities in sedimentary deep subsoil with frequencies of recent roots as well as fossil calcified roots, so-called rhizoliths, and root-derived biopores. Our unique multi-proxy approach revealed significant interrelationships of living microorganisms with recent root quantities, and of fossil microbial communities with frequencies of recent roots and ancient root remains. This indicated that i) the paleoenvironmental signal recorded in terrestrial sediments can be altered by postsedimentary rooting, and ii) roots may entail long-term maintenance of microbial life in deep subsoil. Plant roots entering deeper parts of the soil including the parent material up to several millenia after its deposition are thus an important source of carbon and nutrients, stimulating microbial life far beyond the lifetime of the root. The widespread occurrence of deep roots up to several meters below the topsoil not only at the current study site, but at several sites around the world, suggests that these root effects on deep subsoil microbial communities are not an exception.

Figure 1 .

 1 Figure 1. Overview pictures of the Nussloch profile (a-c) and detail pictures (d-f) of calcified root features (rhizoliths). a) Upper Pleniglacial part of the Nussloch loess-paleosol sequence with a maximum age of 35 ky (Antoine et al., 2009). b) 0-1.8 m depth interval, including the Holocene soil (HS; Calcic Luvisol Siltic). c) 1.8-3.2 m depth interval, with in situ, almost vertically oriented rhizoliths. d) Rhizolith, view at profile wall. e) Rhizoliths indicated as circular white spots on a horizontal level. f) Microrhizoliths (diameter < 1 mm), difficult to observe at the profile wall. Picture shows one microrhizolith in longitudinal view (bottom right) and several microrhizoliths in cross section (e.g. top left).

Figure 2 .

 2 Figure 2. Biomarker classes representing fossil microorganisms (free fatty acids [FAs], core lipid glycerol dialkyl glycerol tetraethers [CL-GDGTs] and bacterial genetic fingerprint) and living microorganism communities (phospholipid fatty acids [PLFAs] and intact polar lipid glycerol dialkyl glycerol tetraethers [IPL-GDGTs]). Diagrams a-d show concentrations of lipids, where in each diagram the upper x-axis refers to lipid contents in the Holocene soil (HS, 0-1 m), and the lower x-axis refers to lipid contents in the deep subsoil 1-9.5 m. a) Free FA contents. The left

  cyclo-and most mono-unsaturated FAs) R P:N was approximately twice as high in deeper parts of the Holocene soil (1 m depth) compared to surface soil (Suppl. Tab. 1). Between 5.5 and 9.5 m, very low R P:N resulted from high contents of Gram -bacterial PFLAs.3.1.2 Distribution patterns of core and intact polar glycerol dialkyl glycerol tetraethersBacterial and archaeal GDGTs were present in all samples (Fig.2c, d), with the ratio of isoprenoid to branched GDGTs (R i:b ;[START_REF] Yang | Correlations between microbial tetraether lipids and environmental variables in Chinese soils: Optimizing the paleo-reconstructions in semid-arid and arid regions[END_REF] ranging between 0.03 and 0.68 for core GDGTs and between 0.19 and 2.65 for intact polar GDGTs (Suppl. Tab. 1). Archaea predominated over bacteria only in topsoil and at the bottom of the sequence according to IPL-GDGT analyses. During recent years, studies emphasized the ubiquitous occurrence of archaea and especially one phylum,

  Fig. 2): (1) organic carbon (C org ) and (2) carbonatic carbon (C carb ) contents; (3) contents of n-alkanes (n-C 15-37 ), deriving from microorganisms and higher plants; (4) alkane carbon preference index (CPI alk ) enabling the distinction between fresh plant aboveground biomass and microbial and degraded OM; frequencies of (5) living roots, (6) rhizoliths and (7) root-derived biopores; (8) dry bulk density; (9) clay contents; (10) color indices a* and (11) L*; (12) magnetic susceptibility measured at 0.3 kHz (κ), and (13) S-ratio of soil or sediment. All data except for alkane content and CPI alk (Gocke et al., unpublished data) were adopted from Gocke et al. (2014b).

Fig

  3.4.5 Bacterial genetic fingerprintBased on the 16S rRNA genetic fingerprint, positive correlation of Actinobacteria with high C org contents (SF 11) supported free FA and PLFA results (SF 2, 3, 5). However, a direct comparison of DNA and PLFA-based community composition needs to be regarded with caution as both datasets have deviating taxonomic accuracy. Actinobacteria and Firmicutes (SF 12) preferred depths strongly affected by seasonal waterlogging (high a*, low κ), and were abundant in depths with rhizoliths, but not in recently rooted depths, which contradicts free FA and PLFA results (SF 2-5). This discrepancy may result either from the smaller size of the DNA sample set excluding the peak root abundances in the Holocene soil, or may demonstrate the time-integrated character of the DNA signal in terrestrial sediments, covering a wide range from potentially fossil to recent microbial communities. The latter hypothesis is supported by the DNA-based SF 12 which is the only statistical factor that correlates with both recent roots and rhizoliths (Tab. 2), corresponding to ancient and recent C input, whereas other statistical factors correlated solely with one of them. 3.5 Root-related long-term alteration of deep subsoil microbial communitiesconsequences and open questions Generally, the microbial community composition in deep subsoil was affected by pedogenic and weathering factors, as shown by correlations with clay, a* and κ. Additionally, our results emphasize

  ongoing microbial dynamics in sedimentary deep subsoil is a widespread phenomenon. As shown by a survey of root and root feature distribution in sedimentary deep subsoils of various age and texture throughout Central and Southeast Europe(Gocke et al., 2014a), deep roots entering soil parent material are not an exception but likely abundant at many sites globally.

  Figure 4.

Table 1 .

 1 Redundancies given in %, describing the portion, to which extent a predictor data set can explain the outcome data set. The data derives from canonical correlation analysis of the five microbial biomarker classes investigated.

	Outcome data set	Free FAs	PLFAs	CL-GDGTs	IPL-GDGTs	Bacterial
						phylotypes
	Predictor data set
	Free FAs		31	52	31	80
	PLFAs	38		38	42	35
	CL-GDGTs	65	38		31	70
	IPL-GDGTs	51	56	41		31
	Bacterial phylotypes	88	45	79	65
	3.4 Influence of environmental setting on deep subsoil microorganisms

  Intact polar glycerol dialkyl glycerol tetraethersArchaeal intact polar GDGTs(GDGT 1292(GDGT ,1298(GDGT ,1300 ; ; SF 9; GDGT 1292 GDGT ,1296 ; SF 10) were most abundant in depths affected by recent rooting, similar to their CL counterparts (SF 6), whereas the significance level was distinctly lower than for SF 6. In a Central Chinese soil profile,[START_REF] Ayari | Distribution of archaeal and bacterial tetraether membrane lipids in rhizosphere-root systems in soils and their implication for paleoclimate assessment[END_REF] observed enriched intact polar GDGTs in close vicinity of living roots. This suggests that the respective source organisms were strongly stimulated by postsedimentary penetrating roots, which is in agreement with our findings. The two bacterial intact polar GDGTs with commonly highest abundance in soil, GDGT 1022 and GDGT 1036 , generally loaded opposite of archaeal GDGTs within SF 9 and SF 10, and thus also behaved contrary to homologues with one (GDGT 1020,1034,1048 ) and two cyclopentyl moieties(GDGT 1018(GDGT ,1032(GDGT ,1046 ; ; SF 7, 8). In summary, core and intact polar GDGTs revealed that source organisms prefer places of stronger reworking, except for GDGT 1022 and GDGT 1036 , potentially deriving from a different group of microorganisms than other GDGTs.

	1018,1032,1046 ; SF 7) occurred mainly in
	depths strongly affected by ancient rooting (abundant rhizoliths, biopores) or by weathering (low S-
	ratio). Homologues with one cyclopentyl moiety (GDGT 1020,1034,1048 ; SF 8) were also more abundant in
	weathered depths (low C carb and alkane content, low CPI alk , abundant biopores, high clay content, a*
	and κ).
	3.4.4

Tech, France], C.-L. Müller [University of Bayreuth, Germany]). Access to microbiology laboratory was provided by H. Drake (University of Bayreuth, Germany).

Acknowledgments

The study was funded by the German Research Foundation (grant number DFG-WI 2810/10-1) and by the Swiss National Science Foundation (grant number 153631). The authors further thank LabEx Matisse for funding, and acknowledge financal support from the French-German PHC/ DAAD PROCOPE program. HeidelbergCement AG (Heidelberg, Germany) enabled sampling in their quarries, assisted by M. Löscher (Leimen, Germany). Several MSc and PhD students as well as technicians supported DNA (S. Hetz, R. Mertel, O. Schmidt, A. Stacheter, A. Wieczorek [University of Bayreuth, Germany]) and GDGT analyses (C. Anquetil [UPMC Paris, France], C. Fosse [Chimie Paris