DOI: 10.5061/dryad.n8pk0p351
title: Data from: A morphometric analysis of starch granules from two Dioscorea species

description: Dioscorea L. is a genus comprised of over 600 species, many of which
 possess edible tubers that are commonly referred to as yams. While
 Dioscorea is a significant crop across the globe, it holds a unique
 cultural significance to the people of Tonga in western Polynesia.
 Presently, Dioscorea is known for its essential role in festivals and
 ceremonies, as well as for its nutritional contribution in Tongan diets.
 To understand and to assess the significance of Dioscorea in the distant
 past, however, archaeologists rely on plant residues (e.g. starch
 granules) preserved on ancient tools. This study provides the necessary
 first step in archaeological starch analysis by examining granule
 morphometrics of two culturally significant Dioscorea species, D. alata
 and D. bulbifera from Tonga. Tubers from three individuals of each species
 were collected on the island of Vava’u and processed for starch granule
 extraction and analysis. Morphometric characters, including two novel that
 describe shape (eccentricity ratio and hilum angle), were measured on 300
 granules per species. When statistically compared, these novel characters
 allow D. alata and D. bulbifera to be readily distinguished from one
 another, and, therefore increase confidence in assigning archaeological
 granules to a specific taxon.
description: Reference Collection Two species of
 <em>Dioscorea </em>that grow on the island nation of Tonga
 were selected for the present study. Plants (including their tubers) were
 collected in Tonga during May and June, 2023 (Table 1). Home visits were
 made to local subsistence farmers, where interviews regarding agricultural
 practices were conducted. When possible, plantation visits were also
 conducted. Square footage of the growing plots was gathered via Garmin
 eTrex GPS, and rough tallies of crop species were collected. <em>D.
 alata</em> samples were collected from a local community garden in
 Vava’u and two additional household gardens<em>.
 </em>Although<em> D. bulbifera</em> is no longer an
 important food crop grown in Tonga, it was introduced to the islands by
 initial settlers and is a persistent volunteer. These specimens were
 collected from forested sections along roadsides.
 <strong>Table 1. </strong>Sample number,
 <em>Dioscorea</em> species, Tongan name, and collection
 location (island) for reference material analyzed in the current
 study. 
 <strong>Lab Sample
 Number</strong> 
 <strong>Species </strong> 
 <strong>Variety/Tongan
 name</strong> 
 <strong>Island</strong> 
 <strong>Context</strong>
 LS1
 <em>D. alata</em>
 <em>‘Ufi Sikalu</em>
 Vava’u 
 Community Garden 
 LS2 
 <em>D. alata</em> 
 <em>‘Ufi Hawaii </em>(purple var.)
 Vava’u 
 Private plantation 
 LS3 
 <em>D. alata</em> 
 <em>‘Ufi Hawaii </em>(white var.)
 Vava’u 
 Private plantation 
 LS5 
 <em>D. bulbifera</em> 
 <em>Hoi</em> 
 Vava’u 
 Roadside, fallow field 
 LS6 
 <em>D. bulbifera</em> 
 <em>Hoi</em> 
 Vava’u 
 Fallow field 
 LS7 
 <em>D. bulbifera</em> 
 <em>Hoi</em> 
 Vava’u 
 Roadside 
 Sample preparation
 A small portion (~1 cm<sup>2</sup>) of the tubers
 were pulverized with a clean glass mortar and pestle and deionized water
 (diH<sub>2</sub>O). Approximately 10 ml of
 diH<sub>2</sub>O was added to the sample, and the mixture was
 strained through a 125 µm Endicott sieve to remove &gt;125 µm debris.
 This mixture was then centrifuged for 3 minutes at 3,000 RPM. The
 supernatant was decanted and discarded. The pellets were resuspended with
 10 ml of diH<sub>2</sub>O using a vortex mixer. Samples were
 once again centrifuged at 3,000 RPM for 3 minutes. The supernatant was
 decanted and discarded. Approximately 7 ml of heavy liquid, lithium
 heteropolytungstate (LST) with a specific gravity of 2.35, was added to
 each vial. The pellets were resuspended in the LST with a vortex mixer.
 The samples were then centrifuged for 20 minutes at 1,000 RPM. This heavy
 liquid solution separates materials of varying densities, with lighter
 organics, such as starch granules, that float to the surface. Organic
 material was extracted (or collected) from the upper 1-2 mm of the vial
 and transferred to a new vial. This material was then rinsed with
 diH<sub>2</sub>O twice, centrifuged for 3 minutes at 3,000
 RPM, and the supernatant was decanted. A final rinse of acetone was used
 to dry and sanitize the samples. Microscopy
 Starch granules were photographed with both polarized light and
 differential interference contrast (DIC) light (Figure 4). Polarized light
 highlights characters such as the hilum and extinction cross. Granule
 characters detected under DIC light include lamellae and fissures. Each
 slide was scanned using a transmitted brightfield microscope fitted with
 polarizing filters and Nomarski optics (ZeissAxioscope2, Zeiss
 International, Göttingen, Germany). A digital camera (Zeiss HRc) with
 imaging and measurement software (Zeiss Zen) was used to capture images
 of, document, and measure starch granules. Approximately 100 starch
 granules from each individual plant ( n = 299 for <em>D.
 alata</em>, n = 302 for <em>D. bulbifera</em>) were
 measured and described. Granules were located via randomly generated
 microscope stage coordinates to avoid granule sorting due to fluid
 transport within the slide medium<em>. </em>
 <strong>Figure 4. </strong>A) <em>D.
 alata</em> granules in DIC (left) and polarized (right) light.
 B)<em> D. bulbifera</em> granules in DIC (left) and polarized
 (right) light. .Granule Measurements and
 Morphology Quantitative and qualitative characters were
 recorded for each starch granule, including size, shape, degree of
 eccentricity, and angle of hilum to width (Table 2; data available from
 Dryad
 http://datadryad.org/stash/share/KKZHwdfkBR2fUUFyxI5odkCx_7sTk9EgpJdlrJ0LURA).  All quantitative measurements were made consistently by a single researcher using Zen software. The maximum length of each granule was measured through hilum. The maximum width was measured at the widest part of the granule perpendicular to the maximum length. <em>Dioscorea</em> spp. starch granules almost exclusively possess an eccentric hilum (Table 2). The degree of eccentricity (eccentricity ratio) was calculated by measuring the length from the hilum to the proximal end (closest to the hilum) divided by the maximum length (Figure 5). The ratio ranges from 1.0, indicating the hilum is far from the proximal end of the granule to 0.0, indicating the hilum is very close to the proximal end. A further quantification of  the differences in shape between <em>D. alata</em> and <em>D. bulbifera </em>is measuring the angle emanating from the hilum to the maximum width of the granule (angle of hilum) (Figure 6).
description: # Data from: A morphometric analysis of starch granules from two Dioscorea
 species
 [https://doi.org/10.5061/dryad.n8pk0p351](https://doi.org/10.5061/dryad.n8pk0p351) ## Description of the data and file structure ### Files and variables A Morphometric Analysis of Dioscorea starch readme.txt file was generated 09-21-2024 by Sara K. Rickett GENERAL INFORMATION 1. Title of Dataset: A Morphometric Analysis of Starch Granules from Two Dioscorea Species 2. Author Information A. Primary Author Contact Information Name: Sara Rickett Natural History Museum of Utah, Department of Anthropology, University of Utah Address: 301 Wakara Way, Salt Lake City, Utah 84108 Email: [sara.rickett@utah.edu](mailto:sara.rickett@utah.edu) B. Principal Investigator Contact Information Name: Lisbeth A. Louderback Institution: Natural History Museum of Utah, Department of Anthropology, University of Utah Address: 301 Wakara Way, Salt Lake City, Utah 84108 Email: [lisbeth.louderback@anthro.utah.edu](mailto:lisbeth.louderback@anthro.utah.edu) 3. Date of data collection (single date, range, approximate date): 2023-07 to 2024-09 4. Geographic location of data collection: Vava'u, Tonga. 18°39′05″S 173°59′01″W / 18.65139°S 173.98361°W 5. Recommended citation for this dataset: Rickett, S.K.: Louderback, L.A.; Bell, A.V. (2024) A Morphometric Analysis of Starch Granules from Two Dioscorea Species. In Plants 2024. DATA & FILE OVERVIEW File List: 1. D_alata.csv Morphological characteristics and measurements of Dioscorea alata granules. Prepared for use in R studio. 2. D_bulbifera.csv Morphological characteristics and measurements of Dioscorea bulbifera granules. Prepared for use in R studio. 3. METHODOLOGICAL INFORMATION 1. Description of methods used for collection/generation of data: Starch granules were photographed with both polarized light and differential interference contrast (DIC) light (Figure 4). Polarized light highlights characters such as the hilum and extinction cross. Granule characters detected under DIC light include lamellae and fissures. Each slide was scanned using a transmitted brightfield microscope fitted with polarizing filters and Nomarski optics (ZeissAxioscope2, Zeiss International, Göttingen, Germany). A digital camera (Zeiss HRc) with imaging and measurement software (Zeiss Zen) was used to capture images of, document, and measure starch granules. Approximately 100 starch granules from each individual plant ( n = 299 for *D. alata*, n = 302 for *D. bulbifera*)~~300~~ ~~per species) ~~were measured and described. Granules were located via randomly generated microscope stage coordinates to avoid granule sorting due to fluid transport within the slide medium*.* 2. Description of CSV data and Column glossary Column A: "Species"- Denotes taxa of granule Column B: "LS Number"- Lab sample number, this column denotes which tuber the sample is from. Column C: "Prep"- Preparation, notes which slide preparation this granule can be found on. Column D: "Photo Num"- All photos are saved with measurements and notation embedded in the file. Column E: "Granule Num"- Photos with multiple granules were labeled with granule numbers to ensure accurate transcription. Column F: "Max Length"- The maximum length of each granule was measured (microns) through the hilum along the long axis of the granule. Column G: "Max Width"- The maximum width was measured (microns) at the widest part of the granule along the short axis. Column H: "Hilum to Prox"- The distance between the hilum and the closest end, measured (microns) along the long axis of the granule. Column I: "Eccentricity Ratio"- The degree of eccentricity (eccentricity ratio) was calculated by measuring the length from the hilum to the proximal end divided by the maximum length. The ratio ranges from 1.0, indicating the hilum is far from the proximal end of the granule to 0.0, indicating the hilum is very close to the proximal end. Column J: "Hilum Angle" - To quantify the differences in shape between D. alata and D. bulbifera, the angle emanating from the hilum to the point of maximum width of the granule was measured in degrees using Zeiss Zen software tools. Column K: "Oval (0=absent, 1=present)"- Oval granules are defined as granules that are rounded with unequal length and width, the ends of the granule are also unequal in width, as defined by the ICSN 2011. Column L: "Triangular (0=absent, 1=present)"- Triangular granules possessed three defined with varied corners. Column M: "Hilum position (0=eccentric, 1=centric)"- Granules with hila outside the estimated center of the granule were noted at eccentric. Column N: "Longitudinal Fissures (0=absent, 1=present)"- Longitudinal fissures are anomalies in the granule progressing along the long axis of the granule. Column O: "Transverse Fissures (0=absent, 1=present)"- Transverse fissures, anomalies in the granule progressing along the short axis from the hilum, note these features were rare and not included in the final analysis. Column P: "Granule curve (0=absent, 1=present)" Curves or bends were noted, frequently occurring at the proximal end of the granule. Column Q: "Lamellae (0=non-lamellated, 1=lamellated)"- Lamellae are visible layers of amylose and amylopectin, which create concentric growth rings emanating from the hilum. Column R: "Notes"- Any additional comments about the granule condition or other features from the researcher. 1. Methods for processing the data: R studio 2. Instrument- or software-specific information needed to interpret the data: Microsoft Excel, R 3. Standards and calibration information, if appropriate: N/A 4. Environmental/experimental conditions: N/A 5. People involved with sample collection, processing, analysis and/or submission: S.Rickett, L.A. Louderback Additional documents: Document titled "Boxplot_totals.PDF" contains boxplots from the total dataset depicting comparisons of granule length (microns), granule width (microns), eccentricity ratio, and hilum angle (degrees), between species.  Documents titled "D_alata_hist.pdf" and "D_bulb_hist.pdf" contain histograms and the results of Shapiro-Wilk tests. The histograms depict the total dataset and top 20%. The Shapiro-Wilk test for the normality 100% of the dataset. Tests and histograms interpret the following values: granule length (microns), granule width (microns), eccentricity ratio, and hilum angle (degrees).  Documents titled "100__frequency_D.alata.jpg" and "100__frequency_D.bulbifera.jpg" depict frequency distributions of presence-absence data recorded across the entire dataset. The presence-absence values are as follows: Lamellae, Longitudinal Fissures, Curve, Triangular, and Oval.