1 Supporting Information for Archaeology in Oceania ...

7 downloads 0 Views 8MB Size Report
Richard Fullagar, Elspeth Hayes, Birgitta Stephenson, Judith Field, Carney Matheson, ... polarised light; (f-‐h) 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          

1  

    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.      

 

2  

    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.        

3  

      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.      

 

4  

    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.        

5  

    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.      

 

6  

    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.      

 

7  

    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.      

 

8  

    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.        

 

9  

  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.      

 

10  

 

    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).        

11  

    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.      

 

12  

    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.      

 

13  

    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.      

 

14  

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