Richard Fullagar, Elspeth Hayes, Birgitta Stephenson, Judith Field, Carney Matheson, ... polarised light; (f-âh) Grinding Surface 2: (f) amorphous organic material ...
Supporting Information for Archaeology in Oceania article: Evidence for Pleistocene seed grinding at Lake Mungo, southeastern Australia Richard Fullagar, Elspeth Hayes, Birgitta Stephenson, Judith Field, Carney Matheson, Nicola Stern, and Kat Fitzsimmons. This file includes: 1. Plates 1-‐13: (Supporting Information pp. 2-‐14) LMGS No. Plate No. 1 1, 2 2 3, 4, 5 3 3 4 3 5 3, 4, 5 6 3, 4, 5 7 3, 4, 5 8 3, 5 9 3 10 6 11 7 12 8 13 9 14 10 15 11 16 12 17 13 2. Chemical Test Protocols: (Supporting Information pp. 15-‐16) Bradford Assay for protein Copper triethanolamine diphenyl-‐carbazide (“Falholt”) test for fatty acids Diphenylamine test for carbohydrates PSA test for carbohydrates IKI test for starch Hemastix® test for ferrous iron
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Plate 1 LMGS1: (a) Grinding Surface 1; (b) Surface 1 at low magnification, showing well-‐ rounded quartz grains and levelled plateaux; (c) Surface 2 at low magnification, showing a levelled but weathered surface; (d) Grinding Surface 2; (e-‐f) Surface 1 at high magnification, showing a bright, reticulated use-‐polish (cf. seed grinding) with arrows indicating orientation of the striations; (g-‐h) Surface 2 at high magnification showing use-‐ polish.
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Plate 2 LMGS1 residues: (a-‐e) Grinding Surface 1: (a) a starch granule photographed at 400x in part-‐polarised (left) and cross-‐polarised (right) light; (b) gelatinised starch and plant material stained with Congo Red; (c-‐d) collagen fibre stained with Picrosirius Red (PSR), photographed at 400x in (c) part-‐polarised and (d) cross-‐polarised light; (e) amorphous collagen and collagen fibres stained with PSR, photographed at 400x in cross-‐ polarised light; (f-‐h) Grinding Surface 2: (f) amorphous organic material photographed at 200x; (g) plant material stained with Congo Red; (h) amorphous cellulose photographed at 400x in part-‐polarised light.
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Plate 3 LMGS 2-‐9, refitting fragments: (a) LMGS2; (b) LMGS3 Surface 1; (c) LMGS3 Surface 2; (d) LMGS4; (e) LMGS5; (f) LMGS6; (g) LMGS7; (h) LMGS8; (i) LMGS9.
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Plate 4 LMGS2, 5, 6 & 7 residues: (a) LMGS2 Surface 1 feather barbule, photographed at 400x in (left) part-‐polarised and (right) cross-‐polarised light; (b) LMGS2 Surface 2 damaged starch stained with Congo Red; (c) LMGS2 Grinding Surface 1 amorphous cellulose, photographed at 200x; (d) LMGS2 Grinding Surface 2 plant tissue, photographed at 200x; (e–f) LMGS5, feather barbules in (e) part-‐polarised light and (f) cross-‐polarised light; (g-‐h) LMGS5, amorphous plant material stained with Congo Red; (i) LMGS7, plant tissue, photographed at 400x, part-‐polarised light; j) LMGS5, minerals and plant fibres, photographed at 400x.
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Plate 5 LMGS2, 5, 6, 7 and 8, showing polish on the highest points of quartz grains, creating a reticular morphology, cf. seed grinding, with arrows indicating orientation of the striations: (a) LMGS2 Grinding Surface 1; (b) LMGS2 Grinding Surface 2; (c) LMGS5; (d-‐e) LMGS6; (f) LMGS7; (g) LMGS8.
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Plate 6 LMGS10: (a) Grinding Surface; (b-‐f) use-‐polish at high magnification on the highest points of quartz grains, showing a reticular morphology cf. seed grinding, with arrows indicating orientation of the striations; (g) a starch granule in (left) part-‐polarised and (right) cross-‐polarised light; (h) plant tissue stained with Congo Red in cross-‐polarised light.
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Plate 7 LMGS11: (a) Grinding Surface 1; (b) Surface 1 at low magnification, displaying well-‐rounded quartz grains and levelled plateaux; (c) Surface 2 at low magnification, showing a levelled surface with deep, interstitial spaces from where quartz grains have been plucked during use; (d) Grinding Surface 2; (e) Surface 1 at high magnification, displaying a bright, reticulated polish cf. seed grinding; (f-‐g) Grinding Surface 2 at high magnification showing use-‐polish, with arrows indicating orientation of striations; (h) Grinding Surface 2, plant fibre; (i) Grinding Surface 2, solidified plant tissue cf. exudate.
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Plate 8 LMGS12: (a) Grinding Surface: (b) Grinding Surface 2 at low magnification, displaying a levelled but weathered surface; (c-‐f) uniform levelled use-‐polish at high magnification cf. file or abrading stone; (g) plant material stained with Congo Red; (h) mineral crystals.
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Plate 9 LMGS13: (a) possible grinding surface; (b) possible grinding surface at low magnification, showing an uneven weathered surface; (c-‐d) possible grinding surface at high magnification showing quartz grains with relatively uniform low wear development (cf. weathering) and no diagnostic traces of use-‐polish.
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Plate 10 LMGS14: (a) Grinding Surface 2; (b) Surface 2 at low magnification showing a levelled surface with interstitial spacing, from where quartz grains have been plucked during use; (c-‐g) bright, reticulated and very smooth use-‐polish at high magnification, showing pitting and arrows indicating orientation of the striations; (g) amorphous organic material (most likely of plant origin).
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Plate 11 LMGS15: (a) Grinding Surface 2; (b) Surface 2 at low magnification showing a levelled surface and interstitial spacing from where grains have been plucked during use; (c-‐f) use-‐polish at high magnification. The polish is most developed on the highest zones of the quartz grains, giving it a bright and reticulated appearance.
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Plate 12 LMGS16: (a) Grinding Surface 2; (b) Surface 2 at low magnification showing levelled and rounded quartz grains; (c-‐f) Surface 2 at high magnification showing use-‐ polish on the highest zones of the quartz grains, giving it a bright, reticulated appearance cf. seed grinding; arrows indicate orientation of the striations; (g-‐h) plant and cellulose stained with Congo Red, in cross-‐polarised light; (i) solidified, brittle mass of organic material, cf. plant exudate.
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Plate 13 LMGS17: (a) Grinding Surface 1; (b) Surface 1 at low magnification showing levelled and rounded quartz grains; (c-‐f) Surface 1 at high magnification showing weathering (evenly smoothed microtopography) and use-‐polish on the highest zones of quartz grains showing slight reticular morphology; (g) a starch granule stained with IKI in (left) part-‐polarised and (right) cross-‐polarised light; (h) collagen fibres and amorphous collagen stained with PSR, photographed at 400x, in cross-‐polarised light.
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Chemical test protocols Bradford Assay test for protein Protein was identified through application of the Bradford Assay following the procedures described by Jones et al. (1989) and Kruger (1994). Five micro-‐litres of water-‐ extracted material was added to 25μL of Bradford assay solution (i.e. 100mg of Coomassie Blue G250, 50mL of 95% ethanol and 100mL of 85% phosphoric acid, made to 1L with distilled water) and mixed for 20min at 1,000RPM at 25°C. Absorbance was then read for 2μL of this solution at 595nm. Copper triethanolamine diphenyl-‐carbazide (“Falholt”) test for fatty acids Fatty acids were detected with the Copper triethanolamine diphenyl-‐carbazide test (Falholt et al. 1973) hereafter referred to as the “Falholt” test. Extractions were freeze-‐dried for 48 hours so that any additional liquid was removed, and then re-‐filled with 10μL of acetonitrile and left to soak for at least 24 hours. Five micro-‐litres of sample were added to 20μL of copper TEA [0.05 mol-‐1 Cu (No3)2 and 0.1 mol-‐1 thiethanolamine pH 8.1] and 5μL of DCP (500μL of 4% 1.5 diphenyl-‐carbazide and 50μL of triethanolamine). After 15min, absorbance was then read for 2μL of this solution at 550nm. Diphenylamine test for carbohydrates Carbohydrates were detected using the diphenylamine test (Kanzaki & Berger 1959). Five micro-‐litres of water extracted sample was mixed with ten micro-‐litres of diphenylamine solution (0.05g diphenylamine MW 169.22, 5mL glacial acetic acid and 0.125mL sulphuric acid) and heated for 10min at a temperature range of 80 to 100°C, bringing the water component of the solution to boil. Following heating, absorbance was then read for 2μL of this solution at 595nm. PSA test for carbohydrates The PSA test was performed as an additional method of carbohydrate detection. This test is credited as the easiest and most reliable method of carbohydrate detection; often used for the measurement of neutral sugars in oligosaccharides, proteoglycans, glycoproteins and glycolipids (Masuko et al. 2005:69; Mecozzi 2005). Five micro-‐litres of the water extraction were mixed with a PSA solution (5μL 4% phenol and 25μL sulphuric acid). The mixture was left for 10min at room temperature. Absorbance was then read for 2μL of this solution at 490nm. IKI test for starch The presence of starch (intact and gelatinised) was assessed using the IKI biochemical test (McCready & Hassid 1943). Five micro-‐litres of the water-‐extracted material removed from each of the used surfaces were mixed to a solution of 5μL potassium iodide (KI) (0.12M) and 5μL of iodine (I) (0.01M). Samples with