Overview and Applications of

Glycerol Dialkyl Glycerol

Tetraether (GDGT)

Yang Yu

1
 Abstract

The understanding of paleotemperature is important for us to conceptualize and

understand the earth’s past. Various biomarkers have been used to assist us in the

reconstruction of the paleoclimate. In recent year one molecule that is commonly found

in microbial membranes have been making significant progress. Glycerol dialkyl

glycerol tetraether (GDGT), is a lipid molecule that can record changes in temperature

as microbes adjust their membranes to their surrounding environment. Once thought to

be exclusive to extremophiles, GDGTs recently have been found in nearly every type of

environment. This newfound versatility allows us to apply GDGTs in a variety of

situation and construct indexes that measures paleotemperature. Some areas of

GDGTs are poorly understood and puts certain constraints on their uses. Future works

will continue to address these ecological and physiological holes in knowledge.

2
Introduction

Biomarkers, specifically lipid based biomarkers have seen an increase in

preferential use in the last two decades. These lipid molecules are can be robust in

environments where skeletal remains are not and are uniquely identifiable. One of the

popular biomarker used to great effect in the Paleocene is the alkenones and its UK37

index. Derived from marine haptophyte algae, it can accurately predict mid-paleocene

aged sea surface temperatures (SST) (Marlowe et al., 1984). Yet its limitation is just

that; the haptophyte algae species the index and biomarker is based on is a recently

evolved species (Marlowe et al., 1984). One cannot accurately assess whether ancient

ancestral algae produced the lipid molecules the same way under the same conditions.

Another limitation with alkenone calibration is the fact that the upper temperature limit is

around 28° C (Marlowe et al., 1984). This can be a problem for studies in the tropics or

paleo-times when the earth was much warmer.

Glycerol dialkyl glycerol tetraether was first discovered in the 1970s via cultures

of Archaea extremophiles (Tierney 2012). These Archaea microbes have large

variabilities, each adapting to their environments. These resilient microbes can be

found in thermal hot springs as high as 90° C or even in high pressure low temperature

environments (Tierney 2012). Their adaptability relies on their ability to synthesize

these tetraether membrane lipids, constantly changing according to the variation in

environments. Robust and nearly impenetrable to ions and protons, these membranes

help them survive such conditions (Tierney 2012). In recent decades, these Archaea

microbes have been found in more environment outside of extreme conditions. These

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GDGTs have been the backbone of studies in the early 2000s by researchers interested

in paleocean conditions. The GDGT structure changes according to fluctuating

temperatures and pH; the reactions to these stressors have been well demonstrated in

both natural and culture samples (Tierney 2012). As environmental stress increases,

the GDGT structure will include more cyclopentane rings (Tierney 2012). One of the

theories as to why this happens is that these rings gives increased stability due to

denser packing of the GDGT molecule and also decreases the general permeability of

the membrane itself (Gliozzi et al., 1983).

GDGT Types

Structurally, there are two distinct types of GDGT molecules: the isoGDGT and

the brGDGT. The isoGDGT is so called due to the inclusion of an isoprenoid functional

group with in their molecular structure as seen in figure 1 (Tierney 2012). isoGDGTs

consists of two head-to-head C40 isoprenoid chains and some variations includes

cyclopentane and cyclohexane rings that are connected by ether bonds to two terminal

glycerol groups (Tierney 2012). This type of GDGTs are produced by the Archaea

group, which contains methanogenic, hyperthermophile and mesophilic species

(Tierney 2012). Each species will often produce different kinds of isoGDGT. The

dominant producer of isoGDGTs recognized by researchers is that of the phylum

Thaumarchaeota (Brochier-Armanet et al., 2008). In most environments that isoGDGTs

are applied to Thaumarchaeota is the ubiquitous producer such as oceans and lakes.

They can make up to 20% of the ocean’s picoplankton (Brochier-Armanet et al., 2008).

Though Thaumarchaeota can be found in soils, they do not produce significantly there

to be of use (Brochier-Armanet et al., 2008). Thaumarchaeota also have a unique

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property in which they will produce GDGT crenarchaeols that has a cyclohexyl ring in

addition to the regular cyclopentane rings (Brochier-Armanet et al., 2008). This usually

happens under moderate to high temperature conditions and is a useful marker for such

events (Tierney 2012).

The other GDGT, brGDGT is a bit less understood than the isoGDGTs. They

were first discovered not from bacterial cultures but from natural samples from a Dutch

peat (Naafs 2017). Unlike the isoGDGTs produces by microbes of the Archaea

Domain, the structure of brGDGTs indicates to researchers that they are more likely to

have been produces by bacteria (Naafs 2017). Even though like isoGDGTs they share

features such as ether bonds on terminal glycerol groups, the branched structure and

the stereochemistry of the glycerol groups points to bacterial origins (Figure 1) (Naffs

2017). Compared to isoGDGTs, brGDGTs are also more commonly found in soils and

peats and conversely, less so in pelagic marine environments (Tierney 2012). Even

though there are hints as to the terrestrial origin of the brGDGT lipids, the primary

producers for the compounds are poorly understood (Ding 2015). So far, one strain of

Acidobacteria have been proven to produce one of nine types of brGDGT (brGDGT-I)

(Tierney 2012).

One of the reasons that GDGTs were not utilized until recent decades is the

technological difficulties surrounding analyzing the compounds. GDGTs were first

discovered in the 70s and observed in modern and ancient sedimentary deposits since

then but were hard to analyze using gas chromatography since the molecules were too

large (Tierney 2012). Recently developed technology such as the high-performance

liquid chromatography mass-spectrometry allowed such studies on GDGTs to be

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possible (Schouten et al. 2007). GDGTs can typically be analyzed in 60-70 minutes

after they are extracted and purified with column chromatography (Schouten et al.

2007). The extraction itself is relatively simple, commonly using dichloromethane and

methanol to get the organic matter (Schouten et al. 2007).

TEX86 and CBT/MBT indices

The way that isoGDGTs respond to changes in temperature is crucial for its use.

GDGTs with higher number of rings will have a higher melting point and more stable at

warmer temperatures (Brochier-Armanet et al., 2008). It has been demonstrated that

Thaumarchaeota produces more cyclic isoGDGTs in higher temperatures and

experiments have confirmed the presence of higher concentrations of cyclic isoGDGTs

in warmer seawater (Brochier-Armanet et al., 2008). The varying concentrations of

higher ringed isoGDGTs were used by Schouten et al. (2003) to construct an index that

reflects the degree of cyclization of isoGDGTs. This index is called the TetraEther index

of 86 carbons, or TEX86 for short. The equation in figure 2 is used to calculate this index;

cren’ being the crenarchaeol regioisomer of isoGDGTs produced at high temperatures

(Schouten et al. 2003). In Table 1, we see the TEX86 relationships with Sea Surface

Temperature (SST) (Tierney 2012). It is a strongly correlative relationship with an r2 of

0.92 for the original Schouten et al. calculations (2003). There are many different

calibrations of TEX86, many based on uniquely different environments. The main

indices used today, shown in Table 1, are the TEX86H and TEX86L developed by Kim et

al. (2010), and Powers’ et al. (2010) for lake surface temperatures (LST).

brGDGT data generally derives from empirical data derived from soil samples.

The lack of culture study hampers more detail studies and calibrations of the molecule.

6
Regardless, Weijers et al. (2007) managed to identify environmental parameters that

have contributed significantly to the brGDGT forms. The cyclization of brGDGT appears

to be specifically governed by soil pH value non-linearly. This relationship is described

in the equation in figure 3. The relationship between brGDGT is called the Cyclization

of Branched Tetraethers (CBT) index (Weijers et al. 2007). With changes in

temperature the molecule will experience methylation (Weijers et al. 2007). The

Methylation of Branched Tetraethers (MBT) index is used to represent this as shown in

figure 3 (Weijers et al. 2007). The correlation is around r2=0.62 for soil temperature

(represented by Mean Annual Air Temperature-MMAT) (Weijers et al. 2007). While this

correlation is not as strong as the TEX86 index for SST recent effort have attempted to

improve upon this general model. Various calibrations, mostly centered around the use

of 5 and 6 methyl brGDGTs have been able to increase the correlation for terrestrial

environments such as seen in the Qinghai-Tibetan Plateau (r2= 0.82) and peats

(r2=0.76) (Ding et al., 2015 and Naafs et al., 2017). brGDGTs have also been found

abundantly in lake sediments (Tierney 2012). Recent studies of lake sediments have

shown promise, with some results even better than the terrestrial MAAT ones (Tierney

2012).

Application of isoGDGT TEX86 Index

One of the earliest uses of the TEX86 index was by Schouten et al. (2003) in

application to the cretaceous era warmhouse conditions. The Cretaceous era was a

period where the temperatures on earth was significantly higher than that of today; sea

temperature was so warm that polar ice caps did not exist (Poulsen et al., 1999).

Previous studies have used stable isotope values such as δ18O to calibrate the sea

7
surface temperatures (Price and Hart, 1998). The oxygen isotope value depended on

fossil remains of forams in the Cretaceous sedimentary rocks (Price and Hart, 1998).

Some uncertainties arose by using the δ18O isotope. The ecological properties of the

forams from that geological time can’t be positively connected with the modern day

forams and the way they incorporate oxygen (Wilson et al., 2001). The seawater pH

values and diagenetic overprinting especially in Ocean Anoxic Events often affect the

accuracy of SST reconstruction too (Wilson et al. 2001).

Schouten et al. (2003) selected the isoGDGT lipids to reconstruct the SST since

they are generously found in the cretaceous rock record, including in organic rich black

shales. They are easily identifiable and well preserved in these rock records. isoGDGT

across 4 different samples were subjugated to the TEX86 proxy which has a correlation

with SST of r2=0.92 (Schouten et al. 2003). The results can be seen in figure 4. The

distribution of isoGDGT species were similar across all the cretaceous rock samples

(Schouten et al. 2003). For isoGDGT I-VII, a high amount of crenarchaeol (V) and a

moderate amount of crearchaeol (VI) were found (Schouten et al. 2003). The 2-3

cyclopentane ringed lipids were also found in moderate abundance while isoGDGT-I,II

were in low abundance (Schouten et al. 2003). Since it has been proven that with

higher temperatures more cyclic GDGTs appears, that the high concentration of higher

cyclic GDGTs in the Cretaceous rocks leads to an interpretation of high SST

temperatures (Schouten et al. 2003). Compared to modern day, the isoGDGT-I,II in the

tropics were of much higher concentrations now than the Cretaceous sedimentary rocks

(Schouten et al. 2003). So, the overall SST of the Cretaceous can exceed even the

warmest regions of modern day oceans. TEX86 values ranged from a lower bound of

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0.69 in Aptian values to as high as 0.96 in samples from the OAE 2 (Cenomanian-

Turonian) event (Schouten et al. 2003). The SST calibrated from these TEX86 values

are from 27-32° C in the Aptian to 32-36°C in the OAE2, very high compared to present

day values as seen in figure 5 (Schouten et al. 2003). δ18O paleothermometry

calculated SST to be around (32-33 ±3°C) are in good agreement with the results of

GDGT based TEX86 index (Price and Hart 1998). Overall TEX86 index was exemplary in

its performance as a SST reconstruction tool in this early study. It was especially

noteworthy in its application to organic-rich sediments such as black shales where

foraminifera can be lacking for δ18O paleothermometry analysis. So, it is a very nice

complimentary tool to be used alongside δ18O techniques to confirm SST values in

ancient warmhouse conditions.

The Tex86 index have also been successfully used in modern day lake sediments

for Lake Surface Temperature (LST) reconstruction. Tierney et al.’s (2010) work on the

Lake Tanganyika successfully showed temperature changes across a time frame of

1500 years. Lake Tanganyika is a region where there are limited recent temperature

reconstruction but abundant GDGTs present in the sediment for TEX86 to be applied

(Tierney et al., 2010).

In figure 6, the LST trend obtained from TEX86 of the isoGDGTs in lake

sediments showed an increasingly faster trend upwards (Tierney et al., 2010). It

amounted to an increase in temperature of up to 2°C in the last 150 years, much higher

than any periods in modern times (Tierney et al., 2010). The LST correlated extremely

well with global temperature recorded in air temperature and indicated global climate

change as the primary factor in LST increase (Tierney et al., 2010). The LST increase

9
was also negatively correlated with the loss of primary productivity in the lake due to an

increasingly stratified water column (Tierney et al., 2010).

Applications of brGDGT MBT/CBT Index

brGDGTs are not found and used as much in marine sediments as isoGDGTs,

but are abundantly found in soil/peat samples where isoGDGTs are lacking. The use of

brGDGTs is great for terrestrial areas such as that shown in the Qinghai-Tibetan

plateau in the 2015 study by Ding et al. the brGDGT index were used as proxies for

mean annual air temperature (MAAT) and soil pH, with methylization of the molecule

being the primary response to temperature changes. In their articles they used a

recently discovered series of 6-methyl brGDGTs along with the previous 5-methyl

brGDGT to calibrate a more accurate MBT/CBT index for brGDGT the equation of

which is seen in figure 7 (Ding et al., 2015). 30 samples across the relatively arid

Qinghai-Tibetan Plateau (QTP) were analyzed and the 6-methyl brGDGTs were found

to be the most abundant brGDGT components (Ding et al., 2015). This is different from

global soil brGDGT levels since the 5-methyl brGDGT is generally the most abundance

(Ding et al., 2015). It shows that brGDGTs will adapt to local changes in conditions

such as moisture as wells temperature (Ding et al., 2015). The soil from which these

results were derived were also leaning towards the alkaline side and thus indicating

Acidobacteria is probably not the producer of these brGDGTs (Ding et al., 2015).

The new brGDGT index was constructed by Ding et al. (2015) after the original

MBT index calibrations showed too much scattering (figure 8). Its inclusion of the 6-

methyl brGDGT improved the overall r2 by 0.2 (Ding et al., 2015). Though Ding et al.

(2015) specified that the calibration for brGDGTs should be done against the local

10
environmental variables. The dry cold environment of the Qinghai Tibetan Plateau and

the abundance of 6-methyl brGDGTs necessitated such a calibration (Ding et al., 2015).

The overall results for MAAT correlation was excellent as seen in figure 9.

brGDGTs in peats recently gained an important research in which a global-

calibration for temperature and pH was introduced. Naafs et al. (2017) analyzed peats

in part because the environmental factors of temperature and pH and its effect on

brGDGT in peats as opposed to soil was not well defined (Naafs et al., 2017). 470

samples across 96 peatlands were obtained to construct this new calibration figure 10

(Naafs et al., 2017).

The majority of brGDGTs found in peats are brGDGT-I and II (up to 99%

brGDGT-I in tropical peats), with some brGDGT-III dominance in high latitude peats

(Naafs et al., 2017). This is interesting as global brGDGTs are dominated by 4,5 and 6

methyl brGDGTs (Naafs et al., 2017). It was found that with increasing depth GDGT

production increases preferentially under the presumably anaerobic conditions. (Naafs

et al., 2017) Yet oxygen content did not affect the distribution of brGDGTs as first

hypothesized based on similar situation in stratified lake systems (Naafs et al., 2017).

In fact, the only affect that anoxic conditions in peat seems to have is to increase the

overall production of brGDGTs (Naafs et al., 2017). This might be because in peat the

primary producers of brGDGTs might be anaerobes, where they are more active in

shallow layers and increasing in abundance deeper into the peat layers (Naafs et al.,

2017).

Discussion and Conclusion

11
 The TEX86 index constructed from isoGDGTs and MBT/CBT index are

demonstrated to be great tools for SST, LST and MAAT reconstruction. GDGTs are still

relatively new and several factors must be considered that can put a constraint on the

accuracy of such indices.

Thaumarchaeota is assumed to be the dominant producers of isoGDGTs in

oceans. Yet there are debates to as how much contribution come from other Archae

family members, of which there are many different types. For example, the mesophilic

Euryarchaeota have also been shown to produce significant amounts of isoGDGTs in

marine environments (Schouten et al., 2008). Special care must also be taken when

observing niche regions where Methanogenic Archae or anaerobic variations can

dominate, since they can produce significant amounts of isoGDGTs 0-3 (Wakeham et

al. 2003). One such problem was examined by Zhang et al. in a 2016 study in a

stratified lake with anoxic conditions showed that the temperature predicted by the

TEX86 index was much lower than the other index techniques. In fact, this is a trend in

anoxic areas across the globe, where there is a certain cold bias when applying the

standard TEX86 index to oxygen deprived areas (Zhang et al. 2016). This is because

the previously mentioned Archaea species will contribute high amounts of isoGDGT 0-3,

inflating its concentration and making the end TEX86 value to be lower than it normally is

(Zhang et al., 2016).

Another concern with Thaumarchaeota is that they can reside in sub-surface

depth (100-200m) as well as sea-surface (Huguet et al., 2007). This creates the

possibility that TEX86 index can sometimes reflect temperatures in the subsurface ocean

rather than the SST (Huguet et al., 2007). The good news is that SST is strongly

12
correlated with subsurface temperature nearly everywhere in the ocean. The problem

areas come around the near-shore and upwelling zones, where TEX86 will biased

toward subsurface temperatures (Huguet et al., 2007). Additionally, there are some

concerns about Thaumarchaeota’s sensitivity to seasonal temperature swings

(Schouten et al., 2008).

For brGDGTs, the main concern is that only one producer for brGDGTs have

been identified, with is Acidobacterias that primarily produce brGDGT-I, which is a

problem since there are 9 different species, of which brGDGT-V and VI are widely

distributed and important in soil temperature reconstruction (Naafs et al., 2017).

Without primary production bacteria cultures from which to experiment different

temperature and pH on, the index accuracy and understanding can suffer (Tierney

2012). brGDGTs distribution is affected by variables other than temperature, such as

pH, moisture level, and oxygen content (Ding et al. 2015). It may be that brGDGTs

always need to be calibrated to local bacterial communities and environmental factors

and a global calibration of soil brGDGTs not as useful (Ding et al. 2015).

Local calibrations are important for lake surface temperatures for both brGDGTs

and isoGDGTs. Both of which are affected by oxygen level in the lake and brGDGTs

affected by the pH values (Zhang et al. 2016). Different species of Archaea living can

also affect isoGDGT distribution when using the TEX86 indices (Zhang et al. 2016).

Though there are recent calibrations accounting for that (Power et al., 2010).

As for degradation of lipid molecules, the GDGTs are quite robust molecules that

do not get altered much as it falls through the water column after the organism dies

(Tierney 2012). Much of which is consumed and deposited rapidly as fecal matter,

13
preserving it in the sedimentary records (Tierney 2012). There is a concern with

thermal maturation, at 240C-300C GDGT will began to crack and degrade (Tierney

2012). Thus, GDGTs should not be used with thermally mature samples (Tierney

2012).

GDGTs overall represent a new and robust way to characterize and reconstruct

paleotemperature profiles. They can be used for terrestrial, marine and lacustrine

environmental across a significantly wide range of temperature and geologic age. Much

of the field remains to be refined, providing space for many future endeavors to

investigate.

14
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Table 1: TEX86 calibrations (Tierney 2012)

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Figure 1: a general overview of the GDGT molecular structures (Tierney 2012).

Figure 2: General TEX86 Index formula (Schouten 2002).

Figure 3: General MBT/CBT Index formulas (Weijers et al.2007)

Figure 4: Holocene TEX86 Index calibrations used with Cretaceous rock sediments (gray

band is Cretaceous values) (Schouten et al. 2003).

Figure 5: Cretaceous SST juxtaposed with present day values as obtained with TEX86

(Schouten et al. 2003).

Figure 6: LST of Lake Tanganyika in the last 1500 years as calculated with TEX86

(Tierney et al., 2010).

Figure 7: New MBT/CBT equation for 6-methyl branch brGDGT heavy soil (Ding et al.,

2015).

Figure 8: Scatterplot showing inaccuracy if 6-methyl branch brGDGT is excluded using

original MBT/CBT equation (Ding et al., 2015)

Figure 9: New plot using new equation shows much better correlation with mean annual

air temperature (Ding et al., 2015).

Figure 10: Equations used for MBT/CBT calculations for brGDGT in peat (Naafs et al.,

2017)

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Figure 1

Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

Figure 10

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