ARTICLE IN PRESS

Quaternary Science Reviews 23 (2004) 893900

The stable isotope analysis of pollen as an indicator of terrestrial

palaeoenvironmental change: a review of progress and
recent developments.
N.J. Loadera,*, D.L. Hemmingb
b

a
Department of Geography, University of Wales Swansea, Singleton Park Swansea, SA2 8PP UK
Department of Environmental Sciences and Energy Research, Weizmann Institute of Science, Rehovot, Israel

Received 5 February 2003; accepted 31 December 2003

Abstract
Few terrestrial archives offer multi-proxy information with temporal and spatial coverage comparable to marine palaeo-records.
Pollen assemblages that are preserved in terrestrial sedimentary systems are, however, one such source. They offer palaeoecologists
an archive from which to gain insight into past environmental change and associated vegetation dynamics, typically extending back
many thousands of years. Recent preliminary results from the stable isotopic analyses of raw and extracted pollen exina suggest that
signicant potential exists to utilise palaeo-pollen records as quantitative indicators of past terrestrial palaeoenvironmental change
over medium- to long-timescales. Moreover, by combining isotopic analyses with conventional pollen assemblages the information
available is greatly enhanced, especially regarding the relative timing of plant community response to external forcing.
In this paper, we review the limited number of studies that have investigated the isotopic analysis of pollen and we highlight the
difculties and current limitations that are involved. We propose some possible solutions to the methodological issues raised and
discuss recent ndings of a pilot study of the carbon, hydrogen and oxygen isotopes in raw pollen grains collected from a network of
north European sites.
r 2004 Elsevier Ltd. All rights reserved.

1. Background and literature review

In addition to the valuable palaeoecological information provided by the presence/absence of single pollen
types, identied by the morphology of its resistant exine,
pollen spectra can also be successfully combined to
provide an insight into past plant community response
or, through statistical models, to provide quantitative
estimates of palaeoenvironmental changes (Birks and
Line, 1993; Huntley, 2001).
Attempts have been made to link long pollen
sequences (Reille and de Beaulieu, 1990; Pons et al.,
1992; Tzedakis, 1994) with marine and ice core records,
based upon correlation of orbitally tuned marine isotope
sequences with rhythmic variations in key pollen spectra
(Tzedakis et al., 1997). Whilst this approach may offer
some validity when viewed over long timescales, at submillennial levels such correlations could mask important
*Corresponding author. Tel.: +44-1792-513693; fax: +44-1792295955.
E-mail address: n.j.loader@swansea.ac.uk (N.J. Loader).
0277-3791/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.quascirev.2003.06.015

temporal differences between cause and effect as

recorded in the marine and terrestrial archives. One
major drawback of conventional pollen stratigraphy is
that the vegetation community responds with inertia to
climate and environmental forcings. Consequently, the
time taken to attain critical pollen sums indicative of a
local presence could blurr or obscure important
information about the magnitude, absolute timing and
rate of terrestrial palaeoenvironmental changes. Such
vegetative inertia is one of the reasons why direct
comparisons of terrestrial, oceanic and ice core sequences are difcult (Lowe and Walker, 1997).
Stable isotopic analysis of plant organic remains has
provided a valuable tool with which scientists have
explored past climate variability and plant physiological
responses to environmental change (Van der Water et al.,
1994; Switsur and Waterhouse, 1998 and references
therein; Conte and Weber, 2002). Although the physical
and physiological processes that regulate the isotopic
composition of plant organic components are extremely
complex, models do exist that provide signicant
insight into the key fractionation mechanisms involved

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N.J. Loader, D.L. Hemming / Quaternary Science Reviews 23 (2004) 893900

(Francey and Farquhar, 1982; Edwards et al., 1985;

Edwards, 1993; Roden et al., 2000). Such models can be
utilised and developed in conjunction with environmental observations and laboratory experiments to further
improve this understanding.
The stable isotopic analysis of pollen and spores for
palaeoenvironmental applications is a relatively new
approach in palaeoecological research. It has emerged
primarily during the last 5 years in response to advances
in mass spectrometry and sample preparation methods.
However, the potential of using these materials as
accurate isotopic standard materials was explored as
early as 1941 by Murphey and Nier (Murphey and Nier,
1941). They demonstrated, through analysis of a sample
of lycopodium spores, that carbon isotopic analysis of
sporopollenin was possible, and that it has a lower 13C
abundance than atmospheric CO2. Technological limitations at the time necessitated that Murphey and Nier
use large samples to ensure reliable isotopic determinations, whereas, today it is possible to use just a few
microgrammes of pollen for the same degree of
accuracy. This serves to highlight the signicant
advances that have been made in sample preparation
and mass spectrometric techniques, without which
isotopic analyses of palaeo-pollen samples would be
unfeasible. The labour intensive nature of the separation
of large numbers of grains required for reliable analyses
has also prevented an earlier uptake and more extensive
exploration of this method. Today, many of the
problems already encountered are only just becoming
possible to address and we are able to start exploring the
potential of this environmental proxy.
A pollen grain is not a single homogenous material,
but comprises a number of distinct physical/chemical
parts including: a central inner nucleus; a cellulose inner
layer or intine; a sporopollenin outer structure or exine
and a polysaccharide coating (Shaw and Yeadon, 1964;
Moore et al., 1991). The term Sporopollenin was rst
used by Zetzsche (1932) to describe the material
comprising the outer structural exine of spores and
pollen (approximately 6580%) (Shaw and Yeadon,
1964). It is the exine that exhibits most of the
morphological information that enables pollen grains
to be identied. Sporopollenin is also a very resistant
compound, and remnants of a similar material have
been identied in some of the earliest geological
formations (Brooks and Shaw, 1968). Chemical analyses
have since shown sporopollenin to be a b-carotenoid
ester of the approximate formula C90H150O33. Potential,
therefore, exists to explore the isotopic signal contained
within the three elements of the pollen grain.
Sixty-six years after Murphey and Nier, Amundson
et al. (1997) explored the potential for using the stable
carbon isotope composition (d13C) of grass pollen to
determine variability in the relative abundance of
grasses using either the C3 or C4 photosynthetic path-

way. Since the distribution of grasses that use the C4

pathway are largely determined by climatic factors such
as precipitation, seasonality and growing season temperature, isotopic analysis of grass pollen assemblages
could provide a proxy for palaeo-aridity (Amundson
et al., 1997). However, it is often not possible to visibly
differentiate species with different photosynthetic mechanisms from the morphology of grass pollen grains.
As C3 and C4 species differ considerably, by B15%, in
their d13C compositions, isotopes provide a powerful
means of partitioning between these species. Amundson
et al. demonstrated the applicability of this method
using modern C3 and C4 grass species. They also showed
that the d13C composition of bulk plant matter was
reected in pollen d13C, supporting assumptions that
models of plantisotopeenvironment interactions
are broadly applicable for pollen material. Further,
they identied that the acetylation (acetolysis) method
(Erdtman, 1960), that is widely employed to remove the
pollen cellulose intine and facilitate species identication, could lead to isotopic contamination through the
incomplete removal of carbon bearing compounds used
in this process.
The composite nature of pollen grains and the
variable susceptibility of these different components to
diagenesis (Benner et al., 1987; Spiker and Hatcher,
1987) prompted investigation into alternatives to
acetylation for the isolate sporopollenin prior to isotopic
analysis. Loader and Hemming (2000, 2002) presented
results that supported the nding of Amundsen et al,
that acetylation can contaminate the d13C signal, and
they developed an alternative, carbon-free acid-digestion method to isolate the sporopollenin component.
The need for removal of the cellulose intine was
considered, and the recommendation was made that
for palaeoapplications, assuming a variable degree of
diagenesis downcore, removal of the intine would be
prudent. Loader and Hemming (2000) also explored the
environmental signal preserved within raw pollen versus
extracted pollen exina and demonstrated that the
sporopollenin exine of pollen grains preserves the d13C
signal of the raw pollen, and by extrapolation to the
work of Amundson et al., the d13C signal of the bulk
organic material.
In 2001, De! scolas-Gros et al. presented a dataset
based upon the natural variability of d13C in pollen
grains from different C3 and C4 species. They identied
a wide range of isotopic variability, between 28.6%
and 21.7% (V-PDB) for C3 species and between
15.9% and 10% for C4. This variability is typically
greater than the observed inter-annual differences
between tree-ring or leaf samples (Leavitt and Long,
1982; Switsur and Waterhouse, 1998; Schleser, 1999;
Rundgren et al., 2000), and it highlights the need to
understand inter- and intra-species variability and to
develop suitable sampling protocols that ensure that the

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environmental signals present in pollen d13C compositions are not obscured. De! scolas-Gros et al. were again
able to demonstrate the role of acetylation in contaminating d13C compositions.
In an effort to examine the inter-species variability of
sporopollenin d13C, Loader and Hemming (2002)
compared the sporopollenin d13C of different species
growing in close proximity under the same macroenvironmental conditions. They noted differences of up
to 3% between just three genera, indicating the need for
the separation and analysis of individual genus from
assemblages if robust environmental interpretations are
to be made (Fig. 1).
In one of only a limited number of published palaeoapplications to date Beerling and Jolley (1999), analysed
the d13C record of bulk pollen preserved within a
composite record of sediments spanning the PalaeoceneEocene transition. They identied an isotopic
excursion in one of the sequences that was broadly
comparable with other marine and terrestrial bulk
isotopic indicators, and they proposed that this departure was linked to the venting of a pulse of isotopically
light CO2 from the ocean to the atmosphere. The
covariance of their record with the other independent
indicators, together with their interpretation of the rapid
nature of the pollen isotope response, supports the
further application of pollen stable isotope analyses and
wider investigation of the palaeorecord.
In the remainder of this paper we present the ndings
of recent studies that aim to address two key limitations
of utilising and interpreting palaeo-pollen isotopic
records. The rst concerns possible methods to isolate
and prepare more efciently pollen samples for isotopic
analyses, and the second focuses on improving our

Raw Pollen Delta 13C VPDB (per mille)

-23
-23.5
-24
-24.5
-25
-25.5
-26
-26.5
-27
-27.5
-28
700

900

1100

1300

1500

Site Altitude (approximate m.a.s.l.)

Fig. 1. Graph depicting the relationship between pollen d13C along an

altitudinal transect in Switzerland for three European tree species
Betula pendula (lled diamonds), Picea abies (open triangles) and Pinus
sylvestris (open squares).

895

understanding of pollen formation processes and their

respective isotopic fractionations.

2. Sample preparation
At present, one of the greatest obstacles to the wider
application of the isotopic analysis of pollen preserved
in sedimentary sequences is the painstaking and timeconsuming preparation and removal of pollen grains
from a sediment matrix. Unfortunately, as in conventional palynology, the exact combinations of methods
required to extract pollen grains from a matrix depend
very much upon the type of material under study,
consequently a single protocol cannot be proposed
(Moore et al., 1991; Charman, 1992). The important
element remains however, that the resulting pollen
concentrate is as free from impurity as possible and
that each step does not fractionate the isotopic
composition of the pollen component under study.
Studies of pollen from trees growing at the same
locations (Loader and Hemming, 2002) and the results
of De! scolas-Gros et al. (2001) demonstrate that,
although selected trees respond in a broadly similar
manner, there are signicant isotopic off-sets between
different tree-genera. This variability is to be expected as
similar ndings were observed in isotope dendroclimatology (Leavitt and Newberry, 1992). Whilst a bulk
pollen signal may be suitable for a 14C determination
(e.g. Regne! ll, 1992; Richardson and Hall, 1994) such an
approach is felt unsuitable for palaeoenvironmental
reconstructions where physiological factors signicantly
inuence the signal. For this reason, it is important that
a single genus, or ideally species, should be isolated and
analysed from a palaeorecord. It is also likely that the
differences observed between plant genera could provide
important palaeoecological information.
The added time and patience required to separate
sufcient pollen grains for a d13C analysis is considerable. However, contrary to the work of Brown et al.
(1989), we have found that approximately 350700
spruce grains (equivalent to 510 mg of carbon) are
required to produce a large enough sample for d13C
analysis (Fig. 2). To isolate such a sample is time
consuming, yet feasible in samples with a high pollen
concentration and low species diversity.
A number of different approaches have been explored
to isolate pollen from a sediment matrix, including dense
media separation, centrifugation, micro-sieving followed
by manual picking (Loader, unpublished). These procedures are based upon published methods and can yield
specic pollen concentrate of high purity (Brown et al.,
1989; Long et al., 1992; Regne! ll, 1992; Richardson and
Hall, 1994; Regne! ll and Everitt, 1996; Kretschmer et al.,
1997; Nakagawa et al., 1998; Morgenroth et al., 2000).
Additional potential may also be realised through the

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896

use of hydrodynamic SPLITT-cell separations that

could theoretically separate out distinct pollen grain
types from a supporting matrix through a dynamic
density separation of the suspended material (Giddings,
1985; Keil et al., 1994; Jiang et al., 1997; T Frasch,
Postnova Analytik GmbH, personal communication).
At present, manual separation of the pollen grains is
the most rate-limiting of the sample preparation stages.
It is likely that a future solution to this problem might
come from two areas. Firstly, through improvements in
the ability to analyse isotopically microgramme quantities of carbon, such as those proposed by Sharp and
Cerling (1996), Wieser and Brand (1999), and secondly,
through advances in the automated separation, as
with the SPLITT system, and computer recognition
of pollen grains (France et al. 2000). Computer
recognition systems may be programmed to recognise
a single pollen type, which could be automatically
picked for isotopic analysis using an integrated
micro-manipulator.

3. The d13 C; d18 O and dD signals in pollen: a pilot study

In order to understand the nature of the signal
preserved within the pollen grains, it is rst essential that
the origin of this signal is accurately determined.
Existing plant/isotope/environment models provide a
useful framework from which to commence (e.g:
Francey and Farquhar, 1982; Edwards et al., 1985;
Roden et al., 2000), however, for pollen, additional
factors such as the inuence of climate and use of stored
photosynthates (Andersen, 1991; Hicks, 1989) must also
be characterised before a complete exploitation of the
pollen archive can be realised. In light of these
requirements we have performed, as a rst step, a
number of simple spatial studies to compare the isotopic
composition of Pinus sylvestris L. pollen grains with
local climatic conditions and meteoric source waters,
using a network of sites across Northern Europe
described in Loader and Hemming (2001).
3.1. d13 C analyses

10
9

Microgrammes Carbon

8
7
6
5
4
3
2
1
0
0

100

200

300

400

500

600

No. Picea Grains

Fig. 2. Relationship between sample size (No. Grains) and equivalent

mass (microgrammes C) for Picea abies pollen.

Pinus sylvestris L. pollen were collected across a

network of living trees during a single growing season
(Table 1). Flowerheads from the lowest 2 m were
sampled from around the circumference of each tree to
minimise the inuence of aspect (sun/shade). At each
site, pollen from a number of owerheads and trees were
combined to reduce variability between individuals. The
species Pinus sylvestris L. was selected because it has a
wide distribution throughout Europe, it produces large
amounts of pollen that are easily collected and
identied, and it is also abundant in the fossil record
(Loader and Hemming, 2001). The d13C values of the
pollen grains were determined using an ANCA GSL
elemental analyser interfaced with a Europa 20/20 mass
spectrometer. Owing to a general paucity of available
meteorological data, comparisons were made with
temperature data collected at each site for the average
development period (AprilJune) during which the pollen
was released. We recognise that temperature is not the
only meteorological inuence on d13C fractionation,

Table 1
Location of the sample sites and stable (C,H,O) isotope composition of pollen analysed across the spatial study area
Site

Latitude
( N)

Murmansk
Rovaniemi
Helsinki
Tallin
Uppsala
Dawyck
Crowthorne
Wroclaw

68.57
66.33
60.18
59.25
59.90
55.25
51.23
51.05

Longitude
( E)
33.02
25.50
24.57
24.47
17.60
02.50
00.49
16.52

d13C
(per mille)
VPDB
28.71
27.83
26.40
25.86
28.00
26.58
25.57
24.27

d18C
(per mille)
VSMOW
3.00
0.79
6.78
0.88
0.33
4.6
4.73
1.32

dD
(per mille)
VSMOW
175.52
128.81
145.23
133.06
126.69
101.69
100.08
99.68

Estimated
development
period

Collected by:

June/July
June
May/June
May/June
May/June
May
May
May

Botanical Gardens, Murmansk.

R. Jalkanen
M. Kotilainen
J. Ellia
K. Bremer
D.Knott
A. Loader
B. Bogacz

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-4
Delta 18-O Precipitation (per mille V-SMOW)

16
Mean April-June Temperature (degrees C)

897

14
12
10
8
6
4

y = 0.41x - 9.0
2
R = 0.79

2
0
-29

-28

-27

-26

-25

-24

Delta 13C raw pollen (per mille VPDB)

y = -0.34x - 8.26
R2 = 0.61

-6

-8

-10

-12

-14
-6

-4

-2

Delta 18-O Pollen (per mille V-SMOW)

Fig. 3. Relationship between mean AprilJune temperature and pollen

d13C for Pinus sylvestris pollen collected from the European spatial
study network.

Fig. 4. Relationship between d18O in precipitation from local GNIP

stations and pollen d18O for raw Pinus sylvestris pollen collected from
the European spatial study network.

however, across much of the study region it is possible

to identify a typical association of summer temperatures
(driven by anti-cyclonic conditions) with enhanced
moisture stress, lower humidity, clearer skies and greater
sunshine. The d13C signal appears most strongly to
reect the conditions during a period of about 46 weeks
before pollen y, suggesting that an environmental
signal is imprinted and preserved within the exine of the
pollen grain during this stage of its development (Fig. 3).
The analytical precision for replicated carbon isotope
analyses of standard materials using this method is 0.1
per mille.

hydrogen atoms comprising the pollen grain are carbonbound and, therefore, incapable of exchange. Even in
raw pollen material where cellulose is present (2035%)
the exchangeable component would only represent a
small fraction of the total hydrogen isotope signal. Since
the aim of these pilot studies was the initial identication
of an isotopic signal within the carbon, oxygen and
hydrogen isotopes of modern pollen grains, we analysed
raw pollen for each site without equilibration. It was felt
that at this initial stage the possible inuence of the
labile hydrogen component of the total membrane
would be tolerably small (ca 1% of total hydrogen
atoms based upon structural estimates of Shaw and
Yeadon, 1964) and that identication of a broad
agreement or disagreement with local isotopic indicators
would be sufcient to support or counter the need for a
more thorough exploration of this isotopic signal in
pollen exina. The dD results should, therefore, be viewed
with this caveat in mind.
For this preliminary analysis the d18O and dD data
were compared with the d18O and dD compositions of
precipitation, monitored at sites within 100 km of the
pollen collection sites as part of the International
Atomic Energy Agencys Global Network for Isotopes
in Precipitation (Schotterer et al., 1996). Signicant
relationships are observed between both the pollen and
precipitation d18O compositions (Fig. 4), and the pollen
and precipitation dD compositions (Fig. 5). However,
whilst pollen d18O is negatively related with precipitation, pollen dD displays a strong positive relationship.
The reasons for this are unclear. In studies of other plant
components the relationship between precipitation and
d18O is typically positive, (Saurer et al., 1997), and this
can be explained as a degree of transfer of the
precipitation signal to the plant component material.
Indeed, this may be the case for the pollen dD signal,

3.2. d18 O and dD analyses

Oxygen and hydrogen isotope analyses (d18C and dD,
respectively) were performed on samples of pollen from
the same sites as those used for d13C analyses. d18C
compositions were determined using a high temperature
pyrolysis conversion to CO, and the resulting gas
measured on a Micromass Optima mass spectrometer
at the Weizmann Institute of Science, Israel. dD analyses
were made by Dr. David Dettman, University of
Arizona. The analytical precision for replicated oxygen
and hydrogen isotope analyses of standard materials
using these methods are 0.25 per mille 2 per mille,
respectively.
When analysing the stable hydrogen isotope ratios of
cellulose, a proportion (30%) of the hydrogen atoms are
labile and capable of exchange following their initial
incorporation into the cellulose structure. To overcome
this potential source of error these labile hydroxyl
hydrogens are normally replaced through derivitisation,
nitration or isotopic equilibration with water of a
known isotopic composition. The b-carotenoid structure
of the pollen exine is such that the large majority of the

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898

Delta D Precipitation (per mille V-SMOW)

-40
y = 0.41x - 9.0
R2 = 0.79
-60

-80

-100
-200

-180

-160

-140

-120

-100

-80

the biochemical fractionations involved in the respective

photosynthetic pathways of the two elements were
identied as a probable cause. Alternatively, the
development of the pollen grain early in the growing
season may involve predominant use of stored photosynthates that were produced during an earlier growth
period or become susceptible to isotopic re-exchange
with xylem waters that reect the isotopic consequences
of metabolic processes during the dormant period. A
more complete physiological/mechanistic understanding
of such processes, including the monitoring of pollen
development, coupled with a time series experiment/
labelling of plant water and O and H isotopes of pollen
would enhance greatly the understanding of this archive
and the condence with which it may be interpreted.

Delta D Raw Pollen (per mille V-SMOW)

Fig. 5. Relationship between dD in precipitation from local GNIP

stations and pollen dD for raw Pinus sylvestris pollen collected from
the European spatial study network.

-20

Delta D per mille (V-SMOW)

-40
-60

y = -5.7462x - 124.71
R2 = 0.6773

-80
-100
-120
-140

y = 7.6918x + 4.628
R2 = 0.9682

-160
-180
-200
-15

-10

-5

10

Delta 18-O permille (V-SMOW)

Fig. 6. X 2Y Plot demonstrating the statistical relationships between

dD and d18O in precipitation from local GNIP stations (triangles) and
pollen (diamonds).

however, it is clearly not applicable for the pollen d18O

signal. This anomaly is well illustrated when the d18O
and dD of both the precipitation and pollen are
compared graphically (Fig. 6). On this gure it is
evident that the relationship between precipitation d18O
and dD follows reasonably closely the well-established
Global Meteoric Water Line (GMWL), in which
precipitation dD has been shown to be proportional to
8d18O+10 (Craig, 1961).
In this preliminary study it is not possible to identify
the causes of these opposing d18O and dD signals
preserved in the pollen. Although uncommon, such
differences have been observed in other plant components (Terwilliger et al., submitted), where differences in

4. Conclusions
The stable isotope analysis of pollen has many
potential applications within palaeoecology. We have
presented some preliminary studies and ndings, which
we hope will be explored more fully in the future.
Sample preparation remains one of the biggest obstacles
to the wider exploration of this archive, and we have
proposed some developments in this area that perhaps
may be explored further by others working in this eld.
Results presented from a pilot study of the pollen d13C,
d18O and dD signals demonstrate that environmental
information appears to be preserved in these isotopic
signals. However, it is clear that there are large isotopic
fractionations involved in the formation of pollen that
are not well understood, especially regarding d18O and
dD. More detailed examinations of pollen genesis are
required and theoretical models need to be developed to
explain these processes before the isotopic composition
of pollen can be utilised as a reliable palaeoenvironmental indicator. Especially useful would be the
application of controlled environment experiments in
which atmospheric and hydrological parameters are
manipulated. By understanding these isotopic signals
and overcoming present limitations imposed by pollen
separation methods, we hope to be able to provide
valuable new information about terrestrial environmental changes and the carbon and hydrological cycles, at
time scales that can provide a terrestrial link between
ice-core and marine isotopic records.

Acknowledgements
We are grateful to all those who assisted in the
collection of pollen for this study. We also gratefully
acknowledge the help and support of David Dettman
and Chris Eastoe, University of Arizona, Tucson; Dan
Yakir, Emanuela Negreanu, Merav Montag and Raya

ARTICLE IN PRESS
N.J. Loader, D.L. Hemming / Quaternary Science Reviews 23 (2004) 893900

Shamis at the Weizmann Institute of Science; Nick

Shackleton, Kathy Willis and Mike Hall at the Godwin
Institute for Quaternary Research, University of Cambridge. NJL acknowledges assistance from the BES
(SEPG 1511) and EU projects ISONET (EVK2-CT2002-00147) & PINE (EVK2-CT-2002-00136).

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