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Simultaneous Extraction of Phytoliths, Pollen and Spores from Sediments
Journal of Archaeological Science (2000) 27, 363–372 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 offer 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 different types of microfossils provide valuable palaeoenvironmental information, the use of separate extraction methods for different 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 microfossils—pollen, spores and phytoliths—by 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 effort, thus enabling palynologists and phytolithologists to adopt more effective 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 pollen analysis (Pearsall & Trimble, 1984; Piperno, Bush & Colinvaux, 1991; Rossen, 1994; Rovner, 1988; Lentfer & Boyd, 1998), and thus offers important archaeological site- and function-specific detail. However, 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 diversifying microfossil catchment areas, it enhances
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 different sedimentological circumstances. Consequently, 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 typically require separate extractions, which may be both time- and resource-consuming. This paper examines the possibility of combining preparation techniques for three commonly encountered microfossils—pollen, spores and phytoliths. *For correspondence. Tel: [61–66] 203 007; Fax: [61–66] 21 2669; Email: [email protected]
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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 dissolved 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 extraction to ensure optimum pollen and spore concentrations. 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. Secondly, it provides assessment of possible additional treatments to concentrate palynomorphs. Thus, a multidisciplinary approach can be achieved with little extra cost and effort. 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 procedure (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 oxidation but including an alkali treatment, similar to the other techniques described for pollen and spore extraction (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 method’s efficacy at concentrating pollen and spores and providing accurate representations of original populations, pollen counts from HLFPol residues used for phytolith analysis (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 (..=3·9%) 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 efficient pollen extraction. The heavy liquid used was cadmium iodide and potassium iodide at 2·35 specific gravity. Samples were stained prior to mounting as per standard pollen techniques, and no evaluation of staining effects 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 Stockmarr’s (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 estimate 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 effect 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 principal component scores and pairwise comparisons using the Bonferroni method to calculate critical mean differences at =0·05, determined significant differences 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
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variance and therefore all variables were included in principal components analysis.
crush sediment with pestle and mortar
Results measure 1 cc sediment
disaggregate sediment, shake 12 hours in 5% Calgon solution
alkali digestion, boil 5 min in 10% KOH sol'n
remove carbonates in 15% HCl
sieve through 250 µm mesh
sieve through 100 µm mesh
remove clays, deflocculate in 5% Calgon sol'n, centrifuge and decant, repeat until supernatant clears
separate light and heavy fraction with heavy liquid flotation
dissolve silica in hydrofluoric acid
remove cellulose with acetolysis
stain with safranin
dehydration with 50% and 100% ethanol
dehydration with TBA
mount in silicone oil
Morphology and clarity 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, palynomorphs from HLFPol residues exposed to heavy liquid separation and dehydrated in ETOH (ethyl 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 effects of grain swelling and contraction related to 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 inferior to that of PolStd residues for all sediments. Pollen and spores were often obscured by small silica particles apparently attracted to them. This attraction was observed during slide mounting; palynomorphs clearly visible immediately following their application to slides were often obscured by silica shortly afterwards. Such aggregation occurred particularly with more viscous media (silicone oil and glycerol) and was much reduced in water. Information retrieval and pollen and spore concentrations Two-way ANOVA tests and pairwise comparisons showed that significant sedimentmethod interaction occurred at =0·05 for: (i) total pollen and spores (including Alnus) count/h; (ii) total pollen and spores (excluding Alnus) count/h; and (iii) total concentrations of pollen and spores (excluding Alnus) in 1 cc sediment. Counts/h (including Alnus) were equivalent for sediments 1, 2, 4 and 5, but were significantly greater from PolStd residues than HLFPol residues for sediments 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
Figure 1. Flowchart summarizing the two pollen extraction methods used in this study: the heavy liquid extraction procedure (HLFPol) and a standard pollen extraction procedure (PolStd). HLFPol 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, acetolysis to remove cellulose and TBA (tertiary butyl alcohol) for dehydration. Heavy liquid flotation is used to separate microfossils from heavy mineral fractions in HLFPol samples and ethyl alcohol is used for dehydration.
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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 1 2 3 4 5 6
Location
Sample description
Deception Bay, southeast Queensland, Australia Coastal heath dominated by Melaleuca quinquenervia (Cav.) S. T. Blake, Blechnum indicum Burm. f., exotic pine and grass Deception Bay, southeast Queensland, Australia Disturbed coastal herbland/shrubland dominated by Phragmites australis (Cav.) Trin., Blechnum indicum and Melaleuca quinquenervia Deception Bay, southeast Queensland, Australia Coastal shrubland dominated by Melaleuca quinquenervia, Blechnum indicum, Phragmites australis, and Imperata cylindrica (L.) Beauv. Paligmete Village, Pililo, south West New Britain, Papua New Guinea Village site with Cocos nucifera (L.), tropical regrowth forest and grass Paligmete Village, Pililo, south West New Britain, Papua New Guinea Village site with Cocos nucifera, tropical regrowth forest and grass Garua Island, north West New Britain, Papua New Guinea Coconut plantation with Cocos nucifera, tropical regrowth forest, ferns 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 different at =0·05. Although most pollen and spore types were present in residues extracted by both methods, there were differences in type presence/ absence between samples. The most notable discrepancies occurred in favour of the HLFPol residues (i.e., types not recorded in PolStd residues were relatively common in HLFPol residues). For sediment 6, Euphorbiaceae pollen was present in HLFPol residues but absent 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 differences 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, 79·5% was explained by the first six principal components. Distribution patterns of samples for methods within sediments, according to PC scores based on the transformed pollen and
Dark brown sandy clay loam, pH 4·17 sample from topsoil, quartz and feldspar Black humic sand, pH 4·28 sample from depth 80–100 cm, quartz and disordered kaolinite Yellow white clay loam, pH 3·79 sample from depth 25–45 cm, quartz, feldspar and montmorillonite Brown clay, pH 7·5 sample from depth 35–40 cm, quartz and well ordered kaolinite Black smectitic clay complex derived from midden matrix, pH7·10 sample from depth 10–15 cm, calcite and quartz Red brown clay, pH 7·13 sample from depth 170–180 cm, moderately kaolinite
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 Euphorbiaceae 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 comparable distributions for both methods. Sediment 1 samples are also contrasted by PC5 but to a lesser degree, and variation between methods for this sediment is not significant. Nevertheless, sample differences 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 extraction 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 nevertheless substantial evidence for differential selection of fossil palynomorphs between the two extraction methods. The major causes for such differential selection are associated with: (i) differential destruction of palynomorphs; (ii) sediment variability and insufficient
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disaggregation for the least oxidized sediments; and (iii) possible differential loss of palynomorphs during flotation. Differential 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
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reasonable, thus, to assume that acetolysis and HF treatment were largely responsible for the differential 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 weathering (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
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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 concentrations, suggesting that insufficient sediment disaggregation 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 highlyorganic 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 lightcoloured 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 necessary. However, it is safer to under-oxidize rather than over-oxidize. Of the many oxidants used by palynologists (e.g., Erdtman & Erdtman, 1933; Brown, 1960; Gray, 1965; Forster & Flenley, 1989; Moore, Webb & Collinson, 1991), acid oxidants are preferred to alkaline 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 complete than oxidation with chloric acids, and KOH can be added to neutralize the acid and stop the reaction.
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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 29·4% of variation in the assemblages and contrasts samples according to regional variation. PC2 explains 16·4% 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 11·7%, 9·4%, 6·6% and 6·0% 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, temperature, 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 flotation, although as indicated here, pollen and spore concentrations can be reliably estimated. To avoid
over- or under-representation, reliability would be dependent on equivalent percentage losses for all pollen and spore types. Moore, Webb & Collinson (1991) and Phipps & Playford (1984) note that better dispersion of particles occur if residues are washed with non-ionic detergents prior to flotation. Phipps & Playford (1984) also note that aggregation occurs less frequently following nitric acid treatment. It is possible, therefore, that aggregation losses can be reduced by additional processing with detergents, oxidation and focused microwave digestion. Furthermore, where silica particles aggregate around palynomorphs on slides, and thus prevent pollen and spore identification, subsamples could be taken and treated with HF. For difficult sediments, HLFPol extraction can be used to determine pollen and spore presence. To ensure reliable results, however, separate extractions specific to pollen and spores may be necessary. Differential loss of palynomorphs during flotation For HLFPol residues, silica aggregation around pollen and spores causes identification and counting difficulties. It may also affect 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). Although 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 differentially 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 sediments with high organic content. However, even without 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 effect 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 identifications of pollen and spores in some situations, exine features can be altered, making enlarged grains difficult 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 differential 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 polytungstate (Munsterman & Kestholt, 1996), and new techniques 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 effort. 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 characteristics. The authors wish to acknowledge Glen
Fredlund’s and Geoffrey Playford’s comments on an earlier version of this paper.
References Andersen, S. T. (1986). Palaeoecological studies of terrestrial soils. In (B. E. Berglund, Ed.) Handbook of Holocene Palaeoecology and Palaeohydrology. Chichester: John Wiley, pp. 165–177. Brande, A. (1976). Zur Anwendung der Schweretrennung in der Pollenanalyse. Flora 165, 95–101. Brown, C. A. (1960). Palynological techniques. Baton Rouge: Brown. Cain, S. A. (1944). Size-frequency characteristics of Abies fraseri pollen as influenced by different methods of preparation. American Midland Naturalist 31, 232–236. Cotter, M. M. (1996). Holocene environmental change in Deception Bay, Southeast Queensland: A palaeogeographical contribution to MRAP Stage II. Tempus 6, 193–205. Dricot, E. & Leroy, S. (1989). Peptization and sieving for palynological purposes. Geobound 2, 114–126. Erdtman, G. (1952). Pollen Morphology and Plant Taxonomy. Angiosperms: An Introduction to Palynology, 1. Stockholm: Almqvist and Wiksell. Erdtman, O. G. E. & Erdtman, H. (1933). The improvement of pollen analysis technique. Svenske Botaniske Tidskrift 27, 347–357. Faegri, K. & Iversen, J. (1989). Textbook of Pollen Analysis. Chichester: John Wiley. Forster, R. M. & Flenley, J. R. (1989). The application of density gradient centrifugation to palynology. Report, School of Geography and Earth Resources, University of Hull. Fredlund, G. (1986). Problems in the simultaneous extraction of pollen and phytoliths from clastic sediments. In (I. Rovner, Ed.) Plant Opal Phytolith Analysis in Archaeology and Palaeoecology: Occasional Papers No. 1 of the Phytolitharien. Raleigh: North Carolina State University, pp. 12–23. Frey, G. D. (1955). A differential flotation technique for recovering microfossils from inorganic sediments. New Phytologist 54, 257–258. Gordon, A. D. (1982). Numerical methods in Quaternary palaeoecology. V. Simultaneous graphical representation of the levels and taxa in a pollen diagram. Review of Palaeobotany & Palynology 37, 155–183. Gray, J. (1965). Extraction techniques. In (B. Kummel & D. Raup, Eds) Handbook of Paleontological Techniques. San Francisco: W. H. Freeman, pp. 530–587. Hafsten, U. (1959). Bleaching+HF+acetolysis: a hazardous preparation process. Pollen et Spores 1, 77–79. Hart, D. M. (1992). A field appraisal of the role of plant opal in the Australian environment. PhD Thesis. Macquarie University. Hunt, C. O. (1985). Recent advances in pollen extraction techniques: A brief review. In (N. R. J. Fieller, D. D. Gilbertson & N. G. A. Ralph, Eds) Palaeobiological Investigations: Research Design, Methods and Data Analysis. Oxford: BAR International Series, 266, pp. 181–188. Johnson, W. C. & Fredlund, G. (1985). A procedure for extracting palynomorphs (pollen and spores) from clastic sediments. Transactions of the Kansas Academy of Science 88, 51–58. Jones, R. A. (1994). The application of microwave technology to the oxidation of kerogen for use in palynology. Review of Palaeobotany & Palynology 80, 333–338. Jones, R. A. (1998). Focused microwave digestion and the oxidation of palynological samples. Review of Palaeobotany & Palynology 103, 17–22. Jones, R. A. & Ellin, S. T. (1998). Improved palynological sample preparation using an automated focused microwave digestion system. American Association of Stratigraphic Palynology Contribution Series 33. Kealhofer, L. (1996). The human environment during the terminal Pleistocene and Holocene in northeastern Thailand: Phytolith evidence from Lake Kumphawapi. Asian Perspectives 35, 229–253.
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Kealhofer, L. & Piperno, D. R. (1994). Early agriculture in southeast Asia: Phytolith evidence from the Bang Pakong Valley, Thailand. Antiquity 68, 564–572. Kealhofer, L. & Penny, D. (1998). A combined pollen and phytolith record for fourteen thousand years of vegetation change in northeast Thailand. Review of Palaeobotany & Palynology 103, 83–93. Knox, E. M. (1942). The use of bromoform in the separation of noncalcareous microfossils. Science 95, 307–308. Lentfer, C. J. (1997). Extraction of phytoliths and other palynomorphs from sediments. Masters Thesis. Southern Cross University. Lentfer, C. J. & Boyd, W. E. (1998). A comparison of three methods for the extraction of phytoliths from sediments. Journal of Archaeological Science 25, 1159–1183. Lentfer, C. J., Gojak, D. & Boyd, W. E. (1997). Hope Farm Windmill: Phytolith analysis of cereals in early colonial Australia. Journal of Archaeological Science 24, 841–856. McIntyre, D. J. & Norris, G. (1964). Effect of ultrasound on recent spores and pollen. New Zealand Journal of Science 7, 242–257. Martin, H. A. (1973). Palynology and historical ecology of some cave excavations in the Australian Nullabor. Australian Journal of Botany 21, 283–316. Moore, P. D., Webb, J. A. & Collinson, M. E. (1991). Pollen Analysis. London: Blackwell. Munsterman, D. & Kestholt, S. (1996). Sodium polytungstate, a new non-toxic alternative to bromoform in heavy liquid separation. Review of Palaeobotany & Palynology 91, 417–422. Pearsall, D. M. & Trimble, M. (1984). Identifying past agricultural activity through soil phytolith analysis: A case study from the Hawaiian Islands. Journal of Archaeological Science 11, 119–133. Penny, D., Grindrod, J. & Bishop, P. (1996). Holocene palaeoenvironmental reconstruction based on microfossil analysis of a lake sediment core, Nong Kumphawapi, Udon Thani, northeast Thailand. Asian Perspectives 35, 209–227. Phipps, D. & Playford, G. (1984). Laboratory techniques for extraction of palynomorphs from sediments. Papers of the Geology Department, University of Queensland 11, 1–23.
Piperno, D. R. (1983). The application of phytolith analysis of the reconstruction of plant subsistence and environments in prehistoric Panama. Microfilm. Ann Arbor, University of Michigan. Piperno, D. R. (1985). Phytolith taphonomy and distributions in archaeological sediments from Panama. Journal of Archaeological Science 12, 247–267. Piperno, D. R. (1994). Phytolith and charcoal evidence for prehistoric slash-and-burn agriculture in the Darien rain forest of Panama. The Holocene 4, 321–325. Piperno, D. R., Bush, M. & Colinvaux, P. (1991). Paleoecological perspectives on human adaptation in central Panama, II. The Holocene. Geoarchaeology: An International Journal 6, 227–250. Piperno, D. R. & Clary, K. M. (1984). Early plant use and cultivation in the Santa Maria Basin, Panama: Data from phytoliths and pollen. In (F. W. Lange, Ed.) Recent Developments in Isthmian Archaeology: Advances in the Prehistory of Lower Central America. Oxford: BAR International Series, 212, pp. 85–121. Powers, A. H. & Gilbertson, D. D. (1987). A simple preparation technique for the study of opal phytoliths from archaeological and Quaternary sediments. Journal of Archaeological Science 14, 529–535. Reitsma, T. (1969). Size modification of recent grains under different treatments. Review of Palaeobotany & Palynology 9, 175–252. Rosen, A. (1994). Identifying ancient irrigation: A new method using opaline phytoliths from Emmer wheat. Journal of Archaeological Science 21, 125–132. Rovner, I. (1988). Micro- and macro-environmental reconstruction using plant opal phytolith data from archaeological sediments. Geoarchaeology: An International Journal 3, 155–165. Sittler, C. (1955). Methods et techniques physicochimiques de preparation des sediments en vue de leur analyse pollinque. Review d’Institut Franc¸ ais Petrole 10, 103–114. Stockmarr, J. (1972). Tablets with spores used in absolute pollen analysis. Pollen et Spores 13, 615–621. Therin, M. (1997). Starch protocol: Explanation. Report, Australian Museum. Wilson, S. M. (1985). Phytolith analysis at Kuk, an early agricultural site in Papua New Guinea. Archaeology in Oceania 20, 90–96.
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 offer 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 different types of microfossils provide valuable palaeoenvironmental information, the use of separate extraction methods for different 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 microfossils—pollen, spores and phytoliths—by 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 effort, thus enabling palynologists and phytolithologists to adopt more effective 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 pollen analysis (Pearsall & Trimble, 1984; Piperno, Bush & Colinvaux, 1991; Rossen, 1994; Rovner, 1988; Lentfer & Boyd, 1998), and thus offers important archaeological site- and function-specific detail. However, 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 diversifying microfossil catchment areas, it enhances
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 different sedimentological circumstances. Consequently, 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 typically require separate extractions, which may be both time- and resource-consuming. This paper examines the possibility of combining preparation techniques for three commonly encountered microfossils—pollen, spores and phytoliths. *For correspondence. Tel: [61–66] 203 007; Fax: [61–66] 21 2669; Email: [email protected]
363 0305–4403/00/050363+10 $35.00/0
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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 dissolved 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 extraction to ensure optimum pollen and spore concentrations. 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. Secondly, it provides assessment of possible additional treatments to concentrate palynomorphs. Thus, a multidisciplinary approach can be achieved with little extra cost and effort. 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 procedure (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 oxidation but including an alkali treatment, similar to the other techniques described for pollen and spore extraction (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 method’s efficacy at concentrating pollen and spores and providing accurate representations of original populations, pollen counts from HLFPol residues used for phytolith analysis (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 (..=3·9%) 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 efficient pollen extraction. The heavy liquid used was cadmium iodide and potassium iodide at 2·35 specific gravity. Samples were stained prior to mounting as per standard pollen techniques, and no evaluation of staining effects 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 Stockmarr’s (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 estimate 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 effect 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 principal component scores and pairwise comparisons using the Bonferroni method to calculate critical mean differences at =0·05, determined significant differences 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 measure 1 cc sediment
disaggregate sediment, shake 12 hours in 5% Calgon solution
alkali digestion, boil 5 min in 10% KOH sol'n
remove carbonates in 15% HCl
sieve through 250 µm mesh
sieve through 100 µm mesh
remove clays, deflocculate in 5% Calgon sol'n, centrifuge and decant, repeat until supernatant clears
separate light and heavy fraction with heavy liquid flotation
dissolve silica in hydrofluoric acid
remove cellulose with acetolysis
stain with safranin
dehydration with 50% and 100% ethanol
dehydration with TBA
mount in silicone oil
Morphology and clarity 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, palynomorphs from HLFPol residues exposed to heavy liquid separation and dehydrated in ETOH (ethyl 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 effects of grain swelling and contraction related to 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 inferior to that of PolStd residues for all sediments. Pollen and spores were often obscured by small silica particles apparently attracted to them. This attraction was observed during slide mounting; palynomorphs clearly visible immediately following their application to slides were often obscured by silica shortly afterwards. Such aggregation occurred particularly with more viscous media (silicone oil and glycerol) and was much reduced in water. Information retrieval and pollen and spore concentrations Two-way ANOVA tests and pairwise comparisons showed that significant sedimentmethod interaction occurred at =0·05 for: (i) total pollen and spores (including Alnus) count/h; (ii) total pollen and spores (excluding Alnus) count/h; and (iii) total concentrations of pollen and spores (excluding Alnus) in 1 cc sediment. Counts/h (including Alnus) were equivalent for sediments 1, 2, 4 and 5, but were significantly greater from PolStd residues than HLFPol residues for sediments 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
Figure 1. Flowchart summarizing the two pollen extraction methods used in this study: the heavy liquid extraction procedure (HLFPol) and a standard pollen extraction procedure (PolStd). HLFPol 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, acetolysis to remove cellulose and TBA (tertiary butyl alcohol) for dehydration. Heavy liquid flotation is used to separate microfossils from heavy mineral fractions in HLFPol samples and ethyl alcohol is used for dehydration.
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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 1 2 3 4 5 6
Location
Sample description
Deception Bay, southeast Queensland, Australia Coastal heath dominated by Melaleuca quinquenervia (Cav.) S. T. Blake, Blechnum indicum Burm. f., exotic pine and grass Deception Bay, southeast Queensland, Australia Disturbed coastal herbland/shrubland dominated by Phragmites australis (Cav.) Trin., Blechnum indicum and Melaleuca quinquenervia Deception Bay, southeast Queensland, Australia Coastal shrubland dominated by Melaleuca quinquenervia, Blechnum indicum, Phragmites australis, and Imperata cylindrica (L.) Beauv. Paligmete Village, Pililo, south West New Britain, Papua New Guinea Village site with Cocos nucifera (L.), tropical regrowth forest and grass Paligmete Village, Pililo, south West New Britain, Papua New Guinea Village site with Cocos nucifera, tropical regrowth forest and grass Garua Island, north West New Britain, Papua New Guinea Coconut plantation with Cocos nucifera, tropical regrowth forest, ferns 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 different at =0·05. Although most pollen and spore types were present in residues extracted by both methods, there were differences in type presence/ absence between samples. The most notable discrepancies occurred in favour of the HLFPol residues (i.e., types not recorded in PolStd residues were relatively common in HLFPol residues). For sediment 6, Euphorbiaceae pollen was present in HLFPol residues but absent 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 differences 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, 79·5% was explained by the first six principal components. Distribution patterns of samples for methods within sediments, according to PC scores based on the transformed pollen and
Dark brown sandy clay loam, pH 4·17 sample from topsoil, quartz and feldspar Black humic sand, pH 4·28 sample from depth 80–100 cm, quartz and disordered kaolinite Yellow white clay loam, pH 3·79 sample from depth 25–45 cm, quartz, feldspar and montmorillonite Brown clay, pH 7·5 sample from depth 35–40 cm, quartz and well ordered kaolinite Black smectitic clay complex derived from midden matrix, pH7·10 sample from depth 10–15 cm, calcite and quartz Red brown clay, pH 7·13 sample from depth 170–180 cm, moderately kaolinite
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 Euphorbiaceae 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 comparable distributions for both methods. Sediment 1 samples are also contrasted by PC5 but to a lesser degree, and variation between methods for this sediment is not significant. Nevertheless, sample differences 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 extraction 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 nevertheless substantial evidence for differential selection of fossil palynomorphs between the two extraction methods. The major causes for such differential selection are associated with: (i) differential destruction of palynomorphs; (ii) sediment variability and insufficient
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disaggregation for the least oxidized sediments; and (iii) possible differential loss of palynomorphs during flotation. Differential 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
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reasonable, thus, to assume that acetolysis and HF treatment were largely responsible for the differential 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 weathering (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
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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 concentrations, suggesting that insufficient sediment disaggregation 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 highlyorganic 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 lightcoloured 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 necessary. However, it is safer to under-oxidize rather than over-oxidize. Of the many oxidants used by palynologists (e.g., Erdtman & Erdtman, 1933; Brown, 1960; Gray, 1965; Forster & Flenley, 1989; Moore, Webb & Collinson, 1991), acid oxidants are preferred to alkaline 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 complete than oxidation with chloric acids, and KOH can be added to neutralize the acid and stop the reaction.
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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 29·4% of variation in the assemblages and contrasts samples according to regional variation. PC2 explains 16·4% 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 11·7%, 9·4%, 6·6% and 6·0% 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, temperature, 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 flotation, although as indicated here, pollen and spore concentrations can be reliably estimated. To avoid
over- or under-representation, reliability would be dependent on equivalent percentage losses for all pollen and spore types. Moore, Webb & Collinson (1991) and Phipps & Playford (1984) note that better dispersion of particles occur if residues are washed with non-ionic detergents prior to flotation. Phipps & Playford (1984) also note that aggregation occurs less frequently following nitric acid treatment. It is possible, therefore, that aggregation losses can be reduced by additional processing with detergents, oxidation and focused microwave digestion. Furthermore, where silica particles aggregate around palynomorphs on slides, and thus prevent pollen and spore identification, subsamples could be taken and treated with HF. For difficult sediments, HLFPol extraction can be used to determine pollen and spore presence. To ensure reliable results, however, separate extractions specific to pollen and spores may be necessary. Differential loss of palynomorphs during flotation For HLFPol residues, silica aggregation around pollen and spores causes identification and counting difficulties. It may also affect 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). Although 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 differentially 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 sediments with high organic content. However, even without 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 effect 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 identifications of pollen and spores in some situations, exine features can be altered, making enlarged grains difficult 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 differential 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 polytungstate (Munsterman & Kestholt, 1996), and new techniques 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 effort. 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 characteristics. The authors wish to acknowledge Glen
Fredlund’s and Geoffrey Playford’s comments on an earlier version of this paper.
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