DOI: 10.5061/dryad.2rbnzs7rh
title: Identification of starch granules on ground stone tools exposed to fire

description: Intense wildfires destroy everything in their path, including
 archaeological sites. Prehistorically, archaeological sites were regularly
 and intentionally burned. In what ways does burning affect those sites?
 With increased wildfire activity, research has begun to describe the
 effects of fire on archaeological materials through post-fire and
 experimental treatment, yet, little is known about the effects of fire on
 microbotanical remains, such as starch granules. Although some studies
 address the impact of fire on starch-rich foods, there is virtually no
 research on the fire effects of starch granules embedded in ground stone
 tools. The current study examines changes in the morphology of starch
 granules embedded in ground stone tools before and after exposure to flame
 combustion. A measurable amount of intact and identifiable starch granules
 was recovered from all of the treated samples. However, significantly
 fewer intact, identifiable granules were found as tools were exposed to
 higher temperatures for longer periods of time.
description: <strong>Starch Extraction and
 Identification</strong> All samples were
 processed for starch following standard lab procedures (see Louderback et
 al., 2015). Large ground stone tools were surface sampled for ~5 min with
 a sonicating toothbrush and DH<sub>2</sub>O ; smaller tools
 were sampled after sonicating in a DH<sub>2</sub>O bath for 3
 min. Each sample was rinsed through an Endecott mesh sieve (125µm) with
 DH<sub>2</sub>O and sample liquid &lt;125µm was retained
 in a 50mL test tube. Sample extract &gt;125µm was discarded. The
 sample liquid (presumably containing starch) was centrifuged for 3minutes
 at 3,000 rpm. The supernatant was decanted and the sample pellet was
 transferred to a sterile 15mL tube with DH<sub>2</sub>O ,
 mixed with a vortex, centrifuged for 3minutes at 3,000 rpm, and decanted.
 Samples were resuspended with ~7mL of heavy liquid (lithium
 heteropolytungstate; specific gravity 2.2), vortex-mixed, and centrifuged
 for 15 minutes at 1,000rpm. Heavy liquid separates the lighter organic
 material, including starch granules, from the heavier content. The lighter
 organics were collected using a pipette and transferred to a new, sterile
 15mL tube. Residual heavy liquid was rinsed from the organic matter with
 ~10mL of DH<sub>2</sub>O , mixed with a vortex, and
 centrifuged for 3 minutes at 3,000rpm; twice. The samples were decanted
 and rinsed with ~7mL of acetone, vortex-mixed, and centrifuged for 3
 minutes at 3,000rpm. After decanting one final time, processed samples
 were left to dry overnight before mounting on glass slides for microscopy
 observation. Starch granules were measured and
 described based on a set of established criteria, including maximum length
 through the hilum (µm), hilum position, two-dimensional shape, clarity of
 the extinction cross, and the presence or absence of surface features such
 as fissures and pressure facets (Brown and Louderback 2020; Holst et al.,
 2007; ICSN, 2011; Joyce et al 2021; Louderback and Pavlik, 2017; Musaubach
 et al., 2013; Piperno et al., 2004, 2009; Reichert, 1913; Torrence and
 Barton, 2016).  These criteria were recorded as absent (0) or present (1)
 and expressed as a percentage of the occurrence. Slides
 were scanned with a transmitted brightfield microscope using polarizing
 filters and Nomarski optics (Zeiss Axioskop Imager M1, Zeiss
 International, Göttingen, Germany). Observations were obtained using
 randomly generated X and Y coordinates on the microscope stage. All starch
 granules observed within each field of view were measured and described.
 Images and measurements at 400X were captured under polarized light (POL)
 with a digital camera (Zeiss AxioCam MRc5) using Zen Core 3.1 imaging and
 measurement software. The presence of surface features was imaged and
 recorded in differential interference contrast (DIC) micrographs.
 <em> </em>
 <strong>Identification and Quantification of Treated Starch
 </strong> The relative proportions and
 arrangements of amylose and amylopectin affect both granule morphology and
 functionality (Vamadevan and Bertoft, 2015). They can also cause various
 mechanical and chemical changes, such as granule swelling
 (gelatinization), pasting, and loss of birefringence in response to
 different food processing methods (Cai et al., 2014; Crowther, 2012; Di
 Poala et al., 2003; Gong et al., 2011; Mason, 2009; Wang et al.,
 2014). Exposing starch granules to heat can cause
 morphological damage such that granules may be difficult to identify.
 Granules exposed to high temperatures have been shown to enlarge in size
 (swell) and gelatinize, losing their birefringence cross, amylose layers,
 and surface characteristics before dispersing away entirely (Crowther,
 2012; Singh et al., 2002; Vamadan and Bertoft, 2015). Congo Red dye
 (empirical formula C32H22N6O6S2Na2) reacts with granules when amylose
 layers are broken down (usually due to cooking or some other form of
 damage), staining them orange to vivid red. Undamaged starch granules
 (with intact amylose layers), however, are hydrophobic and, therefore, do
 not react with Congo Red (Lamb and Loy, 2005).   To
 measure the percent or degree of damage to starch granules, milled and
 burned samples were treated with a Congo Red solution following a protocol
 similar to Lamb and Loy (2005). Dried residue samples were resuspended
 with 25µL of Congo Red and absorbed the stain for 15 minutes before
 diluting with 100µL of DH<sub>2</sub>O (1:4 ratio). Slides
 were prepped with 25µL of the hydrated sample and a cover slip was applied
 but not affixed with fingernail polish (experimentation found that clear
 fingernail polish interfered with Congo Red stain). The liquified stained
 samples dried within ~45 minutes, therefore, all observations were
 photographed immediately. Microscope observations on slides from the
 milled and burned (close proximity) samples were obtained using randomly
 generated X and Y coordinates. Samples exposed directly to flame, however,
 produced fewer measurable granules, so observations were collected by
 scanning the entire slide. Size distributions from the
 control, milled, and burned samples were statistically analyzed with a
 Kolmogorov-Smirnov (K-S) test to determine any significant difference in
 the distribution of median lengths. Boxplot-stripcharts were generated in
 R Statistical Software (v4.0.2; R Core Team 2020) to show the quartile
 summary variation
description: Windows Excel Spreadsheets