ORIGINAL RESEARCH ARTICLEpublished: 08 August 2013

doi: 10.3389/fmicb.2013.00222

Wide distribution of autochthonous branched glyceroldialkyl glycerol tetraethers (bGDGTs) in U.S. Great Basinhot springsBrian P. Hedlund1*, Julienne J. Paraiso1,2, Amanda J. Williams1, Qiuyuan Huang3, Yuli Wei4,Paul Dijkstra 5, Bruce A. Hungate5, Hailiang Dong3 and Chuanlun L. Zhang2,4*

1 School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV, USA2 State Key Laboratory of Marine Geology and School of Ocean of Earth Sciences, Tongji University, Shanghai, China3 Department of Geology and Environmental Earth Science, Miami University, Oxford, OH, USA4 Department of Marine Sciences, University of Georgia, Athens, GA, USA5 Department of Biological Sciences and Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, AZ, USA

Edited by:

Eric Boyd, Montana State University,USA

Reviewed by:

Jeff R. Havig, Penn State University,USAScott T. Kelley, San Diego StateUniversity, USA

*Correspondence:

Brian P. Hedlund, School of LifeSciences, University of Nevada, LasVegas, 4505 Maryland Parkway, LasVegas, NV 89154-4004, USAe-mail: brian.hedlund@unlv.edu;Chuanlun L. Zhang, Department ofMarine Sciences, University ofGeorgia, Athens, GA 30602, USAe-mail: czsrel@uga.edu

Branched glycerol dialkyl glycerol tetraethers (bGDGTs) are membrane-spanning lipidsthat likely stabilize membranes of some bacteria. Although bGDGTs have been reportedpreviously in certain geothermal environments, it has been suggested that they mayderive from surrounding soils since bGDGTs are known to be produced by soil bacteria.To test the hypothesis that bGDGTs can be produced by thermophiles in geothermalenvironments, we examined the distribution and abundance of bGDGTs, along withextensive geochemical data, in 40 sediment and mat samples collected from geothermalsystems in the U.S. Great Basin (temperature: 31–95◦C; pH: 6.8–10.7). bGDGTs werefound in 38 out of 40 samples at concentrations up to 824 ng/g sample dry mass andcomprised up to 99.5% of total GDGTs (branched plus isoprenoidal). The wide distributionof bGDGTs in hot springs, strong correlation between core and polar lipid abundances,distinctness of bGDGT profiles compared to nearby soils, and higher concentration ofbGDGTs in hot springs compared to nearby soils provided evidence of in situ production,particularly for the minimally methylated bGDGTs I, Ib, and Ic. Polar bGDGTs were foundalmost exclusively in samples ≤70◦C and the absolute abundance of polar bGDGTscorrelated negatively with properties of chemically reduced, high temperature springsources (temperature, H2S/HS−) and positively with properties of oxygenated, lowtemperature sites (O2, NO−

3 ). Two-way cluster analysis and nonmetric multidimensionalscaling based on relative abundance of polar bGDGTs supported these relationships andshowed a negative relationship between the degree of methylation and temperature,suggesting a higher abundance for minimally methylated bGDGTs at high temperature.This study presents evidence of the widespread production of bGDGTs in mats andsediments of natural geothermal springs in the U.S. Great Basin, especially in oxygenated,low-temperature sites ( 70◦C).≤Keywords: geothermal springs, membrane-spanning lipids, bGDGTs, thermophiles, Great Basin, lipids

INTRODUCTIONGlycerol dialkyl glycerol tetraethers (GDGTs) are core membrane-spanning lipids that resist delamination and impart membranestability at high temperature and low pH. GDGTs are synthe-sized by a variety of extremophiles and some non-extremophilesand occur widely in nature (Schouten et al., 2000, 2013 andreferences therein). The isoprenoid GDGTs (iGDGTs) containalkyl groups constructed by polymerization of isoprene sub-units and are synthesized by physiologically and phylogeneticallydiverse archaea [reviewed in Schouten et al. (2013)]. A sepa-rate group of GDGTs, the branched GDGTs (bGDGTs), havebeen identified in lipid extracts prepared from a wide varietyof environments, including peat bogs, soils, estuaries, and lakeand river water and sediments (Schouten et al., 2000; Sinninghe

Damsté et al., 2000, 2009; Weijers et al., 2006a, 2009; Zinket al., 2010; Zhang et al., 2012; Liu et al., 2013; Yang et al.,2011, 2013; Wang et al., 2013). bGDGT types vary according tothe degree of methylation of the alkyl chains, with 4–6 methylgroups per GDGT, and the number of cyclopentyl moieties, with0 to 3 rings per GDGT (Figure S1) (Sinninghe Damsté et al.,2000).

Although bGDGTs contain the membrane-spanning featureand ether linkages similar to archaeal iGDGTs, they are notisoprenoidal and differ in the stereochemical configuration ofthe second carbon position of the glycerol backbone; therefore,bGDGTs were proposed to have a bacterial rather than archaealorigin (Weijers et al., 2006b). Following a number of investi-gations focusing on microorganisms inhabiting water-saturated,

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Hedlund et al. Autochthonous bGDGTs in geothermal springs

anaerobic peat bog environments (e.g., Weijers et al., 2009), asingle bGDGT, bGDGT I (Figure S1), was discovered in pure cul-tures of Acidobacteria, specifically Edaphobacter aggregans Wbg-1T and Acidobacteriaceae strain A2 - 4c (Sinninghe Damsté et al.,2011). The origin of the other bGDGTs found in peat bogs andother natural environments (Figure S1) remains unknown. Yet-uncultivated Acidobacteria are candidates for these other bGDGTssince all tested Acidobacteria produce abundant 13,16-dimethyloctacosanedioic acid (iso-diabolic acid), a likely precursor inbGDGT synthesis (Sinninghe Damsté et al., 2011), and since var-ious bGDGTs and yet-uncultivated Acidobacteria co-exist in peatsoils (Weijers et al., 2009).

Although bGDGTs are known predominantly from soils, a fewstudies have documented bGDGTs in terrestrial geothermal envi-ronments. Substantial amounts of bGDGTs, accounting for upto 64% of total GDGTs, were recovered in all nine geothermalspring samples collected by Schouten et al. (2007) in YellowstoneNational Park. However, the authors suggested that the majorityof bGDGTs were from soil runoff (Schouten et al., 2007). A sub-sequent study of bGDGT lipids in soil transects near two springsin Surprise Valley, California, revealed the presence of bGDGTsin geothermally heated soils that were 12–41◦C at the time ofcollection (Peterse et al., 2009). For both springs, bGDGT con-centrations in heated soils near the springs were greatly elevatedover cooler, more distant soils. bGDGTs were also recovered fromtwo alkaline hot springs in Tibet, ranging from 52.0–83.6◦C (Heet al., 2012). The high concentrations of bGDGTs in some sam-ples and a unique lipid profile compared with nearby soils wereconsistent with bGDGT production in the springs. Most recently,a study of GDGTs extracted from natural sediments and incu-bation experiments in Great Boiling Spring (62–82◦C), Nevada,provided multiple lines of evidence demonstrating the in situ pro-duction of bGDGTs in this hot spring (Zhang et al., 2013). Inaddition, cellulosic material that was incubated in situ in bothsediments and the water column at 77 and 85◦C also containedsignificant concentrations of bGDGTs. The bGDGT data werecompared with 16S rRNA gene pyrotag datasets obtained fromthe same samples (Cole et al., 2013; Peacock et al., 2013), revealingthat Acidobacteria were rare in all samples (<0.01% of pyrotags),and suggesting that the bGDGTs were produced by thermophilicbacteria, possibly yet-uncultivated Bacteroidetes, candidate phy-lum EM3 (Rinke et al., 2013), or candidate phylum “Atribacteria”(formerly candidate phylum OP9; Dodsworth et al., 2013; Rinkeet al., 2013).

In this study, we sought to augment the finding by Zhanget al. (2013) and to further test the hypothesis that bGDGTsare produced in natural geothermal environments and to exam-ine relationships between bGDGT composition and abundanceand physicochemical setting. To do this, we examined the dis-tribution and abundance of bGDGTs in natural sediment andmat samples collected from a variety of geothermal springs inthe northwest Great Basin (U.S.A.), along with a large physic-ochemical dataset. bGDGTs were widespread in the springs butwere more abundant in cooler, more oxidized springs withwell-developed microbial mats, defining the optimal habitat forbGDGT-producing thermophiles in habitats ≤70◦C with abun-dant biomass.

MATERIALS AND METHODSSAMPLE SITES AND PHYSICOCHEMICAL MEASUREMENTSSediment and mat samples (top 1 cm of sediment/mat andwater interface) were collected from eight hot springs locatedin northwest Nevada and northeast California (Figure 1). Thesame samples were used for simultaneous extraction of bGDGTsand iGDGTs; the results for iGDGTs are presented separately(Paraiso et al., in review). The sediment/mat water interface (top∼1 cm) was collected. All samples were frozen on dry ice in thefield and were transported and stored frozen (−80◦C) until theywere thawed for lipid extraction and analysis. Field geochemicalmeasurements and samples for laboratory geochemical measure-ments were taken prior to sediment and mat sampling (Costaet al., 2009; Vick et al., 2010). Briefly, temperature, pH, and con-ductivity were determined using a pH probe with temperaturecorrection (LaMotte 5 Series, Chestertown, MD or YSI Model30, Yellow Springs, OH and WTW Model pH330i, Weilheim,Germany). Redox-sensitive analytes (O2 and sulfide) were mea-sured in the field using commercial kits (Hach, USA) withmodifications for high temperature according to Miller-Colemanet al. (2012). Anions and cations were analyzed in the lab by ionchromatography (Dionex DX-500 chromatograph, AS14A col-umn, with 10 μM Na2CO3/NaHCO3 as an eluent, Dionex, USA)and direct current plasma emission spectrometry (DCP-OES,Beckman, USA). Dissolved inorganic nitrogen species (NO−

3 ,NO−

2 , NH3/NH+4 ) were measured by automated colorimetry

(Lachat, USA) as described by Dodsworth et al. (2011a,b).

LIPID EXTRACTION AND LIQUID CHROMATOGRAPHY-MASSSPECTROMETRY (LC–MS)bGDGTs and iGDGTs were extracted from 5 gram lyophilizedsamples using a modified Bligh-Dyer extraction method asdetailed by Lengger et al. (2012). The resulting lipids were sep-arated by silica-gel column chromatography using n-hexane:ethyl

FIGURE 1 | The study area (inset, orange) was located with the U.S.

Great Basin. Hot spring sampling sites (orange diamonds) were locatedwithin California and Nevada.

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Hedlund et al. Autochthonous bGDGTs in geothermal springs

acetate (1:1, v:v) to collect the nonpolar fraction (F1) and MeOHto collect the polar (F2) fraction. The polar fraction was divided.One aliquot, fraction F2A was not processed further. The otherfraction, F2B was hydrolyzed with MeOH:HCl (95:5; v:v) at 70◦Cfor 3 h, extracted with DIH2O and DCM. All three fractionswere dried under N2, dissolved in n-hexane:isopropyl alcohol(99:1; v:v), filtered through a 0.45 μm polytetrafluoroethylene fil-ter, and dried under N2. Dried lipids were dissolved in 600 μLof n-hexane:isopropyl alcohol (99:1; v:v) for analysis. F1 andF2A were run directly on LC–MS and F2B was run on LC–MSafter hydrolysis. F2A was to check whether any core GDGTs maybe present in the polar fraction. If there are any core-GDGTsdetected in F2A, they will be subtracted from F2B and addedto F1.

Each fraction was spiked with a GDGT C46 internal stan-dard and analyzed on an Agilent 1200 liquid chromatographyequipped with an automatic injector coupled to QQQ 6460MS and Mass Hunter LC-MS Manager software. Separation ofGDGT peaks was achieved using a Prevail Cyano column (2.1 ×150 mm, 3 μm; Alltech, Deerfield, IL, USA) as described in Zhanget al. (2012) and GDGTs were quantified using Agilent 6460triple-quadrupole MS with an atmospheric pressure chemicalionization (APCI) source as (Zhang et al., 2012).

STATISTICAL ANALYSESTwo-way cluster analyses and non-metric multidimensional scal-ing (NMS) were completed in PC-ORD (MjM Software Design).Input data for these analyses included the relative abundanceof the polar fractions of individual bGDGTs. Within PC-ORD,lipid data were normalized according to the maximum value ofeach lipid and were used to calculate a Sørensen (Bray-Curtis)distance matrix, which was applied in NMS and two-way clus-tering analyses. For two-way cluster analyses, the flexible betamethod (β = −0.25) was used to create an agglomerative hier-archical clustering tree, with methods modified from those inPearson et al. (2008). NMS was run in PC-ORD’s “autopilotmode,” which determined the optimal number of axes througha Monte Carlo test of 100 runs (50 runs on actual data, 50runs on random data). The final ordination included 99 runs,which were completed for the recommended number of axes.Environmental data were overlaid on the NMS plot to showcorrelation between lipids and geochemistry. Samples with nobGDGTs were removed from NMS and two-way cluster analy-ses to avoid the bias that may occur when a GDGT is assumedto be absent when, in fact, it may be present below detectionconcentrations.

Non-parametric Mann–Whitney tests, Spearman’s rho corre-lation coefficients, and select linear regressions were executed inIBM SPSS Statistics 19 to identify relationships among the GDGTsand geochemical analytes. Analyses were completed at the 0.05level of significance.

RESULTSBRANCHED GDGTs IN HOT SPRING SEDIMENTS AND MATS ANDADJACENT SOILSbGDGTs were detected in 38 of 40 hot springs sampled, span-ning a temperature range of 31–95◦C and a pH range of 6.8–10.7,

across several distinct geothermal systems in the northwest GreatBasin (Table 1; Figure 1; Table S1). Total bGDGT concentrationsranged up to 824 ng/g dry mass in hot springs (Table 1); lowerconcentrations were observed in adjacent desert soils (Table S1;Figure 2). A strong, positive, linear relationship between log-transformed core and polar bGDGTs was observed (r2 = 0.692,sig < 0.001). Similar log-linear relationships existed for indi-vidual bGDGTs in hot spring samples, particularly bGDGT I,Ib, Ic, II, and IIb (Figure S2); however, no such relationshipwas clear for bGDGT III, while polar bGDGT IIc, IIIb, andIIIc were not detected in any hot spring samples. Samples fromdesert soils adjacent to the springs showed a similar relationshipbetween log-transformed core and polar bGDGTs (r2 = 0.475,sig = 0.040); however, significant linear relationships for indi-vidual lipids were only obtained for bGDGT I and bGDGT IIb(Figure S2).

bGDGT I, Ib, and Ic were present in many hot spring sam-ples at concentrations of one order of magnitude or more higherthan in adjacent soils (Figure S2). A two-way cluster analysisbased on the relative abundance of individual polar bGDGTsshowed that hot spring samples had distinct bGDGT compo-sition compared with soil samples, with bGDGT I, Ib, andIc, in decreasing order, being the most common and abun-dant bGDGTs in hot spring samples and bGDGT II and IIIbeing the most common and abundant bGDGTs in soil samples(Figure 3).

RELATIONSHIP BETWEEN bGDGT ABUNDANCE AND TEMPERATURE INHOT SPRING SAMPLESbGDGTs were the dominant GDGTs in some samples, compris-ing up to 99.5% of total GDGTs (bGDGTs plus iGDGTs), whereasiGDGTs dominated other samples, with bGDGTs comprising<1% (Figure 4). The relative abundance of bGDGTs and iGDGTswas related to temperature, with bGDGTs abundant at lower tem-peratures and iGDGTs dominating at higher temperatures (r2 =0.591, sig < 0.001; Figure 4).

The absolute abundance of bGDGTs was also related to tem-perature, with most samples >70◦C containing relatively lowconcentrations of core bGDGTs (≤62 ng/g; Figure 5). This pat-tern was even clearer for polar bGDGTs, which are unstable inthe environment, and therefore, generally interpreted to repre-sent living biomass (White et al., 1979). Polar bGDGTs wereabsent in all but one sample that was >70◦C at the time ofcollection (Figure 5). A Mann–Whitney test confirmed the sig-nificance of 70◦C as a cutoff for both core and polar bGDGTdistribution within this dataset (core sig = 0.046; polar sig <

0.001). Spearman’s rank correlation analysis relating the abso-lute abundance of polar bGDGTs and geochemical measure-ments revealed significant negative relationships between polarbGDGT abundance and temperature (ρ = −0.582; sig < 0.001)and sulfide concentration (ρ = −0.405; sig = 0.014), and sig-nificant positive relationships between bGDGT abundance anddissolved oxygen (ρ = 0.508; sig = 0.001) and nitrate (ρ = 0.650;sig < 0.001) concentrations. Spearman’s rank correlation anal-ysis with the absolute abundance of polar bGDGT I, Ib, andIc, analyzed separately, revealed similar relationships (Table S2).In contrast, the absolute abundance of other polar bGDGTs

www.frontiersin.org August 2013 | Volume 4 | Article 222 | 3

Hedlund et al. Autochthonous bGDGTs in geothermal springs

Tab

le1

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tal

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1018

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Frontiers in Microbiology | Terrestrial Microbiology August 2013 | Volume 4 | Article 222 | 4

Hedlund et al. Autochthonous bGDGTs in geothermal springs

Tab

le1

|C

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8.58

1.26

8.66

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0.73

0.70

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1.25

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635

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FIGURE 2 | Absolute abundance of polar bGDGTs vs. core bGDGTs for

soil and hot spring samples. Samples below the method detection limitfor polar bGDGTs are not shown and were not used for in regressionanalyses (14 hot spring, 4 soil). Samples with <1 ng/g of polar or coreiGDGTs, with negative log10 values are not shown; however, they wereused for regression analyses (3 hot spring, 2 soil).

had few significant correlations with geochemical measurements(Table S2).

The sample from the highest temperature in the Great BoilingSpring geothermal field, GBS-19, contained a low concentrationof polar bGDGTs (2.2 ng/g dry mass), but the highest concen-trations of bGDGT IIb (0.80 ng/g dry mass) and bGDGT III(0.80 ng/g dry mass) among hot spring samples.

CO-VARIATION BETWEEN bGDGTs IN HOT SPRING SAMPLESbGDGT I, bGDGT Ib, and bGDGT II, in order of decreasingabundance, were the dominant bGDGTs in hot spring samples(Table 1), comprising >70% of all polar bGDGTs in all sam-ples except for GBS-19. A two-way cluster analysis based on therelative abundance of individual polar bGDGTs was created togroup geothermal springs and to examine the co-occurrence ofindividual bGDGTs in the environment (Figure 6). The clusteranalysis along the horizontal axis revealed two major groups ofsamples. Group 1 included most low-temperature spring sam-ples, with all except one at ≤51◦C. Group 2, in contrast, includedsites ranging up to 70◦C, with all except one collected from≥50◦C. Several other sites, mostly ≥70◦C, had more uniquebGDGT profiles and clustered separately. No geographical clus-tering was apparent, consistent with non-significant results inthe Spearman’s rank correlation analysis for the majority ofchemical analytes, particularly redox-inactive species. The clusteranalysis along the y-axis showed that the minimally methylatedtetraethers, bGDGT I, bGDGT Ib, and bGDGT Ic, co-varied,leaving a second, less tight cluster for the more highly methy-lated tetraethers bGDGT II, bGDGT IIb, and bGDGT III. Aheatmap associated with the two-way cluster analysis gener-ally supported both cluster analyses and allows visualization

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Hedlund et al. Autochthonous bGDGTs in geothermal springs

FIGURE 3 | Two-way cluster analysis organizes hot spring samples

(black) and soil samples (blue) according to similarity in polar bGDGT

composition (horizontal dendrogram). bGDGTs are organized accordingto their presence and abundance in the samples (vertical dendrogram).Heat map colors indicate minimum (white) to maximum (red) abundance ofbGDGTs, wherein colors are scaled by each lipid’s maximum relativeabundance in hot spring and soil samples.

FIGURE 4 | Percent bGDGTs (core plus polar) vs. temperature. PercentbGDGTs was calculated by dividing total bGDGTs by the sum of all GDGTs(iGDGTs + bGDGTs).

of the distribution of each individual polar lipid within thedataset.

Multivariate relationships between the relative abundance ofindividual, polar bGDGTs and physicochemical factors were

examined using nonparametric multidimensional scaling (NMS;Figure 7). In the NMS plot shown here, distance is proportionalto multivariate dissimilarity in lipid composition (as calculatedby a Sørensen distance matrix). Samples that are highly relatedare plotted closely together, while bGDGT types are plottedaccording to where the centroid of each lipid variable wouldoccur. Physicochemical analytes are plotted as vectors, whereinthe length and direction of a vector is proportional to its relativecorrelation with NMS axes.

The 3-dimensional NMS model was a low-stress and statis-tically significant ordination (min stress = 5.921, p = 0.0196),which suggested strong underlying structure in lipid composi-tion. The NMS plot supported Group 1 and Group 2 identifiedby two-way cluster analysis and indicated a strong correla-tion between the lipid-derived NMS model and temperature(r2 = 0.438, correlation with NMS axis 3). Other strong corre-lations with the lipid-derived NMS model were oxygen (r2 =0.386, axis 3) and pH (r2 = 0.384, axis 3), both of which co-vary negatively with temperature in alkaline geothermal systems(Nordstrom et al., 2005). Individual bGDGTs were spread alonga line originating near the center of the plot, populated by theminimally methylated bGDGT I, bGDGT Ib, and bGDGT Ic,and terminating in the lower right quadrant with the highlymethylated bGDGT III. The trend along axis 3 was in the samedirection as the temperature vector and opposite to the pH andoxygen vectors, demonstrating a possible positive relationshipbetween methylation and temperature and covariates of temper-ature; however, this trend was opposite the pattern of bGDGT I,bGDGT Ib, and bGDGT Ic dominating in most hot spring sam-ples and may have been driven by GBS 19, which was an unusualsample that was dominated by bGDGT IIb and bGDGT III.

DISCUSSIONThe abundance of bGDGTs in geothermal samples reported hereis similar to those in geothermally heated soils at hot spring mar-gins ranging from 18 to 41◦C (9–2,400 ng/g dry mass; Peterseet al., 2009) and in hot spring sediments in Great Boiling Spring(GBS), Nevada, ranging from 62 to 82◦C (15–335 ng/g dry mass;Zhang et al., 2013) and Gulu and Yangbajing hot springs in Tibet,ranging from 52 to 83.6◦C (up to 235 ng/g dry mass; He et al.,2012). These bGDGT concentrations are also similar to thosein non-thermal soils, such as those throughout the Pearl Riverwatershed (10–770 ng/g soil; Zhang et al., 2012) but much lowerthan some terrestrial environments such as decaying peat (up to80 μg/g dry mass; Weijers et al., 2006b).

Our data here support the in situ production of bGDGTsin some Great Basin hot springs based on four lines of evi-dence: (1) the widespread distribution of bGDGTs in Great Basingeothermal springs, (2) the higher concentration of bGDGTs inmany ≤70◦C hot spring samples as compared with nearby desertsoils, (3) the strong relationship between core and polar bGDGTsin geothermal samples (Liu et al., 2011), and (4) the distinctive-ness of soil and hot spring bGDGT profiles (Figure 3). Thesetrends were particularly clear for bGDGT I, Ib, and Ic, provid-ing strong evidence for their autochthonous production in thesprings. However, some hot springs, particularly those >70◦Chad substantially lower abundance of bGDGTs than surrounding

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Hedlund et al. Autochthonous bGDGTs in geothermal springs

FIGURE 5 | Absolute abundance of core and polar bGDGTs as a function

of temperature and pH. Bubble areas (blue) are proportional to the absoluteabundance of the core and polar bGDGTs. Circle in lower right cornerrepresents the maximum absolute abundance in ng lipid g-1 dry mass. Gray

circles represent zero abundance. Pink boxes delimit 70◦C, at and belowwhich bGDGTs are nearly ubiquitous in Great Basin hot springs. Statisticaldata refer to Mann–Whitney tests demonstrating the significantly higherabundance of bGDGTs ≤70◦C.

FIGURE 6 | Two-way cluster analysis organizes hot spring samples

according to similarity in polar bGDGT composition (horizontal

dendrogram). bGDGTs are organized according to their presence andabundance in the samples (vertical dendrogram). Heat map colors indicateminimum (white) to maximum (red) abundance of bGDGTs, wherein colorsare scaled by each lipid’s maximum relative abundance in hot springsamples. Maximum relative abundance (%) of polar lipids is as follows:bGDGT-Ic (21.5), bGDGT-Ib (45.4), bGDGT-I (100.0), bGDGT-IIb (35.7),bGDGT-II (56.0), bGDGT-III (28.6).

soils, particularly polar bGDGTs (Figures 2, 5; Table S1). Theseobservations are consistent with those reported in Schouten et al.(2007) and suggest that soil runoff may be a dominant sourceof bGDGTs in hot springs at temperatures >70◦C. However, thepredominance of autochthonous bGDGTs in lower temperaturesprings is consistent with evidence that bGDGTs can also be pro-duced in other aquatic systems such as lakes (Sinninghe Damstéet al., 2009; Tierney et al., 2010; Sun et al., 2011; Wang et al.,

FIGURE 7 | The NMS plot shows relationships among polar bGDGTs

(relative abundance) and environmental variables. Samples (solidshapes) are arranged accordingly to similarity in polar bGDGT composition.Distance on the plot is proportional to dissimilarity in lipid composition.Lipids (red +) appear where the centroid of that variable would be placedwithin the NMS model. Environmental variables (gray) appear as vectorsthat indicate relative correlation with NMS axes. Colors of samples sitescorrespond to groups identified in the two-way cluster analysis.

2012), rivers and estuaries (Zhu et al., 2011; Zhang et al., 2012),and geothermal springs (Zhang et al., 2013).

All data point to temperature as a possible factor controllingbGDGT production and suggest that most bGDGT-producingthermophiles are moderate thermophiles rather than hyper-thermophiles. However, the effect of temperature on bGDGTproduction may be indirect, since incubation of lignocellulosicmaterials at 77 and 85◦C in GBS led to in situ productionof bGDGTs (Zhang et al., 2013). It is possible that bGDGT-producing thermophiles are members of consortia involved in thedecomposition of complex biomass, which may only be foundat high temperature on rare occasions, such as after delivery of

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Hedlund et al. Autochthonous bGDGTs in geothermal springs

allochthonous plant material into the spring or during degrada-tion of photosynthetic mats after temperature up-shifts. The latterscenario could explain the variable presence of polar bGDGTs in>70◦C sediments in GBS when it was sampled on different dates(Table 1; Zhang et al., 2013). Careful temperature monitoring andtemporal sampling would be required to examine this possibilitymore rigorously. The temporal variability of the GBS geothermalfield has been documented (Anderson, 1978) and is consistentwith our own, more recent observations (e.g., Cole et al., 2013;Peacock et al., 2013). Although polar bGDGTs were present at lowconcentrations in GBS 19, which has a stable source at 95◦C, weurge caution interpreting that bGDGTs can be synthesized at thattemperature since that site has a steep geothermal gradient andtherefore it is difficult to infer the precise temperature at whichlipids may have been produced. Furthermore, this is currently theonly report of polar bGDGTs at temperatures >85◦C and the pre-dominant lipids in that sample are heavily methylated, which isunusual in our dataset.

A few other geochemical measurements correlated with abso-lute or relative bGDGT abundance: nitrate, sulfide, oxygen, andpH, in addition to temperature. Although a direct effect of thesevariables on bGDGT-producing bacteria cannot be ruled out, wecaution against using these data to infer details about the spe-cific habitat and physiology of bGDGT-producing bacteria. Theseanalytes co-vary with temperature in many geothermal outflowsdue to oxidation of reduced compounds present in the sourcewater and gas (e.g., CO2) equilibration with the atmosphere (e.g.,Nordstrom et al., 2005; Swingley et al., 2012) and their rela-tionship to bGDGT composition and abundance is difficult toinfer.

The absence of correlations between bGDGT abundance andany other geochemical analytes and the absence of biogeographicclusters in two-way cluster analysis and NMS plots (Figures 6, 7;Table S3) suggests that geological setting, and the resulting aque-ous and solid-phase geochemistry, are not primary drivers ofbGDGT production within Great Basin hot springs. For exam-ple, no apparent clusters were formed by geographically distinctsprings that are classified as Na-Cl springs (Great Boiling Spring,Sandy’s Spring West, Rick’s Hot Creek) nor Na-HCO−

3 -Cl springs(Surprise Valley, Fly Geyser) (Anderson, 1978; Miller-Colemanet al., 2012). Our data are not consistent with a role of bGDGTsin stabilizing membranes in acidic conditions in these springs. Infact, bGDGT concentrations were directly proportional to pH,which is the opposite of what would be predicted if bGDGTswere serving a role to protect against low pH. This result doesnot rule out possible controls of geochemistry in bGDGT produc-tion in volcanically active geothermal systems such as YellowstoneNational Park or the Rehai Geothermal Field in Tengchong,China, each of which host springs with a much wider pH rangethan the Great Basin (Brock, 1978; Zhang et al., 1987).

The wide range in the relative abundance of bGDGTs andiGDGTs in these springs (Figure 4) demonstrates great differ-ences in relative habitability for bGDGT-producing bacteria andiGDGT-producing archaea. The inverse relationship betweenbGDGT/iGDGT ratios and temperature (Figure 4) is in agree-ment with general trends favoring archaea at environmentalextremes; an increase in the relative abundance of archaea

along temperature transects within GBS has been documentedpreviously (Cole et al., 2013). The dominance of minimallymethylated bGDGT I, bGDGT Ib, and bGDGT Ic amongbGDGTs in this dataset is similar to the dominance of thesethree bGDGTs at higher temperatures in GBS (Zhang et al.,2013) and Yellowstone hot springs (Schouten et al., 2007). Ourresults are also consistent with the high abundance of bGDGTII and III in geothermal soils in Surprise Valley and suggestthat those bGDGTs may be produced by soil microorganisms(Peterse et al., 2009). Tibet hot springs had bGDGT III asthe dominant bGDGT (He et al., 2012). These results sug-gest that factors other than temperature may have some influ-ence on the relative abundance of individual bGDGTs withdifferent degree of methylation in geothermal ecosystems. Thespreading of individual bGDGTs along axis 2 of the NMSplot (Figure 7), perpendicular to the temperature vector, alsosuggests factors other than temperature controlling degree ofmethylation.

CONCLUSIONSThe results presented here, along with previous studies (Schoutenet al., 2007; Peterse et al., 2009; He et al., 2012; Zhang et al.,2013) provide evidence for the production of bGDGTs by ther-mophilic bacteria inhabiting geothermal ecosystems, particularlybGDGT I, Ib, and Ic. Although bGDGTs can be produced up to85◦C (Zhang et al., 2013), the optimal conditions for bGDGT-producing microorganisms appears to be habitats ≤70◦C, includ-ing both heated soils at hot spring margins and in sedimentsand mats within the springs themselves. As such, many bGDGT-producing bacteria appear to be moderate thermophiles, ratherthan hyperthermophiles. Temperature may be a master controllerof the abundance of GDGTs in thermal springs, with moderatetemperatures favoring a high relative abundance of bGDGTs andhigh temperature favoring high relative abundance of iGDGTs.However, the effect of temperature on bGDGT production maybe indirect reflecting the presence of complex organic materialsas incubation of lignocellulosic material at high temperatures ledto production of bGDGTs at 77 and 85◦C (Zhang et al., 2013). Incontrast, geochemistry did not have a strong effect on bGDGTproduction, at least within the context of this study; however,other datasets, such as those from Tibetan hot springs, suggestglobal patterns of bGDGTs in geothermal systems may be morecomplex (He et al., 2012; Wang et al., unpublished data; Li et al.,unpublished data).

ACKNOWLEDGMENTSWe thank Chengling Jia and other members of the Zhang lab atthe State Key Laboratory of Marine Geology at Tongji Universityfor assistance with lipid extraction and analysis. We thank Davidand Sandy Jamieson at Great Boiling Springs, Surprise ValleyHot Spring Resort, and the Bureau of Land Management (BLM)Gerlach, NV office for supporting our sampling efforts.Thisresearch was supported by National Science Foundation grantsDBI REU 1005223, ETBC-1024614, and OISE-0968421 and theNational Natural Science Foundation of China grant 40972211.Additional support was provided by the McNair Scholars Instituteprogram at UNLV. Brian Hedlund acknowledges the generous

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Hedlund et al. Autochthonous bGDGTs in geothermal springs

support of Greg Fullmer through the UNLV Foundation. Thelipid work was supported by the State Key Laboratory of MarineGeology and a National “Thousand Talent Program” award atTongji University.

SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found onlineat http://www.frontiersin.org/Terrestrial_Microbiology/10.3389/fmicb.2013.00222/abstract

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Conflict of Interest Statement: Theauthors declare that the researchwas conducted in the absence of anycommercial or financial relationshipsthat could be construed as a potentialconflict of interest.

Received: 22 May 2013; paper pendingpublished: 17 June 2013; accepted: 21July 2013; published online: 08 August2013.Citation: Hedlund BP, Paraiso JJ,Williams AJ, Huang Q, Wei Y, DijkstraP, Hungate BA, Dong H and ZhangCL (2013) Wide distribution ofautochthonous branched glyceroldialkyl glycerol tetraethers (bGDGTs)in U.S. Great Basin hot springs. Front.Microbiol. 4:222. doi: 10.3389/fmicb.2013.00222This article was submitted to Frontiersin Terrestrial Microbiology, a specialty ofFrontiers in Microbiology.Copyright © 2013 Hedlund, Paraiso,Williams, Huang, Wei, Dijkstra,Hungate, Dong and Zhang. This is anopen-access article distributed underthe terms of the Creative CommonsAttribution License (CC BY). The use,distribution or reproduction in otherforums is permitted, provided the orig-inal author(s) or licensor are creditedand that the original publication inthis journal is cited, in accordance withaccepted academic practice. No use,distribution or reproduction is permittedwhich does not comply with these terms.

Frontiers in Microbiology | Terrestrial Microbiology August 2013 | Volume 4 | Article 222 | 10