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Journal of Archaeological Science (2000) 27, 363372

doi:10.1006/jasc.1998.0374, available online at on

Simultaneous Extraction of Phytoliths, Pollen and Spores from

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

(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



Introduction Phytolith analysis potentially documents more

detailed and local patterns of vegetation change than

icrofossil analysis from archaeological pollen analysis (Pearsall & Trimble, 1984; Piperno,
sediments often provides a basis for Bush & Colinvaux, 1991; Rossen, 1994; Rovner, 1988;
palaeoenvironmental reconstructions. Given Lentfer & Boyd, 1998), and thus oers important
common variation in on-site conditions, preservational archaeological site- and function-specific detail. How-
state of various microfossils also varies. Fossil pollen, ever, phytolithologists are increasingly recognizing the
phytoliths and diatoms, for example, are best preserved weakness of using one microfossil class alone for
under dierent sedimentological circumstances. Conse- palaeoenvironmental reconstruction, and frequently
quently, a range of preparation techniques is now rely on complementary pollen analyses (Piperno, 1983,
commonly used. These largely involve the gradual 1985, 1994; Piperno & Clary, 1984; Wilson, 1985;
removal of unwanted sediment components, either by Kealhofer & Piperno, 1994 Kealhofer, 1996; Penny,
dissolution or physical separation; in pollen analysis, Grindrod & Bishop, 1996; Kealhofer & Penny, 1998).
preparation also involves the removal of remaining This approach facilitates more precise reconstruction
pollen intine. While each class of microfossil provides in several ways. First, it may involve plant taxa lacking
valuable palaeoenvironmental information, they typi- diagnostic phytoliths but with diagnostic pollen (e.g.,
cally require separate extractions, which may be both members of the Araceae family) and vice versa (e.g.,
time- and resource-consuming. This paper examines members of the Poaceae family). Secondly, it increases
the possibility of combining preparation techniques the value of microfossil data bases by using phytoliths
for three commonly encountered microfossilspollen, for identification of specific plant parts. Thirdly, it
spores and phytoliths. provides a means to check identifications of eroded
fossils. Fourthly, it allows inclusion of plants types
*For correspondence. Tel: [6166] 203 007; Fax: [6166] 21 2669; with various dispersal mechanisms. Finally, by di-
Email: versifying microfossil catchment areas, it enhances
03054403/00/050363+10 $35.00/0  2000 Academic Press
364 C. J. Lentfer and W. E. Boyd

assessment of changing past vegetation patterns at with those for residues prepared using the PolStd
both regional and local levels. procedure.
Although extraction methods capable of extracting
pollen and spores and siliceous microfossils were
developed over 50 years ago (e.g., Knox, 1942; Frey, Methods
1955; Hunt, 1985; Fredlund, 1986; Moore, Webb &
Collinson, 1991), these have not been widely adopted Extraction and counting procedures
by phytolithologists or other palynologists. Instead, Four replicates were run for each sediment/method
most analysts use separate procedures. Penny, combination (Figures 1, 2 & 3; Table 1). Sediments
Grindrod & Bishop (1996), for instance, extracted were initially ground with a pestle and mortar, and
pollen using hydrofluoric acid but extracted phytoliths cone quartered (Powers & Gilbertson, 1987) to sample
using liquid flotation and a strong oxidizing agent from 1 cc of each. An aliquot (Powers & Gilbertson, 1987)
lake and swamp sediments. Thus all silica was dis- of 24,559 Alnus pollen grains (..=39%) was added to
solved by HF in the pollen extractions, and pollen and each of the samples for determination of absolute
spores were destroyed by the strong oxidation in the frequency counts. The sediments were sieved through
phytolith extractions. The adoption of separate 250 m and 100 m; the 250 m sieve was used to
procedures can be advantageous for some situations extract as full an assemblage of phytoliths as possible,
such as the preparation of (i) organic-rich sediments whereas the 100 m sieve allowed more ecient pollen
requiring harsh oxidation treatment to release extraction. The heavy liquid used was cadmium iodide
phytoliths from the organic matrix (Lentfer, 1997; and potassium iodide at 235 specific gravity. Samples
Lentfer & Boyd, 1998), or (ii) samples where pollen were stained prior to mounting as per standard pollen
analysis is of prime importance, necessitating extrac- techniques, and no evaluation of staining eects has
tion to ensure optimum pollen and spore concen- been made. Slides were viewed at 400magnification
trations. However, where research focuses primarily on using an optical microscope fitted with a polarizing
phytolith analysis, advantages can be gained by using lens. Non-overlapping transects were viewed and all
single extraction procedures. First, single extraction pollen, spores and phytoliths encountered along
allows pollen and spore presence in sediments to be transects were classified and counted. Counting time
monitored prior to or during phytolith counting. Sec- was limited to 1 h per sample. Concentration of pollen
ondly, it provides assessment of possible additional and spores (microfossils per cm3) was calculated
treatments to concentrate palynomorphs. Thus, a using Stockmarrs (1972) formula: total fossil pollen=
multidisciplinary approach can be achieved with little (fossil pollen countedtotal number markers)/
extra cost and eort. Also, the risk of ignoring valuable (markers counted).
information based on assumptions of unsuitable site
conditions for specific microfossil preservation can be
avoided. Statistical analysis
In this paper, pollen extraction using a heavy Two-way ANOVA and pairwise comparisons were
liquid extraction procedure (abbreviated to HLFPol used for analysis of raw count and concentration data.
in this paper) and a standard pollen extraction pro- To stabilize variance for multivariate analysis, absolute
cedure (PolStd) with HF and acetolysis treatments frequency data was transformed using the arc tan
are compared. The HLFPol technique has been transformation described by Gordon (1982): Xik =arc
shown to be successful for phytolith extraction (see tan (yik/v)1/2, where, Xik denotes the value of the
Lentfer, 1997; Lentfer & Boyd, 1998). If, therefore, transformed variable, yik denotes the calculated esti-
it can also be used for pollen and spore extraction, mate for the number of grains of the kth taxon per
it may be most useful for microfossil extraction at cubic centimetre and v denotes the number of exotic
archaeological sites. The procedure is standard heavy grains added to the sample (i.e., Alnus grains). The
liquid flotation extraction for phytoliths, without oxi- eect of this transformation is to reduce the range of
dation but including an alkali treatment, similar to the values to between 0 and /2. Transformed data were
other techniques described for pollen and spore extrac- analysed using Excel and SPSS programs. Principal
tion (Frey, 1955; Sittler, 1955; Stockmarr, 1972; component analysis (PCA) with Euclidean distance
Brande, 1976; Johnson & Fredlund, 1985; Dricot & measures established patterns of variation within and
Leroy, 1989; Faegri & Iversen, 1989; Moore, Webb & between treatments, and biplots were constructed for
Collinson, 1991). It has the ability to extract various all principal components (PCs) with eigenvalues over 1.
microfossils (including pollen, spores, phytoliths, Two-way analysis of variance (ANOVA) tests on prin-
diatoms and sponge spicules) from sediments in a cipal component scores and pairwise comparisons
single process. To assess this methods ecacy at using the Bonferroni method to calculate critical mean
concentrating pollen and spores and providing ac- dierences at =005, determined significant dier-
curate representations of original populations, pollen ences between the two extraction methods. The use of
counts from HLFPol residues used for phytolith analy- absolute frequency data and transformation reduced
sis (Lentfer, 1997; Lentfer & Boyd, 1998) are compared 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

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

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

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

20 200

+ +



+ +
Pteridophytes/Spores 100

+ +

+ +

Herbs Unknown

50 40
Herbs 20



100 40
Trees and Shrubs Trees and Shrubs 20


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

50 Unknown 1
+ + +


Restionaceae 20
Liliaceae cf.


Asteraceae Liliaceae cf.


+ +
40 Plantaginaceae

Euphorbiaceae 20 Asteraceae


Fabaceae Euphorbiaceae 20



Casuarinaceae 20
Myrtaceae 2

150 Urticaceae/Moraceae


100 Myrtaceae 2


Myrtaceae 1 20
50 Myrtaceae 1



HLFPol Samples
PolStd. Samples
Figure 5. Pollen and spore counts for HLFPol residues showing the
Figure 4. Pollen and spore counts for PolStd residues, showing total total count/type 10 3. Types with fewer than 1,000 grains/cc
count/type 10 3. Types with fewer than 1,000 grains/cc sediment sediment are shown by +.
are shown by +.
reasonable, thus, to assume that acetolysis and HF
disaggregation for the least oxidized sediments; and treatment were largely responsible for the dierential
(iii) possible dierential loss of palynomorphs during destruction of pollen and spores in PolStd samples
flotation. 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
Dierential destruction of palynomorphs likely that pollen and spores within these sediments
Selective destruction of pollen and spores by acetolysis have been exposed to relatively strong oxidative weath-
and oxidation is recorded elsewhere (e.g., Wenner, ering (Andersen, 1986). Also, since exines are more
1947 (cited in Brown, 1960); Erdtman, 1952; McIntyre readily oxidized in alkaline solutions (Faegri & Iversen,
& Norris, 1964; Dricot & Leroy, 1989; Faegri & 1989), the alkaline pH of these sediments would
Iversen, 1989). The composition of pollen and spore have contributed further to the oxidation process
assemblages depends in part on the ability of exines to (cf. Andersen, 1986). The Urticaceae/Moraceae and
withstand harsh chemical treatment, and the degree of Euphorbiaceae pollen and Spore 4 type, observed in
exposure of sediments to oxidative weathering prior to relatively high numbers in HLFPol residues, but absent
treatment. Destruction of pollen and spores occurred from PolStd residues, may have been previously
within the PolStd procedure which employed acetolysis degraded to the point where their exines were
to dissolve cellulose and HF to dissolve silica. It is 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)
1.0 1.0
Mean PC1

Mean PC2

Mean PC3
0.0 0.5
0.0 0.0
1.0 0.5
1.0 2.0
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 acetolysis or oxidation prior to flotation, although
the unidentified type (Unknown 1) in the sediment 1 losses of pollen and spores are likely to increase.
PolStd residues. However, since sediment 1 was Oxidation, especially, should be applied with care
derived from topsoil with the shortest exposure to and the chosen method be as mild as possible. Phipps
weathering, destruction of this type may have been due & Playford (1984) recommended that no or only very
to the composition of its exine and its inability to resist mild oxidation be used with samples containing light-
acetolysis and treatment with other strong acids. coloured palynomorphs associated with small amounts
of organic debris. Where samples have dark-coloured
palynomorphs accompanied by conspicuous amounts
Inadequate disaggregation of organic debris, stronger oxidation would be necess-
Sediment 1, derived from topsoil, represents the most ary. However, it is safer to under-oxidize rather than
recently-deposited sediment and thus has probably over-oxidize. Of the many oxidants used by palynolo-
been exposed to least weathering. It also contains the gists (e.g., Erdtman & Erdtman, 1933; Brown, 1960;
highest pollen and spore concentrations. The HLFPol Gray, 1965; Forster & Flenley, 1989; Moore, Webb &
residues contain, however, significantly lower con- Collinson, 1991), acid oxidants are preferred to alka-
centrations, suggesting that insucient sediment dis- line oxidants, since exines are more easily broken down
aggregation by the HLFPol procedure resulted in in the latter. The commonly-used oxidant, Schulze
palynomorphs being trapped in the colloidal matrices, solution (KClO3 and HNO3), is considered too strong
consequently sinking during heavy liquid flotation, and for Quaternary material, and Erdtman & Erdtman
thus being discarded with the heavy sediment fraction. (1933) used another chloric acid solution (5 or 6 drops
Poor disaggregation was also evident in the phytolith of saturated NaClO3 and 1 ml concentrated HCI). The
extractions with significantly lower ratios of biogenic reaction is violent and should proceed for a few
silica to aggregate material for HLFPol samples seconds only. Phipps & Playford (1984) recommended
(Lentfer, 1997). To increase disaggregation, and thus the use of concentrated HNO3 (carried out in a beaker
produce full pollen and spore spectra from highly- rather than centrifuge tube to avoid spillage from
organic sediments, additional treatments should be violent reactions). This reaction takes longer to com-
included in heavy liquid flotation procedures to ensure plete than oxidation with chloric acids, and KOH can
organic and colloid breakdown. These may entail 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-
sion of particles occur if residues are washed with
non-ionic detergents prior to flotation. Phipps &
1 4a
5b 4b4b 1a
1a Playford (1984) also note that aggregation occurs less

5b 5b 4b 4a
1a 1b 2b frequently following nitric acid treatment. It is poss-
0 5b 5a
5a 5a 3b 3a
1b 2a 2a
ible, therefore, that aggregation losses can be reduced
6a 3b
3a 2b
by additional processing with detergents, oxidation
1 6b 6a 6a3b and focused microwave digestion. Furthermore, where
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
SP1 and spores may be necessary.
0.6 LIL
SP3 Dierential loss of palynomorphs during flotation
0.4 SP4
For HLFPol residues, silica aggregation around pollen
and spores causes identification and counting dicul-


CAS ties. It may also aect flotation during extraction if
PO.C.RS pollen and spore flotation is inhibited by aggregation;
MYRT2 AST UN2 pollen and spore losses due to clumping have been
discussed by Moore, Webb & Collinson (1991). Al-
though the evidence here is unclear, aggregation during
0.2 phytolith extraction may be attributed to the low
URT.MOR pollen and spore counts recorded for sediment 3. The
0.4 abundance of rounded amorphous biogenic silica in
0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 the sediment 3 HLFPol residue (Lentfer, 1997; Lentfer
VPC1 & Boyd, 1998), may reflect under-representation of free
Figure 7. Example of biplot showing the first and second principal
small, flattened particles; the latter may have formed
components of pollen and spore assemblages extracted by the two aggregates which sank dierentially during flotation,
extraction methods (a) PolStd and (b) HLFPol. PC 1 explains 294% and were thus discarded with heavier particles.
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/ Other issues
Moraceae (URT.MOR) pollen and the unknown pollen type (UN4)
with sediments 4 and 5 that have higher numbers of Liliaceae (LIL) It is apparent from the above that the variety of
pollen and the spore identified as SP4. Distribution of samples is sediment types and palynomorphs require a variety of
comparable between the two methods for all sediments except extraction procedures to ensure greatest possible pollen
sediment 6. PCs 3, 4, 5 and 6 (not shown here) explain 117%, 94%,
66% and 60% of the variation in the assemblages, respectively.
and spore concentrations. Disadvantages in both the
PolStd and the HLFPol methods are indicated. Extra
treatments, such as acetolysis and oxidation, may
Faegri & Iversen (1989), however, advise that nitric result in more thorough disaggregation of sediments
acid in concentrations above 15% can completely and could improve heavy liquid flotation for both
destroy exines and, thus, acid concentration, tempera- pollen and phytolith extraction, especially for sedi-
ture, and reaction time should be adapted to individual ments with high organic content. However, even with-
samples. Finally, focused microwave digestion is able out these treatments, heavy liquid flotation has proved
to oxidize samples more thoroughly using relatively to be useful, producing results comparable to the
weak oxidants (Jones, 1994; Jones & Ellin, 1998; Jones, standard pollen extraction for course-and fine-grained
1998), and thus this technique may prove successful for sediments. It should also be noted that procedures
extracting pollen and spores with heavy liquid flotation employing both acetolysis and bleaching have resulted
from organic-rich sediments. in almost 100% destruction of some pollen and spores
Aggregation and subsequent loss of pollen and (Hafsten, 1959); these treatments should not, therefore,
spores could be problematical for heavy liquid flo- be used together.
tation, although as indicated here, pollen and spore The swelling eect of acetolysis on pollen and spores
concentrations can be reliably estimated. To avoid 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 & Fredlunds and Georey Playfords comments on an
Collinson 1991) and it has been noted that pollen earlier version of this paper.
prepared by acetolysis shows more sculpturing and
structural detail than that prepared otherwise (Brown,
1960). While this could be advantageous for identifi- References
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treating type and fossil material in precisely the same Brande, A. (1976). Zur Anwendung der Schweretrennung in der
way (Moore, Webb & Collinson, 1991), palynologists Pollenanalyse. Flora 165, 95101.
using both heavy liquid flotation and standard pollen Brown, C. A. (1960). Palynological techniques. Baton Rouge: Brown.
Cain, S. A. (1944). Size-frequency characteristics of Abies fraseri
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