Organic Geochemistry 61 (2013) 73–84

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Organic Geochemistry

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Comparison of the paleoclimatic significance of higher land plantbiomarker concentrations and pollen data: A case study of lakesediments from the Holsteinian interglacial

0146-6380/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.orggeochem.2013.06.006

⇑ Corresponding author. Present address: Civil and Environmental Engineering,Colorado School of Mines, 1500 Illinois Street, Golden, CO 80401, USA. Tel.: +1 303273 3871; fax: +1 303 273 3413.

E-mail address: jregnery@mines.edu (J. Regnery).

J. Regnery a,b,⇑, W. Püttmann a,c, A. Koutsodendris d, A. Mulch a,b,e, J. Pross a,d

a Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, 60325 Frankfurt, Germanyb Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, 60325 Frankfurt, Germanyc Department of Environmental Analytical Chemistry, Institute of Atmosphere and Environment, Goethe University Frankfurt, Altenhöferallee 1, 60438 Frankfurt, Germanyd Paleoenvironmental Dynamics Group, Institute of Geosciences, Goethe University Frankfurt, Altenhöferallee 1, 60438 Frankfurt, Germanye Institute of Geosciences, Goethe University Frankfurt, Altenhöferallee 1, 60438 Frankfurt, Germany

a r t i c l e i n f o

Article history:Received 1 March 2013Received in revised form 8 June 2013Accepted 17 June 2013Available online 27 June 2013

a b s t r a c t

A sediment core from the Dethlingen paleolake (Holsteinian interglacial, northern Germany) was sub-jected to integrated organic geochemical and palynological/paleobotanical analyses in order to recon-struct past environmental conditions. The fractionated lipid extracts were investigated using gaschromatography–mass spectrometry (GC–MS) and GC–combustion–isotopic ratio-MS. Ring A aromatizedpentacyclic triterpenoids, most likely glutinane derivatives, were the only higher-plant-derived pentacy-clic triterpenoids found. The saturated ring A degraded triterpenoid des-A-lupane was the most abundantaliphatic hydrocarbon throughout the core. Based on published pollen data, which imply the strong pres-ence of Betula and Alnus around the paleolake, the des-A-lupane would be expected to originate mainlyfrom lupane-type precursors synthesized by Betulaceae. The carbon isotopic composition of des-A-lupanevaried from �28.2‰ to �30.6‰ and no significant trend or isotopic shift was observed. However, the dis-crepancy between the variation in pollen data and biomarker concentration (i.e. quasi-absence of diter-penoids) in the core argues for a scenario in which the two proxies represent different components of thevegetation in the catchment area of the paleolake.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The analysis of higher plant-derived biomarkers has the poten-tial for reconstructing past depositional environments (e.g. Castañ-eda and Schouten, 2011). Although diagenesis generally results inreduced structural specificity of biomarkers, some of the moleculartransformations are indicative of the original biological com-pounds and the specific physicochemical conditions that prevailedduring diagenesis of compounds (Hautevelle et al., 2006; Jacobet al., 2007; Medeiros and Simoneit, 2007). In particular, di- andtriterpenoids, as well as their degradation products, can providevaluable information on paleoenvironmental conditions duringsediment deposition and early diagenesis; however, their potentialfor reconstructing past environments is far from being fullyexploited (Otto et al., 1994; Duan and Ma, 2001; Otto and Wilde,2001). Quantification of pentacyclic triterpenoid hydrocarbons

and tricyclic diterpenoid hydrocarbons in terrestrial sediments,including coal, provides a valuable tool for reconstructing the pro-portion of biomass derived from conifers and angiosperms thatcontributed to the organic matter (OM) in sediments (Bechtelet al., 2001, 2002; Widodo et al., 2009). However, decipheringprecise precursor/product relationships often suffers from largelyunknown compound transformation routes, as well as the occur-rence of non-specific precursors in higher land plants. For example,oxygenated pentacyclic triterpenoids of the ursane, oleanane andlupane types occur in almost all angiosperm taxa and their attribu-tion to individual plant families is difficult (Medeiros and Simoneit,2007). With respect to lupanes, only correlation with angiospermsof the Betulaceae family is convincing, since the lupane precursorsbetulin, lupeol and betulinic acid are the dominant pentacyclicconstituents of Betula bark (Hayek et al., 1989). Betulin is alsofound in the bark of other genera belonging to the Betulaceae fam-ily, including Alnus, Corylus and Carpinus (Hayek et al., 1990).

Sediment extracts often also contain tetracyclic hydrocarbonswhose structures allow them to be traced back to pentacyclictriterpenoid precursors that have lost the A ring. These com-pounds are described as des-A-triterpenoids (Trendel et al.,

74 J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84

1989; Logan and Eglinton, 1994). Related hydrocarbons with atetracyclic skeleton are not known in organisms and degradationof ring A has been shown to be microbially induced during thediagenetic transformation of higher plant triterpenoids (Trendelet al., 1989). Des-A-triterpenoids related to pentacyclic triterpe-noids of the oleanane, ursane and lupane type have been foundfrequently in sediments deposited under anoxic conditions (e.g.Logan and Eglinton, 1994; Jaffé and Hausmann, 1995; Jacobet al., 2007). Their potential application in paleoenvironmentalreconstruction, notably with regard to climatic conditions, hasbeen discussed in a number of studies (e.g. Jacob et al., 2007;Huang et al., 2008; Zheng et al., 2010).

To investigate the coherence of the signals from particular bio-markers in terrestrial sediments and the original vegetation, wehave followed an approach using both organic geochemical andpalynological/paleobotanical methods. Such an approach has beenused for the reconstruction of vegetation dynamics and climatevariability during the Miocene on the basis of coals (Bechtelet al., 2002, 2003). An excellent correlation of paleobotanical andbiomarker data was obtained by Otto et al. (1994) for Oligoceneoxbow lake sediments containing well-preserved leaves of Taxodi-um spp. from the quantification of simonellite. However, the paly-nological assemblages in lake sediments are not necessarilyexpected to closely mirror the abundance of higher land plant bio-markers since the composition of pollen assemblages is often af-fected by longer distance transport (e.g. Kotthoff et al., 2008,2011), whereas the biomarker composition most likely reflectsvegetation within, and in the close surroundings of, the lake (e.g.Schwark et al., 2002).

In this study, we have focused on lake sediments representingthe Holsteinian interglacial, which falls within Marine IsotopeStage (MIS) 11, one of the best analogs for the present and futureclimate with regard to orbital climate forcing (e.g. Loutre and Ber-ger, 2003). More specifically, the Holsteinian interglacial is widelyaccepted to be the terrestrial equivalent of MIS 11c in Central Eur-ope (e.g. de Beaulieu et al., 2001; Nitychoruk et al., 2006; Müllerand Pross, 2007; Preece et al., 2007). Paleolake sediments fromthe Holsteinian interglacial are particularly well suited for investi-gating the relationship between higher land plant biomarkers andthe composition of pollen assemblages because the Holsteinianinterglacial is characterized by unique vegetation dynamics inthe Central European lowlands and exhibits a higher proportionof conifer trees than more recent interglacial periods (Ber, 2006).At the same time, it is also marked by two climatically inducedregressive phases in vegetation dynamics, termed Older and Youn-ger Holsteinian Oscillations (OHO and YHO, respectively); theseregressive phases led to the transient development of boreal for-ests during full interglacial conditions (e.g. Kukla, 2003; Koutso-dendris et al., 2010).

2. Material and methods

2.1. Sample description

The core was recovered from the Dethlingen paleolake in theLüneburger Heide region of northern Germany (10� 08.3670 E, 52�57.7800 N). During the Holsteinian interglacial, ca. 11 m of organ-ic-rich diatomaceous sediments were deposited in a rather smalland deep dimictic lake setting (Fig. 1; Koutsodendris et al., 2010,2013). The diatomaceous sediments comprise both annually lami-nated and non-laminated lithological units. The occurrence of var-ved sediments indicates anoxic lake bottom conditions duringdeposition (O’Sullivan, 1983). Fourteen samples were taken fromselected intervals that represent major changes in the conifer/deciduous tree pollen concentrations during the Holsteinian inter-

glacial, including the OHO [ca. 29 m below surface (mbs)] and theYHO (ca. 26 mbs; Fig. 1; Koutsodendris et al., 2010). Each samplewas ca. 1 cm thick, representing ca. 20 yr on average (Koutsodend-ris et al., 2011).

2.2. Extraction and fractionation

Sediment (3–16 g) was freeze dried (20 h) and groundto < 200 lm. An aliquot (1–10 g) was Soxhlet-extracted for 24 husing dichloromethane (DCM)/MeOH (9:1, v:v). S was removedfrom the extract by addition of activated Cu plates. The bulk ofthe solvent was removed using rotary evaporation.

An aliquot (10 mg) of the S-free extract was fractionated usingcolumn chromatography with activated silica gel into three frac-tions (aliphatic, aromatic and polar) by elution with n-hexane, n-hexane:DCM (9:1 v:v) and MeOH. For verification, an aliquot ofthe polar fraction was silylated with bis(trimethyl)trifluoroaceta-mide (BSTFA) in pyridine at 60 �C for 2 h to convert alcohols to tri-methylsilyl (TMS) ethers and fatty acids to TMS esters,respectively. For quantification, a known amount of tetracosane-d50 was added as internal standard to the aliphatic fraction, and1,10-binaphthyl was used as internal standard for aromatic and po-lar fractions. Stock solutions of the internal standards were pre-pared in n-hexane.

2.3. Elemental analysis

Total organic carbon (TOC) content (wt%) was measured using aLECO RC-412 carbon analyzer. The S content (wt%) was determinedusing a LECO TruSpec S analyzer before and after extraction. Ex-tract yield was related to TOC content.

2.4. Gas chromatography–mass spectrometry (GC–MS)

GC–MS analysis of all three fractions was performed using aThermo Scientific Trace GC Ultra coupled to a linear ion trap massspectrometer (ITQ 900) with He (5.0) as carrier gas at a constant1.1 ml min�1 flow. Injections (1 ll) were made in splitless mode(1 min splitless time) through a Thermo Scientific TriPlus autosam-pler. Injector temperature was 280 �C. Separation was achievedwith a TG-5ms column (30 m � 0.25 mm i.d. � 0.25 lm film thick-ness; Thermo Scientific) and the following oven temperature pro-gram: 80 �C (1 min) ? 3 �C min�1 ? 300 �C (20 min). For thepolar fraction the GC oven heating rate was increased to4 �C min�1. The transfer line temperature was 280 �C and the ionsource temperature was 220 �C. The mass spectrometer was oper-ated in positive electron ionization mode (70 eV). Spectra were re-corded in full scan mode (m/z 50–600). MS/MS experiments withthe pulsed q-dissociation technique were carried out for the qual-itative analysis of unknown compounds. The polar fraction wasanalyzed using GC–MS before and after derivatization with BSTFA.Excalibur software (Thermo Scientific, version 2.0.7) was used toprocess the data. Biomarkers were assigned from comparison ofmass spectra and retention times with literature data.

2.5. Compound-specific carbon isotope analysis

The d13C values of individual compounds were determinedusing a Finnigan MAT delta S isotope mass spectrometer coupledto a Hewlett Packard G1530A GC instrument via a Finnigan MATcombustion interface II. A fused silica column (30 m � 0.32 mmi.d.; Thermo Scientific) coated with 5% diphenyl/95% dimethylpolysiloxane (film thickness 0.25 lm) was used with He (5.0) ascarrier gas at 1.4 ml min�1. Samples (1 ll) were injected in splitlessmode using a Finnigan MAT A200S autosampler. The injector con-ditions and the column oven temperature program were the same

Fig. 1. TOC (wt%), TS (wt%), and aliphatic, aromatic and polar fraction (wt%) in Dethlingen core. Major lithological units and C/N ratio according to Koutsodendris et al. (2010,2013).

J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84 75

as for GC–MS. The d13C values are reported in the standard nota-tion against Vienna Peedee Belemnite (VPDB). The system was cal-ibrated using certified stable isotope ratio references comprising amixture of five n-alkanes (C17, C19, C21, C23, C25), phenanthrene andsqualane, with values in the range �20.5‰ to �33.2‰ (A. Schim-melmann, Indiana University, personal communication). The val-ues of the standards correlated highly with those determinedoffline by A. Schimmelmann (R2 0.9999). Ultra-high purity CO2

(Air Liquide) with a known d13C value was pulsed (3�) through areference gas open split at the beginning and end of each sampleanalysis. Regularly performed on/off experiments with directly in-jected CO2 (n = 90) afforded a standard deviation betterthan ± 0.2‰. To assure the accuracy and precision of the system,the mixture of certified stable isotope ratio reference materialswas measured (5�) at the beginning and end of each sample se-quence. The standard deviation for the standards was on aver-age < ±0.4‰. Analysis (4�) of a sample from 24.88 mbs resultedin a precision of ± 0.2‰ for n-C23, ± 0.2‰ for n-C25, ± 0.4‰ fordes-A-lupane and ± 0.5‰ for des-A-olean-13(18)-ene. Data pro-cessing was accomplished using the Isodat NT software (ThermoElectron, version 1.5).

3. Results

3.1. Bulk parameters

TOC content ranged from 5.3% to 17.6%, with the highest valuesfor the non-laminated diatomite (24.63–26.98 mbs; Fig. 1). Thelowest value occurred at the top and bottom of the section. TotalS (TS) content varied between 1.3% and 6.2%, with the highest val-ues in the non-laminated diatomite and the diatomaceous mudsection (23.43–26.06 mbs). The sample from 25.88 mbs had thehighest TOC (17.6%) and TS (6.2%) values, as shown in Fig. 1. TOCand TS values were positively correlated (R2 0.62). For sampleswith high TS values, elemental S dominated. Elemental S contentin the upper part of the non-laminated diatomite and the diatoma-ceous mud section (23.43–26.06 mbs) ranged between 0.7% and1.1%. For all other samples the elemental S content was < 0.3%.

3.2. Aliphatic hydrocarbon fraction (alkanes/alkenes)

A total ion chromatogram (m/z 50–600) from sample 32.25 mbsaliphatic fraction is provided in Fig. 2I. The n-alkanes ranged fromn-C16 to n-C33 with an odd predominance between n-C21 and n-C33

and maximizing at n-C29. In general, the concentration of C25, C27

and C29 n-alkanes showed the same pattern in the core, as indi-cated in Fig. 3. Maximum concentration of n-C25 (0.90 lg g�1

TOC), n-C27 (2.28 lg g�1 TOC) and n-C29 (2.98 lg g�1 TOC) occurredin the non-laminated diatomite at 25.88 mbs. The n-C29 alkane wasmore abundant than n-C27 and n-C31 in the non-laminated upperpart of the section (27.80–24.10 mbs), a typical distribution forlong-chain alkanes in forest areas. The carbon preference index(CPI) for n-C22 to n-C31 alkanes ranged between 3.6 (28.99 mbs;OHO) and 10.8 (29.93 mbs) and the average chain length (ACL)(n-C23 to n-C33) throughout the core was in the range 26.7–28.6(Fig. 3). Paq, the ratio of the abundance of mid-chain (C23 + C25)n-alkanes over the total sum of the abundances of the mid- andlong-chain (C23 + C25 + C29 + C31) n-alkanes (Ficken et al., 2000),ranged between 0.2 and 0.6 and showed a maximum duringOHO (Fig. 3).

In the n-C23 to n-C25 region, seven peaks were tentativelyassigned as being aliphatic des-A-triterpenoids from comparisonwith published data (Fig. 2I). As listed in Table 1, the mass spec-tra exhibited M+� ions at m/z 326, 328 and 330, respectively,referring to tetracyclic terpene and terpane carbon skeletons.Peaks 2, 3, 4 and 5 all displayed M+� at m/z 328 and were tenta-tively assigned as des-A-olean-13(18)-ene, des-A-olean-12-ene,des-A-urs-13(18)-ene and des-A-olean-18-ene on the basis ofpublished mass spectra and relative retention data (Trendelet al., 1989; Logan and Eglinton, 1994; Jacob et al., 2007). Thespectrum and structure of compound 7 (M+� m/z 330) corre-sponded to des-A-lupane (Fig. 2I; Trendel et al., 1989; Borehamet al., 1994). Peaks 1 and 6 (both M+� at m/z 326) showed afragmentation pattern similar to di-unsaturated terrestrialdes-A-triterpenoids of the oleanane and ursane series (Huanget al., 2008; Zheng et al., 2010). Significant ions in the spectraof peaks 1 and 6 were (decreasing relative abundance) m/z311, 326 (M+�), 173, 229, 244, 159, 187, 255, and m/z 136, 311,

Fig. 2. Total ion chromatograms of sample 32.25 mbs aliphatic fraction (I) detailing section between n-C23 and n-C25 alkanes (sample 28.33 mbs), sample 28.48 mbs aromaticfraction (II) and sample 29.93 mbs polar fraction (III); peak identifications in Table 1.

76 J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84

Fig. 3. Profiles of selected n-alkanes and related molecular proxies for Dethlingen core. Concentration is expressed in lg g�1 TOC. ACL, average carbon chain length;ACL = (23 � C23 + 25 � C25 + 27 � C27 + 29 � C29 + 31 � C31 + 33 � C33)/(C23 + C25 + C27 + C29 + C31 + C33). CPI, carbon preference index; CPI = 0.5 � [(C23 + C25 + C27 + C29 + C31)/(C22 + C24 + C26 + C28 + C30) + (C23 + C25 + C27 + C29 + C31)/(C24 + C26 + C28 + C30 + C32)]. Paq = (C23 + C25)/(C23 + C25 + C29 + C31) n-alkanes.

Table 1Peak assignment for compounds discussed in the text.

Nr. Compound MW BP Key ions (m/z) Ref.b

Aliphatic fraction1 Diene des-A-triterpenea 326 311 244, 229, 1732 Des-A-Olean-13(18)-ene 328 189 313, 204, 109, 218 A, B3 Des-A-Olean-12-ene 328 203 218, 189, 313, 95, 231 A, B4 Des-A-Urs-13(18)-ene 328 313 189, 161, 95, 177, 204 C5 Des-A-Urs-12-ene 328 313 231, 203, 189, 95 C6 Diene des-A-triterpenea 326 136 311, 229, 204, 173, 957 Des-A-Lupane 330 163 149, 191, 123, 177, 287 A, D

Aromatic fraction8 Simonellite 252 237 195, 179, 252 E9 3,4,7,12a-Tetramethyl-1,2,3,4,4a,12,12a-octahydrochrysene 292 292 168, 181, 207, 277 F

10 3,3,7,12a-Tetramethyl-1,2,3,4,4a,11,12,12a-octahydrochrysene 292 207 249, 193, 155, 123 F11 3,4,7-Trimethyl-1,2,3,4-tetrahydrochrysene 274 259 229, 244, 215, 202 F12 3,3,7-Trimethyl-1,2,3,4-tetrahydrochrysene 274 218 202, 259 F13 3-Methyl-24-nor-friedela-1,2,5(10)-triene 406 185 391, 205, 171 G

Polar fraction14 3-Hydroxy-24-nor-friedela-1,2,5(10)-trienea 408 187 393, 205, 173

a Tentatively assigned.b A, Trendel et al. (1989); B, Logan and Eglinton (1994); C, Jacob et al. (2007); D, Boreham et al. (1994); E, Philp (1985); F, Freeman et al. (1994); G, Schaeffer et al. (1995).

J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84 77

326 (M+�), 173, 95, 108, 204, respectively. Concentration ofdes-A-lupane ranged from 0.01 lg g�1 TOC to 1.80 lg g�1 TOC(Fig. 4) and in the laminated diatomite (33.35–28.99 mbs)exceeded those of the C27 and C29 n-alkanes (compare Fig. 3).

3.3. Aromatic hydrocarbons

The fraction of aromatic hydrocarbons was low in all samples,varying between 0.2% and 2.4% (Fig. 1). The increased proportionof 7.6% at 24.88 mbs can be explained by the presence of elementalS not totally removed from the sample. Repetition of the Cu treat-ment was not carried out since elemental S did not influence theMS analysis of the aromatic hydrocarbons. The total ion chromato-gram (m/z 50–600) of the aromatic fraction at 28.48 mbs is shownin Fig. 2II.

Some mono-, di- and triaromatic diterpenoids of the abietaneclass, such as dehydroabietins, simonellite (8) and retene, were

present in low abundance (low ng g�1 TOC vs. lg g�1 TOC) in mostsamples. Simonellite was in the range < 1 ng g�1 TOC to 8 ng g�1

TOC as shown in Fig. 5, with a maximum during the OHO. Tetra-and pentacyclic aromatized products of triterpenoid precursors,such as b-amyrin and lupeol, were detected in the aromatic hydro-carbon fractions in much higher concentration than tricyclic diter-penoids. Four tetracyclic aromatic hydrocarbons of the oleananeand ursane series (9, 10, 11, 12) with M+� at m/z 274 and m/z 292,respectively were assigned after comparison with reference spectra(Table 1 and Fig. 2II). The spectrum of 13 (Fig. 6A) showed M+�

at m/z 406 (C30H46) and major fragments at m/z 185, 205, 173,391 (M+� –CH3), and was identical to that of 3-methyl-24-nor-friedela-1,3,5(10)-triene first published by Schaeffer et al. (1995).Quantification of 13 gave a concentration in the range 3 ng g�1

TOC to 137 ng g�1 TOC (Fig. 4). The concentration profile of 13was similar to that of the phenolic compound 14 described inSection 3.4.

Fig. 4. Variation in abundance of des-A-lupane vs. des-A-olean-13(18)-ene, concentration in lg g�1 TOC of des-A-lupane, des-A-olean-13(18)-ene, compound 13, compound14, and proportion of selected deciduous tree pollen along the Dethlingen core. Betulaceae comprise Alnus, Betula, Corylus and Carpinus (Koutsodendris et al., 2010).

Fig. 5. Concentration of simonellite (ng g�1 TOC) as well as conifer/deciduous tree pollen concentration at the exact core depth as geochemically analyzed samples andproportion of all recorded conifer tree pollen along the Dethlingen core (Koutsodendris et al., 2010). Note change in pollen abundance scale for different trees.

78 J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84

3.4. Polar fraction

None of the triterpenoid precursors such as b-amyrin or lupeolwere detected. The most prominent peak (Fig. 2III) was tentativelyassigned as a phenolic derivative of an aromatized triterpenoid ofthe friedelane or glutinane class. To our knowledge, it has not beendescribed before.

The MS fragmentation pattern, both for the silylated and theunderivatized component, are in accord with 14 being 3-hydro-xy-24-nor-friedela-1,2,5(10)-triene (Fig. 6B). It exhibits M+� at m/z408 (C29H44O) and major fragments at m/z 187, 173, 205, 393

(M+� –CH3). The presence of an OH was confirmed by a shift of72 Da to higher mass after derivatization (Fig. 6C) to the TMS ether(M+� at m/z 480). The base peak at m/z 187 for the underivatizedcompound (Fig. 6B) indicated that the oxygen was at ring A or B.In the case of a ring B aromatized hydrocarbon with methyls at C-13 and C-14, characteristic fragment ions at m/z 213, 225, 239would be expected (Budzikiewicz et al., 1963). Their absence arguesfor ring A aromatization. Furthermore, Simoneit et al. (2003) inves-tigated phenolic triterpenes closely related to 14, although not fri-edo-rearranged, and indicated the location of the hydroxyl functionto be generally on ring A. In addition, 14 has the same key and major

185

205

OH

187

205

53 4

1314

Si O

205259

A

B

C

Fig. 6. Mass spectra of (A) compound 13 in aromatic fraction, (B) tentatively assigned compound 14 and (C) its TMS derivative in polar fraction, with interpretation of twomajor fragments.

J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84 79

ions as the ring A aromatized compound 13 found in the aromaticfraction, except for a shift to higher mass by 2 Da, reflecting thepresence of an OH instead of a methyl at C-3.

3.5. Carbon isotope ratio of specific biomarkers

The carbon isotopic composition of n-C23 and n-C25 alkanes var-ied from �24.9‰ to �28.4‰ and �26.5‰ to �30.8‰, respectively,whereas that of des-A-olean-13(18)-ene and des-A-lupane rangedfrom �23.5‰ to �28.1‰ and �28.2‰ to �30.6‰, respectively(Fig. 7). Due to the very low amount of aromatic hydrocarbon bio-markers, attempts to determine their carbon isotopic compositionwere unsuccessful.

4. Discussion

4.1. Origin of biomarkers

The distribution pattern of n-alkanes in lake sediments is gener-ally based on contributions from terrestrial/emergent plants (i.e.C29), floating/submerged vegetation (C23 and C25 alkanes) andaquatic algae and bacteria (C17 and C21 alkanes). Proxies such asACL, CPI and Paq are used to derive climate-induced changes re-corded in lake sediments according to the n-alkane distributionsin vegetation (Ficken et al., 2000; Meyers, 2003; Sun et al., 2013).The distribution from Dethlingen sediments is thought to becharacteristic for land plant-derived n-alkanes (e.g. Eglinton and

Fig. 7. d13C values (‰) of individual biomarkers and n-alkanes in lake sediments from Dethlingen.

80 J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84

Hamilton, 1967; Xu and Jaffé, 2008; Seki et al., 2010). However, thehigher Paq value and the lower ACL value at 28.99 mbs (Fig. 3)suggest a significant input from submerged vegetation into thepaleolake during the OHO.

The mono-, di-, and triaromatic diterpenoids of the abietaneclass, present in low abundance in the lake sediments, are charac-teristic of conifers (Otto et al., 1997; Hautevelle et al., 2006; Stefa-nova et al., 2011). Simenollite concentration vs. all conifer pollenrecorded at Dethlingen paleolake is displayed in Fig. 5. The Pinuspollen abundance most closely reflected the concentration profileof simonellite throughout the core, with increasing concentrationduring the OHO and the YHO.

None of the triterpenoid precursors (b-amyrin or lupeol) weredetected in the polar fractions after derivatization. This arguesfor a complete transformation of the biological precursor com-pounds during early diagenesis, either by biotic or abiotic pro-cesses. The early diagenetic transformation of higher planttriterpenoids leads to des-A-triterpenoids, as well as their aroma-tized derivatives (Stout, 1992; Hazai et al., 1992; Jacob et al.,2007). The presence of similar B- and C-ring aromatized tetracyclicdes-A-triterpenoids has also been described for Late Pleistocene–Holocene sediments (< 20 kyr) from Lake Caçó in NE Brazil (Jacobet al., 2007). However, these transformation processes strongly de-pend on the conditions in the water column of the lake and withinthe sediment (Jaffé and Hausmann, 1995; Jacob et al., 2007).

Dominating the entire record, des-A-lupane was the most prom-inent component, not only among the des-A-triterpenoids, butamong all components in many of the aliphatic hydrocarbon frac-tions of the samples. The transformation of lupane type precursorsto des-A-lupane could have been favored by microorganisms underanaerobic conditions within the paleolake, as suggested by studiesof comparable lake systems (Jacob et al., 2007; Huang et al.,2008). It has been assumed that des-A-triterpenoids of the olean-ane and ursane type are more sensitive to aromatization and fur-ther degradation in the lake water column and sediment thandes-A-lupane (Jacob et al., 2007). The relative abundances of des-A-lupane and mono-unsaturated des-A-triterpenes showed similardowncore trends (Fig. 4). Des-A-lupane has also been reported as

the only saturated ring-A-degraded triterpenoid in all samplesfrom early Holocene peat deposits in southern China (Huanget al., 2008). In contrast to the findings of Jacob et al. (2007) andHuang et al. (2008), the concentration of des-A-lupane correlatedsignificantly with that of des-A-olean-13(18)-ene (R2 0.92) andthe sum of the monounsaturated triterpenes (R2 0.87) as shownin Fig. 8. However, the high des-A-lupane concentration cannotbe explained by the presence of spike-rush (Eleocharis spp., Cyper-aceae family) along the lake shore, as proposed by Jacob et al.(2007) for a small Brazilian lake. If significant Cyperaceae popula-tions had thrived along the shores of the Dethlingen paleolake, ahigh amount of Cyperaceae pollen should have been introducedinto the lake (cf. Shulmeister, 1992). However, Cyperaceae pollenwas present only in low amount in the Dethlingen core (Koutsodendriset al., 2012). Otto et al. (2005) detected considerable amounts ofdes-A-triterpenoids (i.e. 2, 3 and 7) in fossil plant leaf extracts ofBetula spp., most probably generated from triterpenoid precursorsthrough microbial or photochemical processes (Corbet et al., 1980).At Dethlingen, four genera belonging to the Betulaceae family werefound: Alnus, Betula, Corylus and Carpinus (Koutsodendris et al.,2010). Based on the pollen record, which exhibited very high pro-portions of Betulaceae (Koutsodendris et al., 2010; Fig. 4), it couldbe hypothesized that the des-A-lupane originates mainly from thewell-known lupane type precursors (e.g. betulin, betulinic acid, lu-peol, lup-20(29)-en-3b,16b-diol) synthesized by Betula spp.(O’Connell et al., 1988; Cole et al., 1991; Modugno et al., 2006).However, des-A-lupane likely originates only from lupanol and/orlupenol via loss of ring A and reduction of the 20(29) double bond.An origin from betulin, betulinic acid and lup-20(29)-en-3b,16b-diol would require an additional reduction process. Apart from Bet-ula, Alnus also synthesizes lupane-related triterpenoids (Felföldi-Gava et al., 2009); thus, and considering that Alnus is wellrepresented in the palynological record from Dethlingen (Fig. 4),this genus may have been an additional source of des-A-lupane.A recent study by Stefanova et al. (2011) corroborates this assump-tion, as the paleobotanical data therein provide evidence for a sig-nificant role of Alnus as a source of des-A-lupane. In addition, Alnusand Betula typically grow in the immediate vicinity of lakes,

R 0.9241

Des

-A-o

lean

-13(

18)-e

ne (µ

g g-1

TO

C)

Des-A-lupane (µg g-1 TOC)

A

R 0.8683

Mon

oene

des

-A-tr

iterp

enes

(µg

g-1 T

OC

)

Des-A-lupane (µg g-1 TOC)

B

0.0 1.0 2.00.0

1.0

0.0 1.0 2.00.0

0.5

Fig. 8. Correlation between concentration of des-A-lupane and des-A-olean-13(18)-ene (A) and sum of monoene des-A-triterpenes (B), respectively.

J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84 81

whereas Corylus and Carpinus tend to be more abundant in the dis-tant surroundings (Ellenberg, 1988). Therefore, we interpret thepresence of lupane type precursors in the Dethlingen sedimentsas deriving predominantly from Alnus and Betula, although a (morelikely subordinate) input of Carpinus and Corylus cannot be ex-cluded. This interpretation is in line with the carbon isotopic com-position of des-A-lupane throughout the core (�29.2 ± 0.7‰),which is compatible with published data (Schoell et al., 1994)and confirms the assumption that its pentacyclic lupane type pre-cursor originates mainly from higher land plants located aroundthe paleolake. As shown in Fig. 7, no significant trend or isotopicshift could be observed for des-A-lupane along the core.

No evidence for isotopic fractionation due to microbial activityor photochemical alteration was found when comparing triterpe-noids and the structurally related des-A-derivatives (Freemanet al., 1994). Hence, d13C values of these degradation productsare expected to be related mainly to the isotopic composition ofthe carbon source. The n-C23 and n-C25 alkanes, as well as des-A-olean-13(18)-ene, are significantly enriched in 13C (ca. 3–4‰) rel-ative to other lipids known to derive from higher land plants (Sekiet al., 2010). Considering the results of Freeman et al. (1994), thesecompounds might originate partly from aquatic macrophytes witha higher d13C proportion as a result of low CO2 availability in thesubaqueous environment of the paleolake (cf. Hollander and Smith,2001). The n-C25 alkane d13C value averaged �28.6 ± 1.2‰

throughout the core, meaning that it was enriched in 13C by ca.4‰ vs. published data for it in extant higher land plants (Schoellet al., 1994; Lockheart et al., 1997; Xie et al., 2004; Seki et al.,

2010). Moreover, a high concentration of n-alkanes in non-emer-gent (n-C23 and n-C25) and emergent (> n-C29) plant taxa has ledto the suggestion that aquatic macrophytes are among the domi-nant sources of lipids and n-alkanes in lake sediments (Fickenet al., 2000). It is notable that the less depleted d13C values forthe n-alkanes might also be related to the occurrence of microor-ganisms such as bacteria, fungi and algae within the Dethlingenpaleolake. Fungal spores and algae have been found to also containn-C23 and n-C25 alkanes, with less depleted d13C values than higherplants (Xie et al., 2004). Furthermore, a d13C enrichment of n-al-kanes in peat of ca. 2.5‰ after burial has been observed (Fickenet al., 1998).

Aromatized pentacyclic triterpenoids of the glutinane andfriedelane type (13 and 14) were the only higher plant-derivedpentacyclic triterpenoids detected. Schaeffer et al. (1995) proposedan acid-catalyzed enone-benzene transposition for 3-oxygenatedtriterpenes, with a gem shift of a methyl from C-4 to C-3, insteadof its elimination leading to direct abiotic A-ring aromatization.Nuclear magnetic resonance and MS analyses led to the identifica-tion of 13 (Schaeffer et al., 1995). This monoaromatic pentacyclichydrocarbon was suggested to originate from D5- or D5(10)-glu-ten-3-one, a partially rearranged oleanane skeleton. The ring A-aromatization of this compound usually requires acidic conditions(Schaeffer et al., 1995). The precursor of 13 is not related to friede-lane, but to glutinane (Schaeffer et al., 1995). Compound 14 couldbe either a glutinane or friedelane derivative as the loss of eitherthe C-24 (if glutinane) or C-25 (if friedelane) methyl during aroma-tization does not allow distinguishing between the two types ofcompounds. Although friedelin cannot be excluded as a possibleprecursor of 14, glutinane-related triterpenoids might be envis-aged, in particular since 13 and 14 had similar concentration pro-files (R2 0.79) throughout the core (Fig. 4). Most likely, 13 and 14originate from the same glutinane type precursor. However, re-ports of the occurrence of glutanoids in angiosperms are scarce(Mahato et al., 1992; Jacob, 2003). Glutinane type precursors occur(Jacob, 2003) in Asteracea, Euphorbiaceae, Ericaceae, Poaceae,Betulaceae (i.e. Alnus) and Fagaceae (i.e. Quercus). In contrast, frie-delin is widely distributed in many kinds of plant tissues of higherland plants (Chandler and Hooper, 1979; Stefanova et al., 2011). Itsabundance in soils has been related to the input from Quercus spp.bark and roots, respectively (Trendel et al., 2010). Comparing theconcentration profiles of 13 and 14 with the respective pollen datathroughout the core, only a correlation with Alnus pollen is evidentas shown in Fig. 4. Euphorbiaceae pollen have not been recorded inthe lake sediments. As discussed earlier in this section, Alnus is wellrepresented in the palynological record of the Dethlingen paleo-lake. Though both des-A-lupane and compound 14 most likely orig-inate from lupane and glutinane type precursors in Betula andAlnus, the concentration of 14 shows only a weak correlation withthat of des-A-lupane (R2 0.32) within the core and therefore arguesfor different contributions from the respective sources for bothcompounds.

4.2. Representativeness of pollen and biomarkers

The discrepancy between the variation in biomarker concentra-tion (i.e. quasi-absence of diterpenoids) and pollen data along thecore argues for a scenario where each proxy represents differentcomponents of the vegetation surrounding the paleolake. Due totaphonomic processes (including sorting during transport; e.g. Tra-verse, 1988), the pollen abundance in lake sediments does not nec-essarily reflect the composition of the vegetation thriving along thelake shore; instead, it provides a more integrated regional view onthe vegetation in the surroundings of the lake. In contrast, the com-position and abundance of biomarkers in lake sediments representa reliable proxy for the vegetation in the lake itself and its close

82 J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84

surroundings (e.g. Meyers and Ishiwatari, 1993; Ficken et al.,2002). Although the pollen record indicates the occurrence of coni-fers in the vicinity of the Dethlingen paleolake (Koutsodendriset al., 2010; Fig. 5), angiosperms growing close to the lakeshore,such as Betulaceae, most likely produced more OM that was intro-duced into the lake. For instance, upon defoliation in autumn,deciduous trees yield a much larger amount of leaf material tobe transported to the lake than evergreen gymnosperms. More-over, different types of plant material (e.g. roots, stems, bark, pol-len) contain variable amounts of terpenoids, which generallyresults in an underestimate of the biomass from conifers comparedwith that from angiosperms in the sediment (Otto et al., 2005). Inaddition, deciduous gymnosperms contain significantly less n-al-kanes than deciduous angiosperms. Analyzing the n-alkane abun-dance of 46 trees species representing 24 families, Diefendorfet al. (2011) found a 200� higher abundance of n-alkanes in decid-uous angiosperms than in deciduous gymnosperms. This providesanother line of evidence for biomarker records largely reflectingangiosperms if both groups were present.

4.3. Paleoclimatic implications of biomarkers

Based on the notion that biomarkers primarily reflect the vege-tation dynamics close to the lake shore as discussed above, we fo-cus here on the OHO and YHO events to gain insight intovegetation change in the littoral zone during climatically inducedvegetation shifts during the Holsteinian interglacial. While simon-ellite concentration significantly increased during the OHO, theconcentration of all des-A-triterpenoids of the oleanane, ursaneand lupane type decreased, although des-A-lupane concentrationremained at an elevated level (Fig. 4). However, des-A-triterpenoidconcentrations did not decrease during the YHO and simonelliteshowed only a slight increase during this interval. The des-A-lu-pane/des-oleane-13(18)-ene ratio, which increased during theOHO, remained stable during the YHO (Fig. 4). Except for des-A-lu-pane, the concentration of all other des-A-triterpenoids, n-C27 andn-C29 alkanes, and 13 and 14 decreased almost to zero during theOHO cold event (Figs. 3 and 4). The ratio between des-A-lupaneand mono-unsaturated des-A-triterpenes (Fig. 8B) closely reflectsthis discrepancy. Interestingly, des-A-lupane showed a minimumconcentration at 28.48 mbs in the profile (Fig. 4). The ACL valuedropped from 28.0 at 29.93 mbs to 26.7 during the OHO. It hasbeen suggested that ACL values could serve as a temperature indi-cator, as ACL decreases with decreasing temperature (e.g. Zhanget al., 2006; Sun et al., 2013), although the paleoclimatic signifi-cance of this proxy requires further research.

According to the pollen record, only the abundance of Betulapollen increased during the OHO, whereas that of all other Betul-aceae pollen decreased (Fig. 4). Often representing pioneer trees,Betula is more tolerant to environmental change than otherangiosperms (e.g. Ellenberg, 1988). Therefore, it appears plausiblethat Betula remained essentially unaffected by the climaticcooling during the OHO; consequently, its pollen productionremained high, in contrast to that of other deciduous trees(Koutsodendris et al., 2010, 2012). This scenario would explainwhy the des-A-lupane concentration did not collapse with thesame intensity as observed for all other des-A-triterpenoids inthe core due to abrupt climate change. Most probably, des-A-lu-pane represents a mixed signal for Betula and Alnus, both growingin the vicinity of the paleolake. The fact that the des-A-lupane/des-A-olean-13(18)-ene ratio peaked during the OHO (Fig. 4) alsopoints to a significant proportion of des-A-lupane deriving fromBetula. The d13C values of des-A-olean-13(18)-ene point to an atleast partial input of this des-A-compound from macrophytes.Furthermore, the low abundance of total des-A-triterpenes atthe OHO reveals a low input of organic material to the lake, as

the decrease in des-A-triterpenoids was not accompanied by anincrease in the corresponding pentacyclics. Such a scenario of re-duced organic input during the OHO is supported by microfaciesanalysis and micro-X-ray fluorescence data, which indicatechanges in lake productivity but no significant changes in thesedimentation processes (Koutsodendris et al., 2011). In addition,an increased Paq value of 0.6 and a slight decrease in C/N duringthe OHO indicate a higher proportion of submerged/floating veg-etation contributing to the organic lake input (Koutsodendriset al., 2013).

The increase in TOC and S contents towards the younger inter-vals of the Holsteinian interglacial (25.88–26.98 mbs; Fig. 1) sug-gests an increased input of terrestrial OM to the paleolake. LowPaq values and a C/N ratio > 15 indicate a significant contributionof terrestrial OM to the lake sediments during this period (Fig. 1).Changes in sediment microfacies and diatom ecology point todecreasing water depth during this interval (Koutsodendriset al., 2013). The high elemental S content during the youngerintervals might be an indication for suboxic sediment layers,although the mechanisms underlying elemental S formation inlakes are not well known (Holmer and Storkholm, 2001). In gen-eral, S deposition in lakes is higher under eutrophic conditions.During the YHO, the content of des-A-lupane as well as 13 and14 increased, in agreement with an increase in the abundanceof Betulaceae pollen (Fig. 4). In particular, the pollen recordshows an increase in Betula and Alnus pollen abundances,whereas the abundance of Corylus pollen remained rather stableand Carpinus pollen abundance decreased (Koutsodendris et al.,2010). Following the same concept as for the OHO, the increasein Betulaceae could explain the increase in des-A-lupane duringthe YHO. The input of des-A-lupane to the paleolake may havebeen facilitated by an increased input of terrestrial material to-wards the younger interval of the Holsteinian interglacial(Koutsodendris et al., 2013). This is further corroborated by a highamount of biomarkers in the sediments during this interval. Inparticular, the n-C23, n-C25, n-C27, and n-C29 alkanes reachedmaximum concentration during the YHO (Fig. 3), whereasdes-A-triterpenoid concentration remained largely stable. Thevariety of biomarkers reflects a diverse forest growing in thesurroundings of the paleolake during the YHO, in agreement withthe pollen data (Koutsodendris et al., 2010).

Although the Holsteinian interglacial in the Central Europeanlowlands is characterized by a high abundance of conifer pollen,only a low amount of conifer-derived biomarkers was foundalong the core from Dethlingen paleolake, as presented forsimonellite. Such a discrepancy has been observed in otherstudies. Meyers and Ishiwatari (1993) observed in an investiga-tion of Lake Washington that the watershed successioninterpreted from lignin components of the lake sedimentsbasically agreed with the pollen reconstruction, except for aninterval dominated by pine. In a recent study, Stefanova et al.(2011) reported a lower abundance of diterpenoids in a paleo-mire than expected on the basis of pollen data. These findingsimply that further investigations will be necessary to overcomethe above-mentioned limitations of combining both proxies forpaleoenvironmental reconstruction. Quantitative relationshipsbetween organic geochemical and palynological/paleobotanicalindicators of OM deposited in lake sediments need better defini-tion. Measurements of their respective contributions to sedimentOM will allow for balanced source assessment. However, in spiteof the differences between both proxies, des-A-lupane, 3-methyl-24-nor-friedela-1,2,5(10)-triene (13), 3-hydroxy-24-nor-friedela-1,2,5(10)-triene (14) and simonellite, concentrations reflect thevegetation changes in the vicinity of the paleolake during thefocused short term vegetation shifts (OHO and YHO), in agree-ment with the pollen data.

J. Regnery et al. / Organic Geochemistry 61 (2013) 73–84 83

5. Conclusions

Higher plant lipid biomarkers (e.g. des-A-lupane) and pollendata in a sediment core from the Dethlingen paleolake providepaleoenvironmental information about the Holsteinian interglacialin Central Europe. We consider des-A-lupane to be representativeof Betula and Alnus that grew in the vicinity of the paleolake. Fur-thermore, a new phenolic derivative of an aromatized triterpenoidbiomarker of the glutinane or friedelane class was tentatively as-signed. The high abundance of des-A-triterpenoids, as well as theunusual transformation process of the glutinane/friedelane typepentacyclic triterpenoids, was favored by anoxic or at least dysaer-obic conditions in the S-rich lake sediments. The discrepancy be-tween the biomarker-derived information and that derived fromthe pollen is explained by the fact that the biomarkers representthe vegetation growing directly along the lakeshore, whereas thepollen data show a more regionally integrated signal. However,the biomarker concentrations are in line with the pollen-derivedvegetation information during short term cold intervals; forinstance, the increase in Betulaceae can explain the increase ofdes-A-lupane during the YHO. Hence, we conclude that biomarkerconcentrations represent a meaningful proxy for reconstructingand understanding the dynamics of the vegetation that thrivedalong the shore of the paleolake.

Acknowledgements

The study was supported through the LOEWE program (Landes-Offensive zur Entwicklung Wissenschaftlich-Ökonomischer Exzel-lenz) of Hesse’s Ministry of Higher Education, Research, and theArts by funding the Biodiversity and Climate Research Centre(BiK-F). Funding for A.K. and J.P. through the German ResearchFoundation (DFG) is gratefully acknowledged. We thank U.C.Müller for providing core material and J. Jacob and two anonymousreviewers for constructive remarks.

Associate Editor—J.K. Volkman

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