Journal of Archaeological Science (2000) 27, 363372

doi:10.1006/jasc.1998.0374, available online at http://www.idealibrary.com on

Simultaneous Extraction of Phytoliths, Pollen and Spores from

Sediments
C. J. Lentfer and W. E. Boyd*
School of Resource Science & Management, Southern Cross University, Lismore 2480, New South Wales,
Australia

(Received 28 March 1998, revised manuscript accepted 27 November 1998)

Archaeological sediments often oer opportunities to examine local palaeoenvironmental conditions from analysis of
included microfossils. On-site conditions commonly vary, and thus so do the preservation conditions for microfossils.
Consequently, a range of palynological preparation techniques are commonly used. While dierent types of microfossils
provide valuable palaeoenvironmental information, the use of separate extraction methods for dierent microfossil
types may be both time- and resource-consuming, especially where the recovery predicability is low. This paper
examines the possibility of combining preparation techniques for three commonly encountered microfossilspollen,
spores and phytolithsby comparing pollen extractions using heavy liquid extraction and standard pollen recovery
procedures. Although the use of heavy liquids for pollen and spore preparations has been well-documented, for several
reasons it has not been a favoured technique for pollen extraction. The research reported here shows that for most of
the sediments tested, heavy liquid extraction procedures produced comparable results to those arising from standard
pollen extraction techniques. For oxidized sediments, especially, more reliable results are likely to be obtained from
heavy liquid extraction procedures than from those employing acetolysis. Overall, heavy liquid procedures allow
complementary suites of data to be investigated with the least cost and eort, thus enabling palynologists and
phytolithologists to adopt more eective research practices for environmental reconstruction.  2000 Academic Press

Keywords: PHYTOLITHS, POLLEN, EXTRACTION TECHNIQUES, HEAVY LIQUID FLOTATION,

PALYNOLOGY.

Introduction Phytolith analysis potentially documents more

detailed and local patterns of vegetation change than

M
icrofossil analysis from archaeological
sediments often provides a basis for
palaeoenvironmental reconstructions. Given
common variation in on-site conditions, preservational
state of various microfossils also varies. Fossil pollen,
phytoliths and diatoms, for example, are best preserved
under dierent sedimentological circumstances. Conse-
quently, a range of preparation techniques is now
commonly used. These largely involve the gradual
removal of unwanted sediment components, either by
dissolution or physical separation; in pollen analysis,
preparation also involves the removal of remaining
pollen intine. While each class of microfossil provides
valuable palaeoenvironmental information, they typi-
cally require separate extractions, which may be both
time- and resource-consuming. This paper examines
the possibility of combining preparation techniques
for three commonly encountered microfossilspollen,
spores and phytoliths.
*For correspondence. Tel: [6166] 203 007; Fax: [6166] 21 2669;
Email: bboyd@scu.edu.au
363
03054403/00/050363+10 $35.00/0
pollen analysis (Pearsall & Trimble, 1984; Piperno,
Bush & Colinvaux, 1991; Rossen, 1994; Rovner, 1988;
Lentfer & Boyd, 1998), and thus oers important
archaeological site- and function-specific detail. How-
ever, phytolithologists are increasingly recognizing the
weakness of using one microfossil class alone for
palaeoenvironmental reconstruction, and frequently
rely on complementary pollen analyses (Piperno, 1983,
1985, 1994; Piperno & Clary, 1984; Wilson, 1985;
Kealhofer & Piperno, 1994 Kealhofer, 1996; Penny,
Grindrod & Bishop, 1996; Kealhofer & Penny, 1998).
This approach facilitates more precise reconstruction
in several ways. First, it may involve plant taxa lacking
diagnostic phytoliths but with diagnostic pollen (e.g.,
members of the Araceae family) and vice versa (e.g.,
members of the Poaceae family). Secondly, it increases
the value of microfossil data bases by using phytoliths
for identification of specific plant parts. Thirdly, it
provides a means to check identifications of eroded
fossils. Fourthly, it allows inclusion of plants types
with various dispersal mechanisms. Finally, by di-
versifying microfossil catchment areas, it enhances
 2000 Academic Press
364 C. J. Lentfer and W. E. Boyd
assessment of changing past vegetation patterns at
both regional and local levels.
Although extraction methods capable of extracting
pollen and spores and siliceous microfossils were
developed over 50 years ago (e.g., Knox, 1942; Frey,
1955; Hunt, 1985; Fredlund, 1986; Moore, Webb &
Collinson, 1991), these have not been widely adopted
by phytolithologists or other palynologists. Instead,
most analysts use separate procedures. Penny,
Grindrod & Bishop (1996), for instance, extracted
pollen using hydrofluoric acid but extracted phytoliths
using liquid flotation and a strong oxidizing agent from
lake and swamp sediments. Thus all silica was dis-
solved by HF in the pollen extractions, and pollen and
spores were destroyed by the strong oxidation in the
phytolith extractions. The adoption of separate
procedures can be advantageous for some situations
such as the preparation of (i) organic-rich sediments
requiring harsh oxidation treatment to release
phytoliths from the organic matrix (Lentfer, 1997;
Lentfer & Boyd, 1998), or (ii) samples where pollen
analysis is of prime importance, necessitating extrac-
tion to ensure optimum pollen and spore concen-
trations. However, where research focuses primarily on
phytolith analysis, advantages can be gained by using
single extraction procedures. First, single extraction
allows pollen and spore presence in sediments to be
monitored prior to or during phytolith counting. Sec-
ondly, it provides assessment of possible additional
treatments to concentrate palynomorphs. Thus, a
multidisciplinary approach can be achieved with little
extra cost and eort. Also, the risk of ignoring valuable
information based on assumptions of unsuitable site
conditions for specific microfossil preservation can be
avoided.
In this paper, pollen extraction using a heavy
liquid extraction procedure (abbreviated to HLFPol
in this paper) and a standard pollen extraction pro-
cedure (PolStd) with HF and acetolysis treatments
are compared. The HLFPol technique has been
shown to be successful for phytolith extraction (see
Lentfer, 1997; Lentfer & Boyd, 1998). If, therefore,
it can also be used for pollen and spore extraction,
it may be most useful for microfossil extraction at
archaeological sites. The procedure is standard heavy
liquid flotation extraction for phytoliths, without oxi-
dation but including an alkali treatment, similar to the
other techniques described for pollen and spore extrac-
tion (Frey, 1955; Sittler, 1955; Stockmarr, 1972;
Brande, 1976; Johnson & Fredlund, 1985; Dricot &
Leroy, 1989; Faegri & Iversen, 1989; Moore, Webb &
Collinson, 1991). It has the ability to extract various
microfossils (including pollen, spores, phytoliths,
diatoms and sponge spicules) from sediments in a
single process. To assess this methods ecacy at
concentrating pollen and spores and providing ac-
curate representations of original populations, pollen
counts from HLFPol residues used for phytolith analy-
sis (Lentfer, 1997; Lentfer & Boyd, 1998) are compared
with those for residues prepared using the PolStd
procedure.
Methods
Extraction and counting procedures
Four replicates were run for each sediment/method
combination (Figures 1, 2 & 3; Table 1). Sediments
were initially ground with a pestle and mortar, and
cone quartered (Powers & Gilbertson, 1987) to sample
1 cc of each. An aliquot (Powers & Gilbertson, 1987)
of 24,559 Alnus pollen grains (..=39%) was added to
each of the samples for determination of absolute
frequency counts. The sediments were sieved through
250
m and 100
m; the 250
m sieve was used to
extract as full an assemblage of phytoliths as possible,
whereas the 100
m sieve allowed more ecient pollen
extraction. The heavy liquid used was cadmium iodide
and potassium iodide at 235 specific gravity. Samples
were stained prior to mounting as per standard pollen
techniques, and no evaluation of staining eects has
been made. Slides were viewed at 400magnification
using an optical microscope fitted with a polarizing
lens. Non-overlapping transects were viewed and all
pollen, spores and phytoliths encountered along
transects were classified and counted. Counting time
was limited to 1 h per sample. Concentration of pollen
and spores (microfossils per cm3) was calculated
using Stockmarrs (1972) formula: total fossil pollen=
(fossil pollen countedtotal number markers)/
(markers counted).
Statistical analysis
Two-way ANOVA and pairwise comparisons were
used for analysis of raw count and concentration data.
To stabilize variance for multivariate analysis, absolute
frequency data was transformed using the arc tan
transformation described by Gordon (1982): Xik =arc
tan (yik/v)1/2, where, Xik denotes the value of the
transformed variable, yik denotes the calculated esti-
mate for the number of grains of the kth taxon per
cubic centimetre and v denotes the number of exotic
grains added to the sample (i.e., Alnus grains). The
eect of this transformation is to reduce the range of
values to between 0 and /2. Transformed data were
analysed using Excel and SPSS programs. Principal
component analysis (PCA) with Euclidean distance
measures established patterns of variation within and
between treatments, and biplots were constructed for
all principal components (PCs) with eigenvalues over 1.
Two-way analysis of variance (ANOVA) tests on prin-
cipal component scores and pairwise comparisons
using the Bonferroni method to calculate critical mean
dierences at =005, determined significant dier-
ences between the two extraction methods. The use of
absolute frequency data and transformation reduced
levels of bias resulting from variables with high
 Simultaneous Extraction of Phytoliths, Pollen and Spores from Sediments 365

HLFPol PolStd variance and therefore all variables were included in

principal components analysis.
crush sediment with
pestle and mortar

Results
Morphology and clarity
measure 1 cc
sediment Overall, pollen and spores in the PolStd residues,
acetolysed after HF treatment and dehydrated with
TBA (tertiary butyl alcohol), were more swollen than
those in HLFPol residues. In comparison, paly-
disaggregate nomorphs from HLFPol residues exposed to heavy
sediment, shake 12
hours in 5% Calgon liquid separation and dehydrated in ETOH (ethyl
solution alcohol) appeared to be more collapsed. Consequently,
morphological characteristics were more readily
observed and identified in the PolStd residues. While
there appears to be a need for further study of the
alkali digestion, boil eects of grain swelling and contraction related to
5 min in 10%
KOH sol'n preparation techniques, pollen extracted by both
techniques was identifiable in this study.
Furthermore, since the HLFPol method produced
residues with silica and palynomorphs, slide clarity was
remove carbonates inferior to that of PolStd residues for all sediments.
in 15% HCl
Pollen and spores were often obscured by small silica
particles apparently attracted to them. This attraction
was observed during slide mounting; palynomorphs
sieve through sieve through clearly visible immediately following their application
250 m mesh 100 m mesh to slides were often obscured by silica shortly after-
wards. Such aggregation occurred particularly with
more viscous media (silicone oil and glycerol) and was
much reduced in water.
remove clays,
deflocculate in 5%
Calgon sol'n,
centrifuge and Information retrieval and pollen and spore
decant, repeat until concentrations
supernatant clears
Two-way ANOVA tests and pairwise comparisons
showed that significant sedimentmethod interaction
occurred at =005 for: (i) total pollen and spores
separate light and dissolve silica (including Alnus) count/h; (ii) total pollen and spores
heavy fraction with in hydrofluoric (excluding Alnus) count/h; and (iii) total concentrations
heavy liquid flotation acid
of pollen and spores (excluding Alnus) in 1 cc sediment.
Counts/h (including Alnus) were equivalent for sedi-
ments 1, 2, 4 and 5, but were significantly greater from
remove cellulose PolStd residues than HLFPol residues for sediments
with acetolysis 3 and 6. Likewise, for sediments 1, 2 and 3, counts/h
(without Alnus) were significantly greater from PolStd
residues, but were equivalent between the two
methods for sediments 4, 5 and 6. Pollen and spore
stain with safranin

Figure 1. Flowchart summarizing the two pollen extraction methods

dehydration with dehydration used in this study: the heavy liquid extraction procedure (HLFPol)
50% and 100% with and a standard pollen extraction procedure (PolStd). HLFPol
ethanol TBA
samples are sieved through larger mesh to allow extraction of large
siliceous microfossils in addition to smaller palynomorphs. PolStd
samples are treated with hydrofluoric acid to dissolve silica, acetoly-
sis to remove cellulose and TBA (tertiary butyl alcohol) for de-
mount in silicone oil hydration. Heavy liquid flotation is used to separate microfossils
from heavy mineral fractions in HLFPol samples and ethyl alcohol is
used for dehydration.
366 C. J. Lentfer and W. E. Boyd

P.N.G.
0 10
Bribie
Location Island Km New
Map Ireland
1,2,3 Bismarck Sea
Deception
Bay Moreton
Redcliffe Island Willaumez
Moreton Peninsula
Bay Garua
6 Island
Pine Rr
New Britain

N
North 4,5 Pililo
Brisbane Stradbroke Solomon Sea
Rr Redland Island 0 100
(a) (b) Km

Figure 2. Location map of (a) Deception Bay, southeast Queensland, showing collection sites of sediments 1, 2 and 3 (derived from Cotter,
1996: 194), and (b) West New Britain showing collection sites of sediments 4 and 5 at Paligmete Village, Pililo, south West New Britain and
sediment 6 at Garua Island north West New Britain.

Figure 3. Particle size analysis of sediments showing percentage dry weight total organic matter.
 Simultaneous Extraction of Phytoliths, Pollen and Spores from Sediments 367

Table 1. Sample locations, sample depths, pH results and lithology determined in part from XRD analysis (details supplied by Cotter, pers. comm.,
for the southeast Queensland samples)

Site Location Sample description

1 Deception Bay, southeast Queensland, Australia Dark brown sandy clay loam, pH 417
Coastal heath dominated by Melaleuca quinquenervia (Cav.) S. T. Blake, sample from topsoil, quartz and feldspar
Blechnum indicum Burm. f., exotic pine and grass
2 Deception Bay, southeast Queensland, Australia Black humic sand, pH 428
Disturbed coastal herbland/shrubland dominated by Phragmites austra- sample from depth 80100 cm, quartz and disordered
lis (Cav.) Trin., Blechnum indicum and Melaleuca quinquenervia kaolinite
3 Deception Bay, southeast Queensland, Australia Yellow white clay loam, pH 379
Coastal shrubland dominated by Melaleuca quinquenervia, Blechnum sample from depth 2545 cm, quartz, feldspar and
indicum, Phragmites australis, and Imperata cylindrica (L.) Beauv. montmorillonite
4 Paligmete Village, Pililo, south West New Britain, Papua New Guinea Brown clay, pH 75
Village site with Cocos nucifera (L.), tropical regrowth forest and grass sample from depth 3540 cm, quartz and well ordered
kaolinite
5 Paligmete Village, Pililo, south West New Britain, Papua New Guinea Black smectitic clay complex derived from midden ma-
trix, pH710
Village site with Cocos nucifera, tropical regrowth forest and grass sample from depth 1015 cm, calcite and quartz
6 Garua Island, north West New Britain, Papua New Guinea Red brown clay, pH 713
Coconut plantation with Cocos nucifera, tropical regrowth forest, ferns sample from depth 170180 cm, moderately kaolinite
and grass

concentrations/cc were equivalent for all sediments
except for the topsoil (sediment 1), where estimated
mean concentrations for PolStd residues were almost
three times greater than for HLFPol residues (Lentfer,
1997, presents tables of ANOVA tests and pairwise
comparisons).
Composition
The assemblage compositions based on estimates
of frequencies of pollen and spore types in 1 cc of
sediment are shown in Figures 4 & 5. Two-way
ANOVA tests (Lentfer, 1997) show that numbers of
types extracted by each method were not significantly
dierent at =005. Although most pollen and spore
types were present in residues extracted by both
methods, there were dierences in type presence/
absence between samples. The most notable discrepan-
cies occurred in favour of the HLFPol residues (i.e.,
types not recorded in PolStd residues were relatively
common in HLFPol residues). For sediment 6, Euphor-
biaceae pollen was present in HLFPol residues but ab-
sent in PolStd residues. Likewise, an unidentified pollen
(Unknown 1) and an unidentified spore (Spore 4) were
present in the HLFPol residues of sediments 1 and 4,
respectively, but absent in the PolStd residues. Minor
dierences were also recorded for rare types (i.e., those
recorded once only in one or two residues), but, as
above, in almost every case types were found in HLFPol
residues but not in PolStd residues.
Multivariate analysis
Of the assemblage variation, 795% was explained by
the first six principal components. Distribution pat-
terns of samples for methods within sediments, accord-
ing to PC scores based on the transformed pollen and
spores frequencies per cc for each sediment, were
equivalent, except for variation determined by PC4 and
PC5 (Lentfer, 1997) (Figures 6 & 7). For PC4, the
variation between methods is associated with sediment
6 residues, where higher concentrations of Euphor-
biaceae and Urticaceae/Moraceae pollen (labelled
EUPHORB and URT.MOR in the figure) in HLFPol
residues account for the contrast with PolStd residues.
Variation between methods according to PC5 are
largely determined by contrasts between one HLFPol
residue with a relatively high Spore 4 value, and one
PolStd residue with a relatively high Euphorbiaceae
value. All other residues for sediment 4 show com-
parable distributions for both methods. Sediment 1
samples are also contrasted by PC5 but to a lesser
degree, and variation between methods for this sedi-
ment is not significant. Nevertheless, sample dierences
indicated are mainly associated with the presence of
Unidentified 1 type (UN1) in HLFPol residues and the
higher numbers of the Myrtaceae type (MYRT 1) in
PolStd residues.
Discussion
Introduction
For most of the sediments, the standard pollen extrac-
tion procedures (PolStd), using hydrofluoric acid and
acetolysis, are equivalent to heavy liquid extraction
(HLFPol). Variation between treatments was observed
and although some of this variation may be reduced by
increasing pollen counts (Lentfer, 1997), there is never-
theless substantial evidence for dierential selection
of fossil palynomorphs between the two extraction
methods. The major causes for such dierential selec-
tion are associated with: (i) dierential destruction of
palynomorphs; (ii) sediment variability and insucient
368 C. J. Lentfer and W. E. Boyd

20 200
Pteridophytes/Spores

+ +

+
+
+
+

+
+

+
+
+ +
+
150
Unknown
Pteridophytes/Spores 100
150
50
100

+ +

+
+ +

+
+
Herbs Unknown

+
50 40
Herbs 20

+
+

+
+
+
150

+
100 40
Trees and Shrubs Trees and Shrubs 20
50

+
150
+
+

+
+
+
Spore 4
20 100
Spore 3 Spore 4
+
+

+
+

+
+

+
20 50
Spore 2

+
+ +
+
+

+
Spore 1
+
+
+
+

+
+ Spore 3

+
+ +
+

+
+
Cyatheaceae Spore 2

+
Unknown 4 40
Unknown 3 + Spore 1 20
Unknown 2
+
+

+ +

+
Unknown 1 Cyatheaceae

+
150 Unknown 4

+
Unknown 3

+
+

+
Poaceae/Cyperaceae/ 100
Unknown 2

+
Restionaceae
50 Unknown 1
40
Poaceae/Cyperaceae/
+ + +

+
+
+

Restionaceae 20
Liliaceae cf.
+

+
Plantaginaceae
+

Asteraceae Liliaceae cf.

+

+ +
40 Plantaginaceae

+
Euphorbiaceae 20 Asteraceae

+
40
+

Fabaceae Euphorbiaceae 20
+

Urticaceae/Moraceae
+
+

+
+

20
Casuarinaceae 20
Fabaceae
Myrtaceae 2
+

150 Urticaceae/Moraceae

+
Casuarinaceae
+
+

100 Myrtaceae 2

+
+

+
Myrtaceae 1 20
50 Myrtaceae 1
+
+

+
+
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
+
+
+

+
+
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d

PolStd. Samples
Figure 4. Pollen and spore counts for PolStd residues, showing total
count/type 10
3. Types with fewer than 1,000 grains/cc sediment
are shown by +.
disaggregation for the least oxidized sediments; and
(iii) possible dierential loss of palynomorphs during
flotation.
Dierential destruction of palynomorphs
Selective destruction of pollen and spores by acetolysis
and oxidation is recorded elsewhere (e.g., Wenner,
1947 (cited in Brown, 1960); Erdtman, 1952; McIntyre
& Norris, 1964; Dricot & Leroy, 1989; Faegri &
Iversen, 1989). The composition of pollen and spore
assemblages depends in part on the ability of exines to
withstand harsh chemical treatment, and the degree of
exposure of sediments to oxidative weathering prior to
treatment. Destruction of pollen and spores occurred
within the PolStd procedure which employed acetolysis
to dissolve cellulose and HF to dissolve silica. It is
HLFPol Samples
Figure 5. Pollen and spore counts for HLFPol residues showing the
total count/type 10
3. Types with fewer than 1,000 grains/cc
sediment are shown by +.
reasonable, thus, to assume that acetolysis and HF
treatment were largely responsible for the dierential
destruction of pollen and spores in PolStd samples
extracted from sediments 1, 4 and 6. Since sediments 4
and 6 were collected from well-drained sites in tropical
environments with distinct wet and dry seasons, it is
likely that pollen and spores within these sediments
have been exposed to relatively strong oxidative weath-
ering (Andersen, 1986). Also, since exines are more
readily oxidized in alkaline solutions (Faegri & Iversen,
1989), the alkaline pH of these sediments would
have contributed further to the oxidation process
(cf. Andersen, 1986). The Urticaceae/Moraceae and
Euphorbiaceae pollen and Spore 4 type, observed in
relatively high numbers in HLFPol residues, but absent
from PolStd residues, may have been previously
degraded to the point where their exines were
readily destroyed by acetolysis and HF. Natural
 Simultaneous Extraction of Phytoliths, Pollen and Spores from Sediments 369

1.5 2.0 2.0

(a) (b) (c)
1.5
1.0 1.0
1.0
0.5
Mean PC1

Mean PC2

Mean PC3
0.0 0.5
0.0 0.0
1.0 0.5
0.5
1.0
1.0 2.0
1.5
1.5 3.0 2.0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Sediment Sediment Sediment

2.0 1.5 1.5

(d) (e) (f)
1.5 1.0 1.0
1.0 0.5 0.5
Mean PC4

Mean PC5

Mean PC6
0.5 0.0 0.0
0.0 0.5 0.5
0.5 1.0 1.0
1.0 1.5 1.5
1.5 2.0 2.0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Sediment Sediment Sediment

Figure 6. Line graphs of mean principal component scores showing interactions between sediments and methods. The first six principal
component scores are shown. PC1 to PC6 are shown by (a) to (f), respectively. Solid lines represent the PolStd method and broken lines
represent the HLFPol method.

oxidation may also account for the destruction of
the unidentified type (Unknown 1) in the sediment 1
PolStd residues. However, since sediment 1 was
derived from topsoil with the shortest exposure to
weathering, destruction of this type may have been due
to the composition of its exine and its inability to resist
acetolysis and treatment with other strong acids.
Inadequate disaggregation
Sediment 1, derived from topsoil, represents the most
recently-deposited sediment and thus has probably
been exposed to least weathering. It also contains the
highest pollen and spore concentrations. The HLFPol
residues contain, however, significantly lower con-
centrations, suggesting that insucient sediment dis-
aggregation by the HLFPol procedure resulted in
palynomorphs being trapped in the colloidal matrices,
consequently sinking during heavy liquid flotation, and
thus being discarded with the heavy sediment fraction.
Poor disaggregation was also evident in the phytolith
extractions with significantly lower ratios of biogenic
silica to aggregate material for HLFPol samples
(Lentfer, 1997). To increase disaggregation, and thus
produce full pollen and spore spectra from highly-
organic sediments, additional treatments should be
included in heavy liquid flotation procedures to ensure
organic and colloid breakdown. These may entail
acetolysis or oxidation prior to flotation, although
losses of pollen and spores are likely to increase.
Oxidation, especially, should be applied with care
and the chosen method be as mild as possible. Phipps
& Playford (1984) recommended that no or only very
mild oxidation be used with samples containing light-
coloured palynomorphs associated with small amounts
of organic debris. Where samples have dark-coloured
palynomorphs accompanied by conspicuous amounts
of organic debris, stronger oxidation would be necess-
ary. However, it is safer to under-oxidize rather than
over-oxidize. Of the many oxidants used by palynolo-
gists (e.g., Erdtman & Erdtman, 1933; Brown, 1960;
Gray, 1965; Forster & Flenley, 1989; Moore, Webb &
Collinson, 1991), acid oxidants are preferred to alka-
line oxidants, since exines are more easily broken down
in the latter. The commonly-used oxidant, Schulze
solution (KClO3 and HNO3), is considered too strong
for Quaternary material, and Erdtman & Erdtman
(1933) used another chloric acid solution (5 or 6 drops
of saturated NaClO3 and 1 ml concentrated HCI). The
reaction is violent and should proceed for a few
seconds only. Phipps & Playford (1984) recommended
the use of concentrated HNO3 (carried out in a beaker
rather than centrifuge tube to avoid spillage from
violent reactions). This reaction takes longer to com-
plete than oxidation with chloric acids, and KOH can
be added to neutralize the acid and stop the reaction.
370 C. J. Lentfer and W. E. Boyd

4 over- or under-representation, reliability would be

(a) 4b
dependent on equivalent percentage losses for all pol-
3 len and spore types. Moore, Webb & Collinson (1991)
and Phipps & Playford (1984) note that better disper-
4a
2
1b
sion of particles occur if residues are washed with
1b
non-ionic detergents prior to flotation. Phipps &
1 4a
5b 4b4b 1a
1a
1a Playford (1984) also note that aggregation occurs less
PC2

5a
5b 5b 4b 4a
4a
2b
2a
1a 1b 2b frequently following nitric acid treatment. It is poss-
0 5b 5a
5a 5a 3b 3a
2a
1b 2a 2a
2b
ible, therefore, that aggregation losses can be reduced
3a
6a
6a 3b
3a 2b
by additional processing with detergents, oxidation
1 6b 6a 6a3b and focused microwave digestion. Furthermore, where
3a
6b
6b
silica particles aggregate around palynomorphs on
2 6b slides, and thus prevent pollen and spore identification,
subsamples could be taken and treated with HF. For
3 2 1 0 1 2 dicult sediments, HLFPol extraction can be used to
PC1 determine pollen and spore presence. To ensure reliable
results, however, separate extractions specific to pollen
0.8
(b)
SP1 and spores may be necessary.
0.6 LIL
SP3 Dierential loss of palynomorphs during flotation
MYRT1
0.4 SP4
For HLFPol residues, silica aggregation around pollen
FAB
and spores causes identification and counting dicul-
VPC2

0.2 EUPHORB CYATH UN1 SP2

CAS
PLANTAG UN3
PO.C.RS
MYRT2 AST UN2
0.0
0.2
UN4
URT.MOR
0.4
0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8
VPC1
Figure 7. Example of biplot showing the first and second principal
components of pollen and spore assemblages extracted by the two
extraction methods (a) PolStd and (b) HLFPol. PC 1 explains 294%
of variation in the assemblages and contrasts samples according to
regional variation. PC2 explains 164% of the variation and contrasts
6b samples that have relatively high numbers on Urticaceae/
Moraceae (URT.MOR) pollen and the unknown pollen type (UN4)
with sediments 4 and 5 that have higher numbers of Liliaceae (LIL)
pollen and the spore identified as SP4. Distribution of samples is
comparable between the two methods for all sediments except
sediment 6. PCs 3, 4, 5 and 6 (not shown here) explain 117%, 94%,
66% and 60% of the variation in the assemblages, respectively.
Faegri & Iversen (1989), however, advise that nitric
acid in concentrations above 15% can completely
destroy exines and, thus, acid concentration, tempera-
ture, and reaction time should be adapted to individual
samples. Finally, focused microwave digestion is able
to oxidize samples more thoroughly using relatively
weak oxidants (Jones, 1994; Jones & Ellin, 1998; Jones,
1998), and thus this technique may prove successful for
extracting pollen and spores with heavy liquid flotation
from organic-rich sediments.
Aggregation and subsequent loss of pollen and
spores could be problematical for heavy liquid flo-
tation, although as indicated here, pollen and spore
concentrations can be reliably estimated. To avoid
ties. It may also aect flotation during extraction if
pollen and spore flotation is inhibited by aggregation;
pollen and spore losses due to clumping have been
discussed by Moore, Webb & Collinson (1991). Al-
though the evidence here is unclear, aggregation during
phytolith extraction may be attributed to the low
pollen and spore counts recorded for sediment 3. The
abundance of rounded amorphous biogenic silica in
the sediment 3 HLFPol residue (Lentfer, 1997; Lentfer
& Boyd, 1998), may reflect under-representation of free
small, flattened particles; the latter may have formed
aggregates which sank dierentially during flotation,
and were thus discarded with heavier particles.
Other issues
It is apparent from the above that the variety of
sediment types and palynomorphs require a variety of
extraction procedures to ensure greatest possible pollen
and spore concentrations. Disadvantages in both the
PolStd and the HLFPol methods are indicated. Extra
treatments, such as acetolysis and oxidation, may
result in more thorough disaggregation of sediments
and could improve heavy liquid flotation for both
pollen and phytolith extraction, especially for sedi-
ments with high organic content. However, even with-
out these treatments, heavy liquid flotation has proved
to be useful, producing results comparable to the
standard pollen extraction for course-and fine-grained
sediments. It should also be noted that procedures
employing both acetolysis and bleaching have resulted
in almost 100% destruction of some pollen and spores
(Hafsten, 1959); these treatments should not, therefore,
be used together.
The swelling eect of acetolysis on pollen and spores
has been well-documented (e.g., Cain, 1944; Brown,
 Simultaneous Extraction of Phytoliths, Pollen and Spores from Sediments 371
1960; Reitsma, 1969; Martin, 1973; Moore, Webb &
Collinson 1991) and it has been noted that pollen
prepared by acetolysis shows more sculpturing and
structural detail than that prepared otherwise (Brown,
1960). While this could be advantageous for identifi-
cations of pollen and spores in some situations, exine
features can be altered, making enlarged grains dicult
to identify. Since these problems can be avoided by
treating type and fossil material in precisely the same
way (Moore, Webb & Collinson, 1991), palynologists
using both heavy liquid flotation and standard pollen
extraction may require access to variously-treated type
material or knowledge of correction factors specific to
various treatments (Martin, 1973).
Conclusion
Although the use of heavy liquid extraction for pollen
and spore analyses has been well documented, it has
not been a favoured technique. This is due, partly, to
the potential for bias resulting from the retention of
material that may obscure pollen and spores, and
partly to dierential losses during particle separation
and flotation. It is also related to the expense and often
high toxicity of the heavy liquids. The availability of a
relatively new, non-toxic heavy salt, sodium polytung-
state (Munsterman & Kestholt, 1996), and new tech-
niques such as focused microwave digestion, may
revolutionize pollen extraction by allowing extraction
with minimum exposure to dangerous chemicals.
Heavy liquid is already used for starch extraction
(Therin, 1997) as well as phytolith extraction (Lentfer,
Gojak & Boyd, 1997; Hart, 1992), and could be used
with HF for highly siliceous sediments. This study
shows that for most of the sediments tested, heavy
liquid extraction produced results comparable to
standard pollen extraction; for oxidized sediments,
especially, more reliable results are likely to be
obtained using heavy liquid extraction rather than
acetolysis. Overall, heavy liquid extraction allows
complementary suites of data to be investigated with
the least cost and eort. It has, therefore, the potential
to allow palynologists and phytolithologists to adopt
more opportunistic research practices and thereby
increase information bases for environmental
reconstruction.
Acknowledgements
Sample collection in Papua New Guinea was
supported by grants from the Australian Research
Council. The laboratory research described here was
supported in part by a grant from the Australian
Research Council and largely by a Southern Cross
University Research Scholarship held by Lentfer.
Maria Cotter kindly supplied the southeast
Queensland sediment samples and details of site char-
acteristics. The authors wish to acknowledge Glen
Fredlunds and Georey Playfords comments on an
earlier version of this paper.
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