Principles and methods of pollen analysis

Introduction

Pollen analysis is the principal technique used to reconstruct Quarternary environments. This is
so for several reasons.

1. Pollen is usually the most abundant fossil preserved in Quarternary sediments. Consequently it
can be counted, and the resulting pollen spectrum should be a statistical representation of all the
pollen grains in the sediment of that age at that place. Pollen assemblages can then be compared
from different points in space and time.

2. Pollen grains are resistant to decay in non-oxidizing situations. Because they are abundantly
produced by the plants, they tend to be deposited universally as the ‘pollen rain’, and thus they
have the chance of being preserved in many sedimentary situations.

3. The taxonomy of pollen grains is relatively well known, and the major types are readily
identifiable under the light microscope.

4. Because pollen grains are small (5-100µm) and abundant, only small amounts of sediment are
needed for an adequate sample.

5. Pollen grains originate from plants which originally grew together as the vegetation of an area.
Therefore pollen can be used to reconstruct the vegetation, including both local vegetation, such
as aquatic and wetland communities, and more distant, regional vegetation growing around the
site of deposition.

Because pollen analysis is such an important technique, a large amount of work has been done
on its various aspects. These include the study of pollen taxonomy and structure, pollen
production, dispersal, and deposition, the representation of different plant species and vegetation
types of contemporary pollen spectra, the preservation of fossil pollen, the interpretation of
pollen diagrams in terms of flora, vegetation, and environment, and the comparison of pollen
diagrams from different areas.

Historical development of pollen analysis

The development of Quaternary pollen analysis parallels that of descriptive ecology. Qualitative
descriptions of vegetation and pollen floras were made during the last century. At the beginning
of this century, quantification in ecology developed, and vegetation began to be described in a
quantitative way. Similarly, in his classic paper of 1916, (reprinted in English in 1967), the
Swedish geologist Lennart von Post put forward the method of quantitative pollen analysis. He
had the idea of presenting pollen spectra as percentages of the sum of the pollen grains counted,
and representing the results as stratigraphic diagrams, with pollen spectra plotted against their
stratigraphic position through the sediment. Von Post showed similarities in pollen diagrams
from a small area, and differences between different areas. He was thus able to add a time
dimension to the study of vegetation. Although pollen analysis has been developed and refined
through the years since 1916, the basic method remains the same.

Basic principles of pollen analysis

1. Pollen or spores are produced in great abundance by plants.

2. A very small fraction of these fulfill their natural reproductive function, and the majority fall
to the ground.

3. Pollen and spores rapidly decay, unless the process of biological decomposition are inhibited
by lack of oxygen. This occurs in places such as bogs, lakes, fens, and the ocean floor, where
pollen is preserved.

4. Before reaching the ground, pollen is well mixed by atmospheric turbulence, which results in a
more or less uniform pollen rain over an area.

5. The proportion of each pollen type depends on the number of parent plants, and hence the
pollen rain is a function of the composition of the vegetation. Therefore a sample of the pollen
rain will be an index of the vegetation at that point in space and time.

6. Pollen is identifiable to various taxonomic levels.

7. If a sample of the pollen rain is examined from a peat or mud of known age, the pollen
spectrum is an index of the vegetation surrounding that place at a point of time in the past.

8. If pollen spectra are obtained from several levels through the sediment, they provide a picture
of the vegetation and its development at that place through the length of time represented by the
sediments.

9. If two or more series of pollen spectra are obtained from several sites, it is possible to compare
changes in vegetation through time at different places.

Techniques of pollen analysis

After the site has been chosen, and a core or other samples obtained and the lithology described,
the pollen sample must be treated in order to concentrate the pollen and to remove as much of the
sediment matrix as possible.
Preparation of pollen samples

The matrix of the sediment is removed by physical and chemical processes, which, as far as
possible, do not affect the pollen grains and spores. The standard method is described in Faegri
and Iversen (1975). It is outlined below.

i). Boil sediment with 10% NaOH or KOH to remove soluble humic acids and to break the
sediment down. Subsequent repeated washes in distilled water are essential.

ii. Sieve to remove coarse debris.

iii. Treat with cold 10% HCl to remove carbonates.

iv. Treat with either hot 60% HF for a short time (up to one hour) or cold 60% HF for a long
time (up to 24 hours) to remove silicates (silt, clay, diatoms, etc)

v). Treat with hot acetolysis mixture (9 parts acetic anhydride: 1 part concentrated H 2SO4), to
hydrolyse cellulose.

vi). After neutralization, stain the residue with safranin or basic fuhsin.

vii). Mount the residue in a suitable medium with a refractive index less than 1.55, which is that
of pollen grains. An excellent medium is silicone oil (refractive index 1.4), because the slides last
well, provided that the pollen has been thoroughly dehydrated, the grains can be turned readily
for identification, and they hardly alter in size over time. Glycerol and glycerine jelly are also in
common use. The first is short lived, and the slides are easily damaged. The second is easy to
use, but the grains are fixed and difficult to turn over, and there is a tendency for grains to swell
after a time and lose their features for some unknown reason, making their identification
difficult, and the use of size statistics impossible.

Other preparation techniques involving oxidation and the use of flotation liquids may be used for
some sediments, but in general they have been found to be less reliable than the standard
procedure outlined above.

Morphology of pollen and spores

Before being able to identify pollen, the analyst should understand their basic function and
structure.

1. Function of pollen grains

A pollen grain contains a male gamete of the plant, and its function is to transfer it to the female
gamete via the stigma and style of an angiosperm, or the nucellus of a gymnosperm. Pollen is
formed in the anthers. Most pollen is liberated as single grains, but in some families, such as
Ericaceae and Orchidaceae, the grain remain in tetrads.
Pollen ranges in size between about 5-100µm, the commonest size being about 30 µm. The
winged grains of Pinaceae are some of the largest, apart from the 350 µm grains of Annonaceae.
The important component of pollen to the palaeoecologist is the sporopollenin found in the
pollen wall. Sporopollenin is a very inert substance, which accounts for the common
preservation of pollen grains and the relative safety with which they can be treated chemically
during preparation procedures.

Identification of fossil pollen

The identification of fossil pollen is obviously of crucial importance, because all further
palaeoecological interpretations will hang upon the identifications. However, successful
identifications of pollen depend upon several factors;

1. The range of the reference material used.

2. Variability within species should be represented in the reference material, by having several
collections of each from different localities, which cover the range of its natural variation.

3. The morphology of the grain. Some grains are very distinctive, whereas others have to be
studied very carefully to find differential characters.

4. The preservation of the fossil grains. Some grains can be identified even though they are badly
deteriorated, but others lose their definitive characteristics readily. Thus a count from badly
preserved material will tend to be biased towards the more robust and distinctive pollen types.

5. The experience and knowledge of the analyst

6. The selection and scoring of morphological characters

Pollen counting

A few words on pollen counting are appropriate here, because numeracy is a basic feature of
pollen analysis, being one of the main reasons why pollen analysis is such an important
palaeoecological method.

Counting should be carried out along regular traverses of the microscope slide, most
conveniently at a magnification of x300 or x400. Higher magnification (x1000) using an oil-
immersion objective is necessary for the identification of some grains.

The number of grains counted depends on the problem being investigated. However, enough
should be counted to achieve a random sample of the pollen grains present, otherwise the count
will not be reproducible. Therefore traverses should be positioned evenly over the whole slide
and not concentrated near the edge or the middle. This is because smaller pollen grains tend to
travel towards the edges of the coverslip more readily than larger ones. Enough grains should be
counted to maintain constant percentages of the pollen sum. This is usually in the region of 300-
500. It depends largely on the number of taxa in the sample.

Presentation of pollen analytical data

Having obtained a stratigraphical sequence of pollen counts, the next stage is to present the
results in the form of a graph or other figure, prior to interpretation of the data. Pollen analytical
data are complex and are most effectively presented in the form of a pollen diagram. This is a
series of graphs of the values for different pollen and spore taxa plotted against their stratigraphic
depth or, sometimes, against their age.

Having identified and counted all the pollen and spores in the different levels of the sequence,
the investigator then has to decide what to plot on the pollen diagram. Mostly, pollen analysts
consider the relative proportions of the different pollen types, which are generally expressed as
percentages of a pollen sum. In addition it is now possible to estimate ‘absolute’ numbers of
pollen grains, in which case the numbers of different taxa are independent of each other.

The pollen sum

The choice of which taxa should be included in the pollen sum should be based on the principle
that all the members of the population under study should be included.

After the percentages have been calculated, the diagram can be drawn. There are very many
different styles of pollen diagram. It should be remembered that the data should be visible in the
clearest possible way to the reader.

Nowadays, computers can be used to speed up the calculations of pollen data and the drawing of
pollen diagrams. Some of the pollen diagrams are shown below.
Figure 4.5 Arboreal pollen taxa (from Muller et al., 2005).

Zonation of pollen diagrams

Pollen diagrams are complex, consisting of many levels and many taxa. Some form of
simplification is often needed. This can be most readily achieved by drawing horizontal lines,
separating sections or zones of differing pollen composition. Such a subdivision aids in the
description of the diagram, the discussion and interpretation of its contents, and its comparison
and correlation with other diagrams. A pollen zone has been defined as ‘a body of sediment with
a consistent and homogenous fossil pollen and spore content that is distinguished from adjacent
sediment bodies by differences in the kind and frequencies of its contained fossil pollen and
spores’. A pollen zone falls into the category of an assemblage zone, which is a biostratigraphic
unit. Previously, there had been much confusion about the criteria used in pollen zonation.

Pollen zones have, in the past, been defined on various criteria.

a). The observed pollen stratigraphy
b). Units of inferred past vegetation
c). Units of inferred past climate
d). Units of sediment lithology
e). Units of inferred time.

Only the observed pollen stratigraphy should be used in strict pollen zonation, as it is free of
interpretive implications.

When pollen assemblage zones are constructed, they should be properly defined. The definition
should include, where appropriate:
a). Type locality and section
b). Description of the fossils in the zone
c). Description of the contacts with other zones
d). The thickness of the zone and its age, if known.
e) The name of the zone, e.g. Betula-Juniperus regional pollen assemblage zone.
f). Other occurrences and general notes.

A pollen diagram should be divided as carefully as possible into pollen assemblage zones. As
such, the zones will be unique to that site, and they can be called site zones or local zones. If they
can be matched at other sites, regional pollen assemblage zones can be defined, and fully
described.

If the regional pollen assemblage zones are adequately radiocarbon-dated, they can be mapped in
space and time.

Comparison of pollen diagrams

Zonation of a pollen diagram is not an end in itself. The zones which are delimited are site or
local zones. The pollen analyst is usually interested in comparing his sequence with others. If the
zones can be matched, regional pollen assemblage zones can be defined and mapped temporally
and geographically.
Pollen production, liberation, dispersal, deposition and preservation

Pollen production

It is well known that different plants produce different amounts of pollen. In general, wind-
pollinated species (anemophilous species) produce very much more than insect-pollinated
species (Entomophilous species), whereas cleistogamous species, such as Viola, in which the
flowers do not open, have a very low pollen production. Entomophilous species often have
highly ornamented pollen grains which tend to stick together, whereas anemophilous species
tend to have smooth, light, and round or winged pollen grains. However, some entomophilous
species are high pollen producers, e.g. Calluna vulgaris and Tilia cordata. These are both good
sources of honey.

It is difficult to estimate the actual pollen production of a species, but this was attempted by Pohl
(1937). He counted the number of pollen grains in an anther, and multiplied this by the number
of anthers in a flower, the number of flowers in an inflorescence, the number of inflorescences
on a herbaceous plant or on a ten-year old branch of a tree, and the number of branch systems on
the tree. Some of his results are shown in Table 9.1.

The major forest trees fall in order of decreasing pollen production: Pinus sylvestris, Alnus
glutinosa, Corylus avellana, Betula verrucosa, Quercus robur, Picea abies, Populus
Canadensis, Tilia cordata, Fagus sylvatica, and Aesculus hippocastanum. It has been estimated
that Pinus sylvestris produces 10-80 kg of pollen per hectare annually.

Pollen liberation

1. Seasonal variation
There is a well marked seasonal variation in pollen liberation with some plants producing their
pollen during specific months in a year as shown in Figure 9.1.

2. Diurnal Variation

Studies on pollen liberation of two grasses Holcus lanatus and Festuca rubra through the day
have shown that early morning and evening are often times of anthesis (production of pollen
from anther), related to air temperature, light intensity, and low relative humidity (RH). Pollen
liberation into the atmosphere is related to RH and atmospheric turbulence, suggesting that
anthesis is an active process, whereas liberation is purely mechanical. Grass – pollen
concentration in the atmosphere is positively correlated with air temperature and hours of bright
sunshine.

Pollen dispersal and deposition

a). Pollen dispersal into the atmosphere
Apart from those grains transported by insects or water, or expelled forcibly by some active
process, pollen grains are dispersed in the atmosphere following the laws governing all small
particles.

The dispersion of pollen depends principally on:

i). turbulence of the atmosphere
ii). Wind speed and direction
iii). Terminal falling velocity, which depends on the weight and shape of the pollen
iv). Height and strength of the pollen source.

Long-distance pollen dispersal in the atmosphere

Pollen can be transported great distances in the atmosphere. Samples of air over the oceans many
hundreds of miles from land contain pollen grains. Samples 450 km from the Norwegian coast
deposited in the summer of 1949 contained the following numbers of pollen.
Pinus 5.5 grains cm-2
Betula 4.7 grains cm-2
Quercus 0.8 grains cm-2
Gramineae 5.5 grains cm-2

This assemblage was recorded some 1700 km from the nearest oak tree. Many studies of pollen
deposition in arctic regions have recorded pollen of temperate trees, long distances from the
nearest trees.

The transport of tree pollen over long distances in the atmosphere can be related to the
trajectories of air masses with their contained particles. The occurrence of long distance transport
must be borne in mind, particularly when interpreting pollen assemblages from environments
with a low local pollen production.

Pollen deposition in forest

Pollen deposition in a forest comprises three components:
i). deposition during flowering
ii). Redeposition of pollen by rain
iii). Deposition of pollen with litter.

Pollen deposition within a tropical forest

There are several differences in pollen deposition in a tropical forest as opposed to a temperate
forest arising due to the differing biology of the two systems.
i). There is a very low pollen production, equivalent to that in arctic tundra sites.
ii). The pollen rain is dominated by species with a high canopy, suggesting that the canopy
component is the most important
iii). Seasonal variation is pronounced, as in a temperate forest, both in the taxonomic
composition of the pollen rain, and in its amount. Highest deposition occurs at the end of the dry
season.
iv). There is little evidence for filtration, although it undoubtedly occurs, because flowering is
continuous throughout the year.

Pollen dispersal in water

1. Freshwater
Although it has long been realized that pollen can be transported in suspension by streams and
rivers, it is only recently that attempts have been made to construct a pollen budget for a lake.

Aerial deposition of pollen in a lake is a small proportion of the total deposition and the rest
come via the streams and from slope run-off. Additional apparent deposition can also come from
pollen resuspended from the upper sediments due to water turbulence.

Streams carry a more or less constant amount of pollen through the year, of a consistent
taxonomic composition, whereas air traps showed marked seasonal variations, depending upon
what was in flower.
The main sources of stream-borne pollen are:
i). Direct fall of pollen from plants growing along the banks;
iii). Bank erosion. This becomes important during floods;
iii). Surface run-off. This appears to be the major source. During floods, pollen influx to streams
increased 142-311 folds, from about 150 grains l-1 to 22-48000 grains l-1.

Pollen sedimentation
When a pollen grain lands on a surface, it is then subject to the processes of deposition, before it
finally becomes incorporated and preserved in the sediment.

Pollen sedimentation in lakes.

Pollen can be redistributed both horizontally and vertically in lake sediments.
Medium to large sphaeroidal pollen grains of deciduous trees (e.g. oak like Quercus) are evenly
distributed over the lake. They are regarded as non-local in origin, and therefore they would have
fallen evenly onto the lake surface. Locally produced pollen such as that of Salix and aquatic
plants are deposited in higher amounts near the source, in the shallow water.

The movement of littoral sediments rich in pollen to the lake centre tends to equalize the absolute
influx of pollen throughout the basin.

In addition to horizontal movements of pollen in lakes, pollen in lake sediments is usually mixed
vertically by the activities of burrowing animals, such as midge larvae, worms, etc. Such animals
can redistribute pollen over a vertical distance of up to 15 cm, although most disturbance is
between 3-4 cm.

Pollen preservation and deterioration

Physical, chemical, and biological processes affect pollen grains from the moment they are
liberated from the plant. Although the pollen exine is very resistant to decay, due to its
sporopollenin content, it can be destroyed in various ways before being examined by pollen
analyst. Badly preserved or deteriorated pollen may still be identifiable, but in some cases it is
too badly damaged to be recognizable.

Four categories of deterioration in fossil pollen grains are commonly observed:

i. corrosion, where the exine is pitted or etched;

ii. broken, where grains are ruptured or split, or where pieces have completely broken away;

iii. crumpled, where grains are folded, twisted or collapsed; and

iv. degraded, where the structural elements are fused together presenting a ‘solid’ or ‘fossilized’
(waxy) appearance to the grain.

The reconstruction of past floras and past plant populations

Local vegetation reconstructions

Tracing the course of local vegetation developments has been, and still remains, a central theme
of pollen analysis. Pollen records from lake cores enable inferences to be made about the history
of a particular lake ecosystem, while in sites where pollen diagrams have been obtained from a
number of different profiles, changes in the local vegetation cover can be mapped through time.
Understanding the nature of local vegetation changes is a prerequisite to establishing the scale
and pattern of regional changes in vegetation, as well as the impact of humans on vegetation
communities.

Regional vegetation reconstructions

For many years, pollen data have constituted one of the principal lines of evidence for
reconstructing vegetational history at the regional and extra-regional scales. Not only does the
technique enable large-scale vegetation patterns to be established, it allows the history of both
individual species and entire vegetational assemblages to be traced through time.