Journal of Archaeological Science (2001) 28, 875886 doi:10.1006/jasc.2000.0623, available online at http://www.idealibrary.

com on

A Comparative Analysis of Wet and Dry Ashing Techniques for the Extraction of Phytoliths from Plant Material
J. F. Parr, C. J. Lentfer & W. E. Boyd*
School of Environmental Science & Management, Southern Cross University, Lismore, New South Wales 2480, Australia (Received 8 August 1999; revised manuscript accepted 5 October 2000)
Two methods are commonly used for the extraction of phytoliths from plant material to be used as reference in the analysis of archaeological phytolith samples: (1) spodograms or dry ashings; and (2) acid digestions or wet ashing. It has been suggested that these techniques may modify the resultant samples in dierent ways. Dry ashing, in particular, has been implicated as a cause of shrinkage and warping in phytolith assemblages when incineration occurs at d450 C. The results of a morphometric comparative analysis between the dry ashing and wet ashing methods do not support these claims. This study establishes that dierences in patterns of dimension and curvature of short bilobate phytoliths and of elongate phytoliths both subjected to dry and wet ash preparation are not statistically signicant. There is, therefore, no detectable evidence of morphological impact as a result of these methods. This nding implies that any dierences that do occur in phytolith size and curvature are typical, possibly random permutation within assemblages, or that they are the result of variation in leaf cell structure rather than the consequence of a particular extraction procedure. This suggests that the practice of using dierent methods of preparation of reference samples for fossil analysis can be reliably continued.  2001 Academic Press Keywords: PHYTOLITH ANALYSIS, SAMPLE PREPARATION METHODS, REFERENCE MATERIAL, DRY ASHING, WET ASHING, PHYTOLITH MORPHOLOGY, MORPHOLOGICAL CHANGE.

Introduction

arious methods have been employed for extracting phytoliths from plant material in the preparation of reference samples for archaeological fossil phytolith analysis. These may be broadly grouped into two main categories: the use of spodograms or dry ashing; and acid digestion or wet ashing (Royner, 1983: 238; Bowdery, 1989: 172174). Both approaches are well established, and rened variations of these techniques are currently widely used for the preparation of phytolith reference collections for plant classication, palaeoenvironmental reconstruction and archaeological applications. However, there is some suggestion that these techniques modify the resultant samples in dierent ways (Jones & Milne, 1963: 207 220; Lanning, Hopkins & Loera, 1980: 549554; Rovner, 1983: 238; Piperno, 1988: 126; Pearsall, 1989: 376). This paper reports on the results of a comparative analysis between the dry and wet ashing techniques. Phytolith research has become increasingly more important and widely used in the eld of palaeoenvironmental analysis (Brown, 1984; Piperno, 1985;

*Author for correspondence: School of Environmental Science & Management, Southern Cross University, Lismore, New South Wales 2480, Australia. Tel.: [61-2] 6620 3007; E-mail: bboyd@scu.edu.au

Piperno, Bush & Colinaux, 1991; Wang & Hill, 1995; Golyeva et al., 1995; Kealhofer & Penny, 1998; Kealhofer & Piperno, 1988), particularly in situations where pollen preservation is poor (Boyd, Lentfer & Torrence, 1998). The potential for phytolith analysis to contribute to the better understanding of prehistoric resource management, food processing and tool function is already evident (Rovner, 1983; Pearsall & Trimble, 1984; Wilson, 1985; Bozarth, 1987; Ball, Brotherson & Gardner, 1993; Piperno & Pearsall, 1993; Fullagar, 1993; Rosen, 1994; Pearsall et al., 1995; Kealhofer, 1996; Albert & Weiner, 1997; Owens, 1997; Field & Fullagar, 1998; Kealhofer, Torrence & Fullagar, 1999). Moreover, the role of phytolith research in plant taxonomy and physiology is well established, and although not as conspicuous perhaps as in other elds of research, it is becoming increasingly popular (Richardson, 1920; Cliord & Watson, 1977; Ollendorf, Mulholland & Ropp, 1988; Jones & Bryant, 1992; Ball, Brotherson & Gardner, 1996). The integrity of processed samples of fossil phytoliths is, therefore, fundamental to any research in the areas of palaeoenvironmental reconstruction and archaeological applications and, ultimately, the main criterion for any phytolith extraction technique must be that it will consistently provide morphologicallyintact phytoliths which accurately represent original
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assemblages (Lentfer & Boyd, 1998: 1159). Nevertheless, potential dierences in the preparation of phytoliths for analysis have been suggested to occur between extraction methods. In particular, the dry ashing technique has been implicated as a cause of shrinkage, warping and changes to the refractive index of phytoliths (Jones & Milne, 1963: 207220; Jones & Handreck, 1967: 125126; Pearsall, 1979: 146147, 1989: 375). Such an eect of the extraction method may seriously impede identication and accurate analysis of fossil phytoliths. Although this hypothesis has been questioned (Lanning, Hopkins & Loera, 1980: 550; Piperno, 1988: 126127; Runge, pers. comm.), there has been no systematic study carried out to test the assumption of these eects. Dry ashing was the original method employed to separate phytoliths from their surrounding organic matter. This technique has evolved from Mohls (1861) experimentation on the retrieval of phytoliths from the remnant ash of burnt plant material. Subsequent researchers, such as Schelenberg (1908), Molisch (1920), Richardson (1920) and Policard (1923), examined a range of dry ashing techniques (described by Uber, 1940: 204, and Netolitsky, 1929; both cited by Jones & Handreck, 1967: 125; Piperno, 1988: 126; Bowdery, 1989: 172). The procedures that are fundamental to dry ashing include the pre-treatment and/or posttreatments of samples by washing with distilled water and/or hydrochloric acid (HCl); additional products have also been used such as hydrogen peroxide (H2O2) (see Bowdery, 1989: 174). Samples are placed in crucibles, in a mue furnace and ignited at a desired temperature. Incineration rates are usually maintained at about 500 C (Stewart & Arthur, 1935: 905; Jones & Handreck, 1967: 125; Piperno, 1988: 126; Pearsall, 1989: 377). Nevertheless, Rovner (1983: 238) points out that higher temperatures up to 1000 C have been applied; an example is provided by Lanning, Ponnaiya & Crumpton (1958: 339) who have used temperatures in the range of 700900 C. The concept of wet ashing was introduced early this century by Zimmerman (1901), although it was not widely recognized until the late 1950s (e.g. Parry & Smithson, 1957: 976) and increased in popularity with the work of Jones & Milne (1963) and Rovner (1972). Wet ashing also requires the pre- and/or posttreatments described above, while the incineration stage is substituted by the use of acid digestion as a means of oxidation (e.g. Jones & Milne, 1963: 209). Bowdery (1989: 174175) provides tables outlining the ranges of temperatures, times and chemicals used in these techniques and their associated references. Over the last four decades there has been ongoing debate regarding the advantages and disadvantages of these techniques (Jones & Milne, 1963: 207220; Lanning, Hopkins & Loera, 1980: 549554; Rovner, 1983: 238; Piperno, 1988: 126; Pearsall, 1989: 376). Piperno (1988: 126127), for example, has questoned the need for concern in relation to the dry ashing

process, suggesting that previous research (e.g. Lanning, Hopkins & Loera, 1980: 550) demonstrated that if ring temperatures do not exceed 500 C, morphological changes in phytoliths do not occur. A recent study testing a range of temperatures on phytoliths from African arboreal species supports this proposition (Runge, E., pers. comm.). In contrast to the dry ashing debate, some researchers have found diculties in applying the wet ashing technique, in particular, commenting on diculties in the use of Schulze solution (a combination of nitric acid (HNO3) and potassium chlorate (KClO3) or sodium chlorate (NaClO3); (Pearsall, 1989: 381; McWeeney, pers. comm.). One of the authors (Lentfer) has also encountered complications with the use of sodium hypochlorite (bleach) for digestion. The problematic areas associated with wet oxidation appears to revolve around dierential digestion rates of phytoliths, cellulose, and other plant tissue by the chemicals used for wet ashing, and apparent loss of phytolith residue (Pearsall, 1989: 381). Rosen (pers. comm.) has found that dry ashing yields around 20% more phytoliths than wet ashing. Finally, a further area of concern for some phytolith researchers is the use of noxious chemicals necessary for the wet ashing process (Piperno, 1988: 126; Runge, pers. comm.). It is clear, however, that both techniques have their strengths and weaknesses. Their application will vary depending on the nature of the research question and the personal preferences of the analyst. Rosen (pers. comm.), for example, has suggested that dry ashing may be more applicable for comparative reference material in relaiton to archaeological sites because many of their phytoliths are retrieved from ash deposits. Alternatively, Rovner (pers. comm.) points out that not all phytoliths recovers from archaeological deposits have been subjected to re, and in these cases wet ashing is a more appropriate method. Following research initiated by Lentfer (1995), this paper examines both ashing techniques in order to assess the nature of evidence for phytolith modication, especially in regard to shrinkage and warping. A systematic comparative analysis of phytolith morphology after dry and wet ash extractions will assist in the understanding of their individual capacities, and most importantly, the ability of these extraction techniques to produce comparative results. It is considered essential amongst phytolith analysts that this issue be addressed to clarify any ambiguity that may aect phytolith analyses (Hodson, Rovner & Rosen, pers. comm.).

Methods
Both extraction methods applied in this assessment are based on frequently-used methodologies with the aim of allowing replication using standardized procedures for phytolith analysis. Leaf samples were cut up

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Table 1. Description of dry and wet ash phytolith extraction from plant material Dry ashing Extraction method using fume hood burning with distilled water, 10% HCl and 15% H2O2 for washing, the removal of carbonates, and organics respectively. (1) Weigh test tube and record. (2) Weigh dried plant material and record. (3) Rinse plant material in distilled water and transfer to crucible. (4) Make template for fume hood. (5) Transfer lidded crucibles to fume hood. (6) Heat to 500 C and hold for 6 h. (7) Switch o fume hood and leave overnight to cool. (8) Remove crucibles and transfer contents to test tubes. (9) Add 10 ml of 10% HCl to test tubeheat in water bath@70 C for 20 min or until reaction stops. (10) Centrifuge@3500 rpm for 5 min and decant. (11) Rinse with distilled water and centrifuge@3500 rpm for 5 min and decant. (12) Add 10 ml of 15% H2O2heat in water bath@70 C for 20 min or until reaction stops. (13) Centrifuge@3500 rpm for 5 min and decant. (14) Rinse with 10 ml of distilled water and centrifuge@3500 rpm for 5 min and decant. Repeat rinse. (15) Add 1 ml of ETOH and leave overnight to dry. (16) Weigh dried material, calculate phytolith weight and transfer to labelled vials with as little 100% ETOH as possible. Wet ashing Extraction method using Schulze solution i.e. nitric acid (HNO3) and potassium chlorate (KClO3). Washing with distilled water.

(1) Weigh test tube and record. (2) Weigh dried plant material and record. (3) Rinse plant material in distilled water and transfer to ask. (4) Add 10 mls of HNO3 and a pinch of KClO3. (5) Place on hotplate set to simmering temperature. Stir regularlywhen contents changes from its initial brown or orange state to clear yellow transfer contents to test tube. (6) Centrifuge@3500 rpm for 5 min and decant. (7) Rinse with 10 ml of distilled water and centrifuge@3500 rpm for 5 min and decant. Repeat rinse two times. (8) Add 1 ml of ETOH and leave overnight to dry. (9) Weigh dried material, calculate phytolith weight and transfer to labelled vials with as little 100% ETOH as possible.

nely, mixed to ensure homogeneity, and then divided into equal weight portions so that both dry and wet oxidation extraction techniques could be applied. A description of the extraction methods is provided in Table 1. The dry ashing technique used in this study was a slightly modied version of that used by Twiss, Suess & Smith (1969: 111), with the addition of centrifuging, hydrochloric acid (HCl) and hydrogen peroxide (H2O2) after initial ashing (Hart, 1998: 9). The reason for the additional use of HCl and H2O2 was to provide clean phytolith assemblages and to assist lucidity during morphometric analysis. Wet ashing was achieved through the use of a method similar to that of Rovner (1972: 591), with Schulze solution (nitric acid and potassium chlorate) employed for digestion (Table 1). The plant materials used in this study were obtained from the Herbarium at the Lae Forestry Research Institute, Morobe Province, Papua New Guinea. All samples are voucher specimens from that Institute, with species identication and accession numbers provided; only very small amounts of plant materials were available for samples (Table 2). The specimens comprise the leaf sections of 10 dierent Poaceae species. However, due to the lack of suitable phytolith types in some species, from these 10 species only eight were selected for each of the two analyses, shrinkage and warping. The selection criterion for species in each study was that they comprise sucient quantities of short and long celled phytoliths respectively.

To test the assumption that dry ashing and wet ashing extraction techniques produced comprative results, two separate analyses were conducted assessing patterns of morphometric change following processing. The rst analysis compared the size of bilobate (dumbbell) shaped phytoliths (Twiss, Suess & Smith, 1969: 111), and the second measured the curvature of elongate phytoliths after extraction procedures. Eight species were chosen for each analysis according to yields of short cells and long cells. Only those species with high yields were suitable for study. Phytolith residue assemblages were mounted on to glass slides in Eukitt mounting medium. Slides were viewed at 400 magnication with an Olympus CH-2 compound microscope tted with a polarizing lter and a camera lucida. From each slide, one area containing phytoliths was photographed for later reference using an Olympus camera mounted to a photo tube on an Olympus BH.2 microscope at 400 magnication. Phytoliths were then traced in plain view using the camera lucida, and divided into two categories (i.e. short cells and long cells). The overall sample comprised 1600 phytoliths, with 50 from each of the two extraction methods analysed for each species. Two separate analyses were conducted to assess the nature of phytolith morphology following dry and wet ashing extraction methods. The aim was to test the assumption that both dry and wet ashing techniques produced comparative results. The rst analysis compared the size of bilobate (dumbbell) phytoliths (Twiss, Suess

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Table 2. Summary of Poaceae species, authority, accession numbers Herbarium Lae Forestry Research Institute, type, fresh weight, residue weight and extraction method Species Bambusa sp. Brachiaria brizantha (Hoscht. ex A. Rich) Stapf. Buergersiochloa macrophylla S.T. Blake, Blumea Supp. Heteropogon triticus (R.Br) Stapf. ex Craib Imperata exaltata (Roxb.) Brogn. Polytoca macrophylla Benth. Saccharum robustum Saccharum ocinarum (L.) Themeda arguens (L.) Hack. Thysanoleana maxima (Roxb.) O.K. Accession no. LH. 103886 LH. 242415 LH. 229126 LH. 18775 LH. 81921 LH. 243442 LH. 18598 LH. 94173 LH. 12057 LH. 207698 Sample type Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Sample wt 01000 g 01000 g 00610 g 00610 g 01000 g 01000 g 01000 g 01000 g 01000 g 01000 g 00768 g 00768 g 00772 g 00772 g 1000 g 1000 g 00267 g 00267 g 01000 g 01000 g Residue wt 00065 g 00107 g 00036 g 0061 g 00037 g 00112 g 00018 g 00145 g 00024 g 00213 g 00088 g 00113 g 00087 g 0003 g 00001 g 0011 g 00012 g 00013 g 00055 g 00177 g Method DA WA DA WA DA WA DA WA DA WA DA WA DA WA DA WA DA WA DA WA

Figure 1. Illustration of parameters measured for the size analysis of bilobate phytoliths.

& Smith, 1969: 111), and the second measured the curvature of elongate phytoliths after extraction procedures. Width and length comparisons Bilobate phytoliths were selected to represent the short cell category because they were present in sucient quantities for statistical analysis in eight of the 10 species assessed. The short cells were designated to test for shrinkage only, because they are compact forms and less likely to warp. Measurements were taken of the length (i.e. maximum dimension (MD) and the widths across the two widest points at each end of the phytolith. The two width measurements were reduced to mean width (MMW) for the analysis (Figure 1). Comparison of mean angles Long cells, mostly spiny elongates (Twiss, Suess & Smith, 1969: 111), were selected to test for warping. It

Figure 2. Illustration of parameters measured for the angle of distortion analysis of elongate phytoliths using the Image Tool software.

was anticipated that warping would be more pronounced in elongate phytoliths and thus that they would provide a clearer evidence for distortion. The elongate phytoliths were traced using a camera lucida, digitized using a Sharp JX-320 atbed scanner and then imported into the program Image Tool on a Power Macintosh PC. Image Tool parameters were set to measure the degree of curvature. Calibrations were made for angles at each end of elongate phytoliths (Figure 2). Statistical analysis Statistical analyses are based on a comparative morphology approach similar to that used by Lepofsky, Kirch & Lertzman (1998: 10061007). A two-tailed t-test using SPSS for statistical analysis compares the mean dimensions of bilobates, the mean angles of

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Figure 3. Examples of a dry ashed sample, showing clear sheets of elongate phytoliths with in-situ bilobates and stomates.

Figure 4. Example of a dry ashed sample, showing disarticulated short cells, including bilobates and crosses.

elongate phytoliths, and boxplots are used to provide visual comparisons of the mean maximum width (MMW), maximum dimension (MD) length, and mean angle distribution after dry and wet ashing procedures. Each box denes the interquartile range i.e. iqr=upper quartile plus lower quartiles. The whiskers extending from above and below the iqr represent the extent of the 100th and 0 percentiles respectively. Outlying values denoted by diamonds and asterisks represent values that all fall further than 15 times and 3 times the interquartile range from the end of the box respectively (Devore & Peck, 1993: 9799).
Figure 5. Example of a wet ashed sample, showing aggregated sheets of unclear elongate phytoliths with in-situ bilobates.

Results
Processing times based on actual duration spent working in the laboratory was comparatively similar for dry and for wet ashing, i.e. around 3 h. The data presented in Table 2 shows that all the corresponding samples started with the same plant material weight. After phytolith extraction the dry ashing method resulted in consistently lower residue weight than the wet ashing. The wet ash residues had a mean average weight of 637% greater than those for dry ashed samples. Microscopic analysis at 100 and 400 magnications found that in the dry ashed samples, elongate phytoliths were mainly in sheets with in-situ bilobates, stomates, and bulliforms, as well as disarticulated cells such as cross shapes, tribolates, and trichomes (Figures 3 & 4). Most phytoliths in the dry ashed samples were suciently lucid and free of residual material. The use of polarized light at 400 and 100 magnications failed to detect calcium oxalate crystals or starch grains in these samples. Wet ashed samples had comparable disarticulated elongates and short cells, but in-situ phytoliths mainly occurred in large, relatively undiagnostic, amber-coloured clumps (Figures 5 & 6). Under crossed polarized light at 400 magnication, these clumps comprised elongate phytoliths bound in white matted material. At 100 magnication the

Figure 6. Example of a wet ashed sample, showing disarticulated short cells, including bilobates and crosses.

white matted areas were shown to consist of individual elongated strips, not raphides, but cellulose-like material that apparently had not been oxidized. Occasional raphides and starch grains were observed randomly throughout wet ashed samples. During the microscopic analysis it was also noted that many elongate phytoliths in both wet and dry ashed samples had a large degree of curvature when in association with stomates.

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Table 3. Summary statistics of analysis testing degree of shrinkage in bilobate phytoliths for dry ashing and wet ashing extraction methods Mean maximum width ( m) Species Heteropogon triticus Brachiaria brizantha Buergersiochloa macrophylla Imperata exaltata Saccharum robustum Saccharum ocinarum Themeda arguens Thysanoleana maxima Method DA WA DA WA DA WA DA WA DA WA DA WA DA WA DA WA x 108 94 126 126 134 146 95 106 112 132 147 157 93 114 115 117 S.D. 20 14 20 14 38 34 15 21 14 21 21 20 12 17 22 20 Range 75150 98125 75175 100175 62250 75225 75137 62162 81150 81187 100200 112200 62133 62150 62175 75162 N 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 Sig. 0000 0887 0100 0005 0000 0018 0000 067 x 181 162 192 194 220 214 156 167 281 208 277 254 208 214 192 194 Maximum dimensions ( m) S.D. 22 27 28 27 50 43 27 125 36 26 48 37 36 37 23 28 Range 125225 112225 125250 125250 100300 150312 112225 125237 203375 162275 200375 162337 150312 150300 125237 125250 N 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 Sig. 0000 0652 0564 0048 0000 0009 0404 069

Width and length comparisons for shrinkage The results of the two-tailed t-tests and boxplots comparing dry ashing and wet ashing and testing for shrinkage are shown in Table 3 and Figure 7. Dierences in the MMW for Heteropogon riticus, Imperata exaltata, Saccharum robustum, Themeda arguens, and Saccharum ocinarum samples were signicant at P=005 (Table 3). The dry ashed MMWs are signicantly larger for H. triticus and S. robustum (P=0000), yet smaller for I. exaltata (P=0005), T. arguens (P=0000) and S. ocinarum (P=0018). No signicant dierences occur in MMW between the wet and dry ashed samples of Buergersiochloa macrophylla (P=0100), Brachiaria brizantha (P=0887) and Thysanoleana maxima (P=0670). The boxplots show that dry ashed samples of H. triticus, B. brizantha, B. macrophylla and T. maxima have signicantly large width distributions in comparison to wet ashed samples. Alternatively, I. exaltata, S. robustum and T. arguens have comparatively small width distributions (Figure 7). Results for the MD (Table 3) show that the dry ashed samples are signicantly larger for H. triticus (P=0000) and S. ocinarum (P=0009), but smaller for I. exaltata (P=0048) and S. robustum (P=0000). There are no signicant dierences between methods for T. arguens (P=0404), B. macrophylla (P=0564), B. brizantha (P=0652) and T. maxima (P=0690). The boxplots demonstrate that the MD distributions for the dry ashed samples of H. triticus, B. brizantha, S. robustum, S. ocinarum and T. arguens are large in comparison to wet ashed samples. The dry ashed samples of B. macrophylla, I. exaltata, and T. maxima have comparatively small distributions to wet ashed samples (Figure 7).

Comparison of mean angles for warping The results of the two-tailed t-tests and boxplots comparing dry ashing and wet ashing and testing for warping are shown in Table 4 and Figure 8. Statistical analysis found that the angle of curvature for three of the eight species examined had signicantly dierent P-values (Table 4). Mean angles were signicantly larger for the dry ashed sample of H. triticus (P=0020), but smaller for P. macrophylla (P=0002) and S. ocinarum (P=0044). No signicant dierences occur in the results for Bambusa sp. (P=0051), I. exaltata (P=0110), S. robustum (P=0239), T. arguens (P=0111) and T. maxima (P=0378). The boxplots show that, if the few outliers that occur are discounted, the curvature and angle distribution is less for dry ashed specimens in ve of the eight species assessed (Figure 8). These ve species comprise I. exaltata, P. macrophylla, S. robustum, S. ocinarum and T. maxima. Of the remaining species, T. arguens and Bambusa sp. are comparatively proportional, while H. triticus has a relatively larger angle distribution than the wet ashed samples.

Discussion
Residue quantities Residue weights for the wet ashed samples are consistently larger than those for dry ashing (Table 2), a result which is in contradiction to results reported by Rosen (pers. comm.), who found that dry ashing provided around a 20% increase in phytolith retrieval when compared to wet ashing. Here wet ashing has provided an average of 637% more residue weight than dry ashing. Hodson (pers. comm.) has suggested that there

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Heteropogon triticus 30 Length (microns) Length (microns) Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method Length (microns) Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method Length (microns) Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method Length (microns) Dry Ash Wet Ash Dry Ash Wet Ash MD MD MMW MMW Method Brachiara brizantha 30

20

20

10

10

Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method

Buergersiochloa macrophylla 40 Length (microns) 30 20 10 0

Imperata exaltata 30

20

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Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method

Saccharum robustum 40 Length (microns) 30 20 10 0

Saccharum officinarum 40 30 20 10 0

Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method

Themeda arguens 40 Length (microns) 30 20 10 0

Thysanolaena maxima 30

20

10

Dry Ash Wet Ash Dry Ash Wet Ash MMW MMW MD MD Method

Figure 7. Boxplots illustrating sample distributions according to mean maximum widths of bilobates (MMW) and maximum dimensions (MD) for dry ashing and wet ashing. The central box showd the median and the lower and upper quartiles of the distribution. Outlying values shown by diamonds and asterisks represent values that fall further than 15 times and 3 times the interquartile range from the ends of the box respectively.

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Table 4. Summary statistics of analysis testing degrees of curvature in elongate phytoliths for dry ashing and wet ashing extraction methods Mean curvature Species Bambusa sp. Heteropogon triticus Imperata exaltata Polytoca macrophylla Saccharum robustum Saccharum ocinarum Themeda arguens Thysanoleana maxima Method DA WA DA WA DA WA DA WA DA WA DA WA DA WA DA WA x 39 58 50 32 45 58 30 58 45 57 42 62 30 43 446 52 S.D. 42 49 42 33 28 48 34 49 53 51 46 53 30 45 40 49 Range 001168 001182 000159 005115 0091208 009189 007152 003228 002277 004191 005213 000192 004117 0001828 0021805 002185 N 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 Sig. 0051 002 011 0002 0239 0044 0111 0378

is a greater possibility of loss in the number of lightly silicied cell wall deposits as a result of acid digestion. The higher yield of compound plates with dry ashing procedures in comparison to wet ashing (Raeside, 1970: 125) supports Hodsons view. However, an important question follows: if it is assumed that dry ashing produces larger phytolith yields, why are the residue weights in this study consistently greater in the wet ashed samples? The white matted areas of cellulose-like material mentioned in the results only occurred in the wet ashed samples. The failure of this material to oxidize using the Schulze solution may account for the higher residue weight for wet ashed samples. The ability of some plants to withstand the wet oxidation process has previously been recognized as a problematic area (Pearsall, 1989: 381). It is possible that the wet ashing method may have needed to be applied for a longer duration and/or additional KClO3 applied to promote greater reaction during oxidaton. This implies that this stage of the wet ashing process requires a combination of regular monitoring and chemical adjustment to suit individual samples. On the other hand dry ashing produces consistent results according to a standardized set of procedures, and is therefore the simpler method of the two. Shrinkage Unlike the results for residue weights, there was no evidence that either dry ashed or wet ashed samples consistently produced larger or smaller MMW or MD values. Five of the eight species assessed in this analysis produced signicantly dierent results according to the treatments. Nevertheless, these results appear to be random rather than the consequence of a particular extraction procedure. For example, statistical analysis

for MMW indicated that dry ashed samples are signicantly larger for Heteropogon triticus, smaller for Imperata exaltata, larger for Saccharum robustum, smaller for Themeda arguens, and smaller for Saccharum ocinarum. The analysis of MD resulted in a similarly random pattern: for dry ashing, samples for H. triticus and S. ocinarum are larger, I. exaltata and S. robustum are smaller, with there being no signicant dierence between methods for T. arguens. Although comparison between dry and wet ashing results for shrinkage reveal signicant dierences both between individual samples and groups of assemblages, the lackof recurring patterns in the results from either method indicates that both dierences in width and length probably do not represent size modication during extraction procedures. The inconsistent pattern of variation, therefore, most likely reects the natural range of variability in the phytolith residues themselves, rather than modication due to the application of the processing treatments. Although the samples from each species were cut up nely and were acquired from the same portion of leaf, it is relevant to note that the leaf segments have a range of variation across their cell structure and that some species have notable variability in cell morphology. Whang, Kim & Hess (1998: 465) have found, for example, that in the leaves of some Poaceae species there are signicant dierences in phytolith morphology depending on their proximity to midribs or lateral veins. Similar results in other species are reported by Ball, Brotherson & Gardner (1993), Ball, Gardner & Brotherson (1996) and Whang & Hill (1995). Thus it is possible that the results shown here are an artefact of such variation and occurred despite eorts to ensure homogeneity of samples prior to treatment during their preparation. This emphasizes, therefore, the importance of selection of plant material

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Heteropogon triticus 20 Bambusa sp. 20

Angle of curvature

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Dry Ash Method Wet Ash

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Imperata exaltata 30

Polytoca macrophylla 30

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Saccharum robustum 30

Saccharum officinarum 30

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Themeda arguens 20

Thysanolaena maxima 30

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Figure 8. Boxplots illustrating sample distributions according to degree of curvature measured in elongate phytoliths. The layout is as for Figure 7.

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for reference collections, and the need for carefully planned, systematic sampling to ensure the full range of morphological types are accounted for. Warping This analysis assessed the elongate phytoliths for evidence of warping as a result of preparation technique. The computer-assisted image analysis was a particularly ecient way to measure microscopic features such as the curvature of phytoliths and transfer this data directly into statistical packages. Statistical testing found that only three of the eight species examined were signicantly dierent according to the treatments. The statistical analysis for these three species show that the degree of curvature of phytoliths in the dry ashed sample was signicantly greater for H. triticus signicantly greater for H. triticus, but lesser for P. macrophylla and S. ocinarum. Again this appears to be random patterning similar to that observed in the previous analysis. However, comparisons made between dry and wet ashed samples using the boxplots in Figure 8, indicate that with the exception of two relatively extreme outliers in S. robustum and S. ocinarum, only one of the eight dry ashed species assessed in this analysis, H. triticus, had a signicantly larger distribution and degree of curvature. Dry ashed samples Bambusa sp. and T. arguens had no signicant dierence to their corresponding wet ashed boxplots. The remaining ve species had both a signicantly larger distribution of curved elongates and exhibited a larger degree of individual curvature in wet ashed samples. Again this may be the result of variation in leaf cell structure (Ball, Brotherson & Gardner, 1993; Ball, Gardner & Brotherson, 1996; Whang & Hill, 1995); plates within a number of publications show, for example, curved elongate phytoliths in-situ with stomates and other features from both dry and wet ashed samples (e.g. Jones & Handreck, 1967: 128; Cliord & Watson, 1977: 4577; Lanning, Hopkins & Loera, 1980: 554). The results, therefore, from these analyses do not support the claim that dry ashing results in shrinkage and warping of phytoliths at around c500 C as proposed by Jones & Milne (1963: 207220). As suggested by Lanning, Hopkins & Loera (1980: 550), Piperno (1988: 126127) and Runge (pers. comm.), there appears to be little need for concern in relation to the dry ashing process, if ring temperatures do not exceed 500 C.

is highly desirable for comprehensive reference collections, and in particular for those which will be used to provide detailed information on phytolith location and variation within plants. Both methods are comparable in time taken to carry out the phytolith extraction procedures, and importantly, it is indicated here that wet ashing also produced comparable results to those of dry ashing for disarticulated phytoliths. However, wet ashed samples do not allow the in-situ material to be clearly distinguishable due to the aggregation of residual matter that apparently had not been fully oxidized. This implies that a combination of regular monitoring and chemical adjustment to suit individual samples during preparation is necessary to achieve satisfactory results with the wet ashing process. In contrast, the dry ashing treatment produces consistent results and can be standardized for all plants and plant parts. This study has, importantly, established that dierences in patterns of dimension and curvature of short bilobate phytoliths and longer elongate phytoliths subjected to dry and wet ash preparation are not statistically signicant. There is no detectable evidence from this study, therefore, of morphological impact as a result of the application of methods. This implies that any dierences that do occur in phytolith size and curvature may be considered to be typical, possibly random permutations within the original assemblages of phytoliths, in part probably assigned to variation in leaf cell structure rather than as a consequence of a particular extraction procedure. Such conclusions imply, therefore, that while care may be needed in the selection of sample preparation method in terms of the eventual use of the reference sample, in terms of providing morphologically comparable reference material, the practice of using various methods of preparation of reference samples for fossil analysis can be reliably continued, and that the concerns expressed by various practitioners may be allayed.

Acknowledgements
The authors also wish to acknowledge with gratitude, the contributions to this discussion of members of the e-mail discussion list PHY-TALK, who promptly commented to an initial question regarding the topic of this paper transmitted over this e-mail list. In particular, the following people are thanked for their specic contributions: Freya Runge, Lucinda McWeeney, Arlene Rosen, Iv Rovner and Martin Hodson. The Herbarium at the Lae Forestry Research Institute, Morobe Province, Papua New Guinea made an invaluable contribution to this work in its supply of herbarium plant specimens. This project was in part funded from an ARC Large Grant awarded to Boyd to support the Prehistory of Garua, Papua New Guinea Archaeological Project of which the research reported here is but a small part.

Conclusion
This comparison of the results for wet and dry ashing procedures has shown that the dry ashing method produces less residual matter than the wet ashing technique. Moreover, it provides clean, lucid, disarticulated, and in-situ phytolith assemblages more eectively than does the wet ashing method. This outcome

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