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83
IX/1/2018
InterdIscIplInarIa archaeologIca
natural scIences In archaeology
homepage: http://www.iansa.eu
Thematic review
Microscopic Analysis of Starch Grains and its Applications in the
Archaeology of the Stone Age
Jaromír Kovárník
a*
, Jaromír Beneš
a
a
Laboratory of Archaeobotany and Palaeoecology, Faculty of Science, University of South Bohemia, Na Zlaté stoce 3, 370 05 České Budějovice, Czech Republic
1. Introduction
Archaeobotany ofers a series of analytical methods (
e.g.
Evans, O´Connor, 1999). These various methods can be
sorted according to the function and character of material
studied, which may include seeds, stones of fruit, wood,
charcoal and other botanical macro-remains on the one
hand, and a large group of botanical microremains on the
other (Jacomet, Kreuz, 1999). Integrated archaeobotanical
approaches, which combine analyses of several biological
indicators, began in the 1970s (Scott and Lewis, 1981;
Cummings, 1994) and have become widespread due to
their efectiveness as a tool for understanding the past
environment, human diet, and the function of particular
archaeological objects (Pető
et al.
, 2013; García-Granero
et al.
, 2015). The analysis of starch grains is a suitable,
though still rather uncommon, archaeobotanical technique
in archaeological research. However, starch grain analysis
has been employed in archaeological research for more than
the last two decades. Starch grains are part of the group of
plant microremains that includes phytoliths, pollen, spores
and other
“
non-pollen” objects. Examination of these plant
remains can elucidate changes in the environment, both
natural and anthropogenic (Lentfer
et al.
, 2002; Evans,
Ritchie, 2005; Roosevelt, 2016). Starch occurs as insoluble,
semi-crystalline granules in plant tissue that store energy in
specifc parts of the plant, such as seeds, roots and tubers
(storage organs) (Hardy
et al.
, 2016), and as transitory starch,
which is usually not recovered or identifed in samples. The
analysis of starch grains is connected with investigations
into plant use and plant processing in the past and also the
composition of the herbaceous component of the human diet
(Barton, White, 1993; Hall
et al.
, 1989; Fullager
et al.
, 1998;
Henry
et al.
, 2014; Corteletti
et al.
, 2015; Tromp, Dudgeon,
2015; Shillito
et al.
,
2018; López, 2018; Primavera
et al.
,
2018). This technique is also suitable for research into
the use and function of artefacts and for deciding issues
of plant domestication and vegetation history (Loy
et al.
,
1992; Hardy
et al.
, 2009; Denham
et al.
, 2003; Fuller
et al.
,
2014; López, 2018, Cagnato, 2018; Albert
et al.
, 2018).
Volume IX ● Issue 1/2018 ● Pages 83–93
*Corresponding author. E-mail: jkovarnik@jcu.cz
ARtiCLe iNFo
Article history
Received: 22
nd
August 2017
Accepted: 11
th
September 2018
DOI: http://dx.doi.org/ 10.24916/iansa.2018.1.6
Keywords:
archaeobotany
starch morphology
amylase
amylopectine
soil
dental calculus
grinding stones
plant subsistence
Stone Age
ABStRACt
Archaeobotanical micro-residuals are today a major focus in artefactual and bioarchaeological
investigations. Though starch grains analysis may be regarded as marginal, it can be a useful analysis
for archaeological research, being a method suitable for the investigation of stone artefacts and ceramic
vessels. Soil samples and dental calculus can also be examined. Through the use of various extraction
methods it is possible to answer questions of diet composition and purpose of stone tool use. As
documented in recent studies examining the composition of the human diet, starch grain research
should be one of the main areas of archaeobotanical investigation. Its applicability can be seen in
studies where it is useful to defne the role of plants in human subsistence. New evidence of plant use
in archaeological contexts in the Stone Age, beginning in the Palaeolithic and ending in the Neolithic,
has been presented in recent papers. Current archaeological studies, including those using starch grain
analyses, have particularly indicated the higher ratio of plants in the diet during the Palaeolithic period.
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However, damaged starch grains can hinder the use of this
particular technique. The results from starch grain analysis
are suitable as complementary analyses to other techniques,
such as palynology, phytolith analysis or plant macroremains
(García-Granero
et al.
, 2015; Pestle, Lafoon, 2018).
The examination of starch grains has improved along
with improvements in microscopic technique. Antonia van
Leeuwenhoek (1632–1723) was the frst scientist to publish
an illustration of starch grains. This Dutch scientist and
microscopist engaged in the observation of natural materials
and created a record of the starch grains of common species
of plants such as wheat, barley, rye, oats, beans, peas, rice
and corn (Hogg, 1854; Britannica, 2016). The work of
Fritzche continued that of Leeuwenhoek. He also recognized
the potential of the heterogeneity of starch grains and its
use for determining the genus and species of plants. It was
only a short step towards the creation of taxonomic keys
and atlases (Torrence, Barton, 2006). The German botanist
and cofounder of cell theory, Matthias Jakob Schleiden
(1804–1881), created a key with his own classifcation based
on starch shape and hilum position. Karl Wilhelm von Nägeli
(1817–1891) continued the study of the structure of starch
(Britannica, 2016; Torrence, Barton, 2006). This Swiss
botanist built on the work of J. M. Schleiden and created a
modifcaton of the starch-grain sorting system (Britannica,
2016), among others we could mention, such as Henry
Figure 1.
Illustration of
Poaceae
starch
grain (Reichert, 1913).
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Kraemer (1868–1924) (Kraemer, 1907). Edward Tyson
Reichert was a physiologist from Philadelphia who created
a comprehensive work summarizing the knowledge of the
study of starches (Figure 1), and described their properties
and use for starch grain identifcation (Reichert, 1913;
Torrence, Barton, 2006). These works found use in everyday
life, especially in the pharmaceutical and food industries,
where there was a need to check the quality and origin of
food and plant products from medicinal plants. For example,
in 1978, William Sedgwick Saunders (London’s Medical
Ofcer for Health) inspected the four sold in London to
prevent it being mixed with gypsum (Stevenson, 2014).
Subsequently, starch atlases were created to recognize
individual species by noting diferences in starch grains
(Loy
et al.
, 1992). Starch analysis has then been put into
use in archaeological research over the last twenty-fve
years. In 2006, Robin Torrence and Huw Barton published
a comprehensive account of starch analyses in archaeology
(Torrence, Barton, 2006). One of the most prominent results
of starch analysis connected with Stone Age artefacts can
be traced to 2007, when the frst results of the human plant
diet at the Palaeolithic Gravettian site were published,
describing the important role of plants in the Palaeolithic diet
(Aranguren
et al.
, 2007). Before Palaeolithic people were
usually regarded as only hunters of large animals. In the last
thirty years, we can see the expansion of starch studies in
archaeology: as described in a recent account by Barton and
Torrence (2015).
In this paper, we summarize some basic knowledge
of starch analyses from the Palaeolithic, Mesolithic and
Neolithic period. Our main focus is on the starch grain itself,
its biology and mode of identifcation of stone implements in
particular. A special section summarizes some of the results
of starch analyses at specifc archaeological sites and with
certain objects.
1.1 Starch
Starch is a polysaccharide used as a reserve energy store
in the majority of autotrophic plants. The exception is
presented by the families Asteraceae, Campanulaceae,
Liliaceae, and others, which store inuline as their reserve
polysaccharide. Starch is a ready source of glucose for
plants, suitable for long storage. It is a composition of
two homopolysaccharides (amylose and amylopectine),
originating from α-D-glukopyranose. Amylose and
amylopectine occur in a weight ratio of 1:3. In some crop
plants, cultivars have been bred with an elevated one or the
other component of starch (amylose or amylopectine) (Prugar,
2008). Amylose is a linear homoglycan consisting of up to
4500 (more often 1000–2000) glucose units. Amylopectine
is a multiple-branched polysaccharide consisting of chains
of 50,000–100,000 D-glucose units. Amylose is a linear
α-D(1-4)-glucane of disaccharide maltose; the branching of
the chain is limited to approximately ten loci per molecule
(Velíšek, 1999; Bemiller, Whistler, 2009).
Starch is synthesised in the green parts of the plant – in
the chloroplasts. There, small starch grains, about 1 µm in
diameter, which are called temporary or transitory starch,
are created. These are further used or transported. Starch
is further stored in special organelles – amyloplasts. Major
quantities of starch are stored in reserve organs in specialized
cells of the seeds, roots and tubers (Bemiller, Whistler, 2009).
Premature fruits also contain starch, but with the ripening
process the starch content decreases and in ripe fruits the
starch hardly occurs. However, there are exceptions, such as
bananas, where high amounts of starch are contained in the
fruit (Velíšek, 1999). Starch is stored in amyloplasts in the
form of starch grains, which are species-specifc and difer
in shape, size and polysaccharide ratio. These starch grain
characteristics are, for the most part, given genetically, but
are also infuenced by external infuences (Selvam, 2013).
According to the crystallinity level of the granules, the starch
can be divided into four forms, designated A, B, C and V.
The variability is due to the internal spatial arrangement
of the molecules. The most stable is form A, which occurs
in cereals, and the least stable is form B, which is found
in root crops and potatoes. The C form is characteristic of
leguminous seeds (it is a mixture of starch form A and B),
whereas gelatinized starches occur in the V form. From a
chemical point of view, starch grains can also contain small
quantities of other substances that occur in plant cells, such
as proteins and lipids (Velíšek, 1999; Ahmed
et al.
, 2016;
Bemiler, Whister, 2009).
1.2 Starch grain
Starch granules occur in various shapes and sizes: round,
kidney-shaped, oval-elongated and polygonal shapes are
common. Granules can be separated or coagulated into
aggregates. Starch grains also difer in size. It is possible to
distinguish several structures and formations, all of which are
used in the identifcation process. For example, the visibility
and position of the hilum is observed: whether it is located in
the centre of the starch granule or of-centre (Lentfer
et al.
,
2002). Sometimes lamellas are visible. These are concentric
lighter and darker stripes on the granule that are circular from
the centre towards the starch edge (Czaja, 1969). They are
more recognizable in larger starch grains and are connected
to the gradual growth of starch grains. They can be divided
into crystalline (a denser part) and semi-crystalline (a softer
part with darker colouring). On the surface of starch grains,
fssures or other superfcial structures can be distinguished.
Furthermore, the bevelling of the starch grain can be
considered (Gott
et al.
, 2006, Bemiller, Whistler, 2009). The
efect of outside, natural or anthropogenic, infuences on
the starch granule can lead to its injury or even destruction
(starch modifcation, swelling, gelatinization). Starch can be
damaged mechanically (
e.g.
broken in the course of milling).
A limiting factor for its preservation can be temperatures
above 50°C, when gelatinization takes place in the presence
of water. The starch grain starts to deteriorate with exposure
to enzymes, the efect of the amylase enzymatic group. Other
harmful infuences are, for example, long water exposure,
low temperatures and charring (carbonization) (Messner
et al.
, 2008; Lentger, 2012).
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2. Methodology of starch analysis
When the starch grain has a certain optical appearance,
the starch can be identifed with optical microscopy in
polarized light by observing the extinction cross on the
starch granule (Figure 2). Further approaches to starch
identifcation encompass chemical methods and staining
methods (Haslam, 2004). Lugol’s solution stains the
starch grains dark blue (Reichert, 1913). Using CongoRed
colouring can be useful for identifying starch damaged by
processing (
e.g.
cooking). After the pigment application, the
starch turns red according to the degree of damage (Lamb,
Loy, 2005). Another staining solution is Trypan blue. Trypan
blue stains damaged starch grains and damaged starch that
has a damaged natural shape, whereas undamaged starch
granules do not stain. (Torrence, Barton, 2006) In some
cases of archaeological investigation the various forms of
starch do not stain well. The starch of individual (plant)
genera and species can also be identifed due to diferent
rates of enzymatic decomposition or observing diferences
in their reaction with acids (Reichert, 1913). For the precise
identifcation of starch grains, a specimen from a reference
catalogue should be used (Haslam, 2004). A reference
catalogue consists of starch grains from the individual parts
of contemporary vegetation. It should contain samples from
crop plants, medicinal plants, commercially-used plants
and other important species occurring in the surrounding
environment. Fresh samples considered for cataloguing
should not be dried at temperatures higher than 35°C.
After drying they can be ground and used to create slides
(Therin
et al.
, 1997, Lentfer 2009, Hart, 2014). The prepared
sample should be examined microscopically and described.
Measured and detected values are then stored electronically,
and the starch grains are also photographed. Results from
the measurement and description of starch grains can then
be employed in creating a statistical method of starch
identifcation from archaeological samples, especially for
determining statistically-signifcant traits and the correlation
between them (Lentfer
et al.
, 2002).
2.1 Sampling of starch from archaeological objects
Starch extraction is conducted by various means, most often
with a pipette directly from the object’s surface, or with the
use of an ultrasonic bath. In the pipetting method a small
quantity of distilled water is placed directly onto the surface
of the examined object (Figure 3), though the place needs
to be carefully selected. The most promising locations for
starch grain conservation are scratches and fssures on the
Figure 2.
Recent grains under diferent
lights (J. Kovárník) a, b) Starch grains
from spring barely (
Hordeum vulgare
) in
cross polarised light. c, d) Starch grins of
Pisum sativum
(Fabaceae) in cross polarised
light. e) Starch grains from spring barley in
normal light. f, g) Starch grain hydrolised by
enzymes.
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surfaces of various objects, or pores in the structure of dishes
(Messner
at al.
, 2008, Li
et al.
, 2013; Pagán-Jiménez
et al.
,
2015, Copeland, Hardy, 2018). The water drop containing
the residues is collected by the pipette, from which it is
possible to create a sample for microscopic analysis. This
technique is suitable for determining the purpose and use of
various objects and their individual parts (Li
et al.
, 2013;
Tao
et al.
, 2011; Yang
et al.
, 2014; Yasui, 2015), allowing an
examination of an object’s specifc function or mode of use.
The other analytic approach is obtaining the sample from the
examined object using sonication. The object is placed into
a clean (sterile) beaker/test tube along with distilled water
and the container inserted into the ultrasonic bath for fve
minutes. The distilled water with the relieved sample particles
is transferred into a test tube and centrifuged at 2500 rpm for
15 minutes. The excess solvent is then removed. Separation
from the denser liquid (ρ=1.8 g.cm
–3
) then takes place, the
lighter fraction containing the starch grains coming to the
surface with the heavier (mineral) fraction remaining on the
test tube bottom. The mixture of sample and dense liquid is
centrifuged for 5 minutes. After that the supernatant (approx.
2 ml) is extracted and subsequently diluted to make the starch
grains form a sediment on the bottom. The microscopic
sample can then be created (Messner
at al.
, 2008; Liu
et al.
,
2010; Marcadert
et al.,
2007). These techniques can, of
course, be combined: it is possible to point-collect samples
with the pipette and subsequently extract residues from the
whole object in the ultrasonic bath (Lantos
et al.
, 2015; Yang
et al.
, 2015; Copeland, Hardy, 2018).
The extracted samples can be stored in a dry condition or in
a tube with an ethanol solution (Therin
et al.
, 1997). One way
for making starch samples are by mounting on a microscopic
slide using only water (in the case of temporary slides) or by
storing dry samples of starch grains on a slide with a cover
glass afxed using clear nail polish. This sample can be stored
in a dry condition and a water drop can be added under the
Figure 3.
Grinding stone from Hrdlovka,
during sampling in the laboratory.
(J. Kovárník).
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cover glass before observation. This method is simple and
allows the observation of starch grains in polarized and non-
polarized light. It also allows repeated observations after
drying and rehydration of the sample. (Piperno, 2006; Coil
et al.
, 2003). Another way is to mount the sample in media to
make a permanent preparation. Mounting media suitable for
starch samples are glycerol (which can be diluted by water
1:1),
Permount
, Euparal, or immersion oil, etc., and the slide
can then be sealed with clear nail polish (Yeung
et al.
, 2015;
Coil
el al.
, 2003). However,
Permount
, for example, has
some disadvantages: the optical properties of
Permount
do
not allow the extinction cross to be observed on small starch
grains. Starch placed into this medium for long periods also
loses its typical refractions and slowly degrades. Starch has
a refractive index of 1.52 to 1.53; for optimal observation
in polarized light mounting media with a higher or lower
refractive index than starch are suitable (Coil
et al.
, 2003;
Piperno, 2006; Wang
et al.
, 2017).
2.2 Sampling of starch from dental calculus
Dental calculus is a layer that forms on teeth, especially
on the tooth base. This layer is formed from biological
plaque after mineralization. This material also contains
other components such as organic materials or chemical
components. It is a rich source of the tracks left by an ancient
life style (Leclerc
et al.
, 2018, Copeland, Hardy, 2018;
Eerkens
et al.
, 2018). Calculus must be carefully pulled
down onto metal foil. After collection, the calculus is placed
in a test tube with 0.6 M HCL for a short time. This step is
for cleaning the calculus and for assuring whether it really is
calculus or another sediment type. The next step is similar
to the previous treatment, the dental calculus is dissolved in
HCl for 5 days at a low temperature (Hardy
et al.
, 2009).
To speed up this extraction procedure it is possible to
crush the larger parts of dental calculus and work with the
resulting powder (Buckley
et al.
, 2014). The fnal solution is
vortexed, centrifuged at 15 min, washed in ultra pure water
and centrifuged again (three times) (Hardy
et al.
, 2009).
Another extraction possibility is using the ultrasonic bath
(Hardy
et al.
, 2012; Madella
et al.
, 2014). A similar method
for dental calculus is described by Cristiani
et al.
, 2016.
2.3 Sampling of starch from soil samples
Starch grains can also be extracted from soils. This
procedure can be complicated because it is necessary to take
the soil samples from a specifc place such as a pore, small
depression or split/crack. However, in such locations there is
a high probability of taking a rich sample. In below-ground
conditions it is harder to preserve intact starch grains. In a
feld experiment that compared the survival rate of starch
grains on grinding stones placed on the ground surface and
below ground, it was shown that the samples taken from the
experimental grinding stones on the soil surface contained
better preserved starch grains (Barton, 2009; Vranová
et al.
,
2015). Despite such observations, starch granules are well
preserved at many archaeological sites (Fullagar
et al.
, 1998;
Therin
et al.
, 1997). The frst step for sample preparation is to
dry the soil in the laboratory. After that, the process is similar
to phytolith extraction including the heavy-liquid fotation
procedure. For separating starch grain from sediment, a
heavy liquid such as cesium chloride (CsCl) or sodium
polytungstate (SPT) should be used. Other chemicals with a
high density can also be used. Starch grain analysis from soil
samples can be usefully applied for land-use reconstruction
(Dickau,
et al.
, 2007; Balme, Beck, 2002; Parr, Carter, 2003).
3. Starch identifcation
Identifcation is carried out by direct observation and by
comparison with specimens from the reference collection.
In a vast collection the most important part is the thorough
description of starch grains, as this will accelerate the
comparison process (Cosgrove
et al.
, 2007; Torrence
et al.
, 2004; Hou
et al.
, 2016). Direct observation can be
accompanied by a statistical evaluation (Cosgrove
et al.
,
2007; Herzog, 2014; Mayle, Iriarte, 2014), for example
multi-dimensional analytic methods, which facilitate
starch-grain identifcation from archaeological fndings
(Lentfer
et al.
, 2002; Torrence
et al.
, 2004). The next step
would be automatic classifcation of starch grains provided
by specialized software with Image analysis modules and
statistics outputs. (Arráiz
et al.
, 2016)
The sample must be prepared carefully and accurately in
a clean laboratory environment and using only starch-free
equipment and chemicals. It is good procedure to: control
the level of contamination in the laboratory; add a control
sample into the set of samples during the whole procedure;
and examine the possibilities of transfer (Bufngton
et al.
,
2018). These simple precautions can reduce the risk of
contamination, but not completely. Other ways to reduce
contamination are complicated and expensive to purchase.
Within this group can be included air fltration, sticky mats,
restricted access, barrier curtains and airlocks (Crowther
et al.
, 2014; Herzog, 2014; Ma
et al.
, 2017).
3.1 Microscopic structures that can look like starch
In nature there exist several structures with similar optical
properties to those of starch. Here is a list of common
structures that look like starch: spherulites, fungal spores,
bordered pits, damaged pollen, wall thickening, algae
(diatoms and coccoliths), and microscopic air bubbles on
the slide. Many of these structures are of the same size as
a starch granule and make a similar optical signal under
polarised light (Torrence, Barton, 2006). Two things that
may help in diferentiating between starch and non-starch
structures are staining the starch grains (iodine staining)
and rotating the birefringence extinction cross (Yeung
et al.
,
2015; Moss, 1976).
3.2 Implications of starch analysis in archaeological
research of the Stone Age
Perhaps the oldest identifcation of starch grains from the
archaeological context of the Stone Age comes from Qesem
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Cave, Israel. The archaeological investigation revealed here
eight hominid teeth in the context of the Amudian industry,
which belongs to the blade-dominated Lower Palaeolithic
Acheulo-Yabrudian Cultural Complex (AYCC) and predates
the Middle Palaeolithic Mousterian traditions (400–250 ka,
Hershkovitz
et al.
, 2011; 2016). The authors present evidence
for potentially inhaled and ingested material in the dental
calculus that was extracted from three of these teeth (Hardy
et al.
, 2016). The identifcation of starch granules and
specifc chemical compounds in the dental calculus samples
from Qesem Cave points to deliberate ingestion of essential
dietary components, most likely in the form of concentrated
sources such as seeds or nuts. The phytoliths and plant fbres
found in the calculus could be the result of non-dietary
activities, including raw material processing, oral hygiene or
food remains.
Interesting evidence regarding a complex of starch
grains was found in Shanidar Cave and Spy Cave (Henry
et al.
, 2011), where the plant residues from tooth calculus
originating from Neanderthal teeth were investigated. The
two sets of samples were compared. The frst came from
Shanidar III, the Shanidar Cave in Iraq. The second set of
samples came from Spy Cave in Belgium (Spy I and Spy
II). In all, seven teeth were sampled: three teeth came from
Shanidar III, two from Spy I and two from Spy II. The
Shanidar Cave samples contained 73 starch grains, and the
Spy Cave samples contained a total of 136 starch grains
(Henry
et al.
, 2011). The Spy Cave samples were 36,000
years old and the samples from Shanidar Cave were about
46,000 years old (Reader, 2011). In addition to the starch
analysis, phytoliths were also analysed. With the results of
both analyses completed, it was found that there had been
heat modifcation of the plant food. The starch granules
were typically damaged by heat. It is considered that larger-
scale plants were used, based on the seasonality of their
maturation. Maturation is an important growing phase for
maturing seeds or starch-rich organs, making them suitable
for harvesting, storing and processing as food (Henry
et al.
,
2011). An important message from this study is the direct
evidence of plant use among the Neanderthals and the role
of plants in their subsistence, a group traditionally regarded
as strongly dependent on hunted animals. The results show
the diference in diet caused by the diferent environments.
But Neanderthals from both the sites had a quite varied diet
(grass seeds, plant underground-storage organs, legumes,
and dates). Henry found that Neanderthals were adaptable,
had good knowledge about their surroundings, and used their
environment very efectively. This is highlighted by their
complicated food behaviour, including heat preparation of
food for better digestibility. The data collected from sediment
could not clearly show which plant material was used for
food. Calculus micro-analysis can also be used for ancient
diet reconstruction. The results from the calculus analysis
also showed that samples taken from diferent teeth from the
same person could provide signifcantly diferent amounts
of information. Two teeth from the same individual were
compared: the calculus from the frst tooth contained a large
amount of starch grains, while the second tooth was poor in
starch grains. This comparison was made at the Spy I site
and repeated at Spy II with the same result. The subsistence
of Neanderthals seems to be much more balanced given the
perspective of starch analysis.
Starch analysis had already been used efectively in a
series of entire archaeological studies of the Gravettian
population belonging already to
Homo sapiens
. Residue
analyses on a grinding tool from Grotta Paglicci in southern
Italy (32,614 ± 429 calibrated (cal) BP), recorded rich
assemblages of starch grains, most probably of
Avena
(oat)
caryopses, and this has substantially enriched our knowledge
concerning the food plants of Palaeolithic Europe in the
context of the Early Gravettian period (Lippi
et al.
, 2015).
The quantitative distribution of the starch grains on the
surface of the grinding stone furnished information about the
tool being used as a pestle grinder. The particular state of
preservation of the starch grains suggests the use of thermal
treatment before grinding, possibly to accelerate the drying
of the plants, making the following process easier and faster
(Lippi
et al.
, 2015). One of the most important results of
starch analysis in archaeology has been made in the case of
Gravettian (the Upper Palaeolithic period) site Bilancino.
The authors have identifed starch grains on the surface
of a grinding stone from the hunter-gatherer campsite of
Bilancino (Florence, Italy), dated to around 25,000 BP.
Analysis has identifed the remains of starches of the wild
plants
typha
and Graminae cf.
Brachypodium.
The stone can
be seen as a grindstone and the starch has been extracted
from locally-growing edible plants. This evidence can be
claimed as implying the making of four – and presumably
some kind of bread – some 15 millennia before the local
“agricultural transition” (Aranguren
et al.
, 2007).
The above-mentioned study from Grotta Paglicci,
Bilancino, and other archaeological sites like the Upper
Gravettian site Dolní Věstonice, clearly indicate the
predisposition of Upper Palaeolithic modern humans for
sophisticated plant exploitation long before the spread of
agricultural knowledge (Revedin
et al.
, 2010; 2015). The
last decade of research has fundamentally changed our view
on how the Paleolithic people exploited plants. Phytolithic
studies, the analysis of starch granules on stone tools, as well
as isotopic and nutritional studies, have newly demonstrated
the signifcant role of plant food in their diet (Beneš, 2018).
This picture seems to be confrmed by micro-remains
analyses of starch from the Palaeolithic period from other
regions of the world. Interesting evidence of ancient starch
has been published by Loy
et al.
(1992), who presented
residue analysis of stone artefacts from the Solomon Islands.
This study described the use of starch analysis on a set of
stone tools from Kilu Cave on Buku Island (Solomon Islands
in Melanesia). In the Kilu Cave profle, evidence was found
of a dual settlement, the oldest occupation being dated at
28,000 years old. Starch analysis was used as direct evidence
of the spread of plant species and to specify the use of tools.
In all, a set of 47 stone tools and fragments were examined,
twenty of them showing no trace of starch grains. Of the
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27 tools that contained starch grains, 8 stone tools came from
younger layers and 17 stone artefacts from older layers (up
to 28,000 years old). Stone artefacts from the older layers
contained starch grains derived from plants of the
Colocasia
and
Alocasia
genera (Loy
et al.
, 1992). This micro-remains
evidence supports our view of the balanced role of plants in
the local Palaeolithic diet in Oceania, despite the fact that
the tropical environment of the Solomon Islands favoured a
plant-based subsistence in general (Bellwood, 2005).
The Late Palaeolithic and Early Neolithic archaeobotanical
knowledge from China concerning starch analysis is very
important. China belongs to the centres of agricultural
beginnings (Bellwood, 2005; Barker, 2006; Beneš, 2018),
where a vast amount of archaeobotanical research has been
recorded in the last decade. China has a large area that
includes boreal landscape and semi-deserts in the north
of the country, temperate deciduous forests, and regions
of subtropical forests in southern China. The two key
developments of the Epipalaeolithic and Early Neolithic
centres were situated in the middle Yangtze basin and in the
upper and middle Yellow River Basin of northern China. A
functional study of two grinding stones (a slab and a grinding
stone) near a burial dated to 9220‒8750 BC cal. (before the
onset of the Neolithic), at Donghulin, investigated the range
of plants exploited during this early occupation period.
Starch residues indicate that the grinding stones were used
for processing plants, and confrm the processing of acorns,
which is consistent with the incidence of oak (
Quercus
) in
the pollen record (Liu
et al.
, 2010b). Similar observations
concerning a younger period have been made in the case of
the Early Neolithic Peiligang culture sites Shigou and Egou
in the catchment of the Yellow River, where starch analysis
on grinding stones has documented the presence of acorns
and other plants, probably
Cyclobalanopsis
,
Lithocarpus
,
Dioscorea
,
trapa
and others (Liu
et al.
, 2010a). The above-
mentioned examples indicate the unchanged plant processing
customs of people in the Late Palaeolithic and Early Neolithic
period refecting a continual development from a hunter-
gatherer economy to the stages of early agriculture. The use
of archaeological artefacts in this context plays an important
bioindicative role as indicated by the starch analysis.
In recent years some new observations have been made in
the feld of Stone Age research concerning Mesolithic and
Neolithic Europe. The most interesting evidence associated
with starch grains has been made during an investigation
of the dental calculus of Mesolithic human teeth at the
site of Vlasac in the Danube Gorges of the central Balkans
(Cristiani
et al.
, 2016). The research has provided direct
evidence that already by 6600 cal. BC, if not earlier, the Late
Mesolithic foragers here consumed domestic cereals, such
as
triticum monococcum
,
triticum dicoccum
, and
Hordeum
distichon
. These plant species were the main crops found
among the Early Neolithic communities of southeast Europe.
This investigation indicates the great value of starch grain
identifcation in the Mesolithic human population and their
interaction with the Neolithic people from the southernmost
Balkans several centuries before the Neolithic package of
domesticated plants and (probably) Neolithic people reached
the inland areas of the Balkans around 6200–6000 BC cal
(Kreuz
et al.
, 2005; Brami, 2017).
Another interesting example, again from the Balkan
region, combines starch and phytolith analysis. At the
late Middle and early Late Neolithic site of Stavroupoli,
Thessaloniki, Greece, a series of organic remains from
ceramic sherds were studied. The research focused on matter
recovered from cooking vessels, where charred food crusts
had adhered to the inner walls of 17 late Middle and early
Late Neolithic vessels (ca. 5600–5000 BC cal.). Starch
grains were very scarce or absent in most of the samples, but
samples ST129 and ST192 presented a high concentration
of small Panicoideae and Triticeae grains (García-Granero
et al.
, 2017), as well as big and small types of Panicoideae.
With supporting phytolith analysis, the research authors
concluded that the analysis of starch grains and phytoliths
from charred food crust on the ceramics added valuable
information to previous studies on the diet in Neolithic
Stavroupoli. Especially remarkable is the identifcation of
the use of the potentially wild weed
Setaria
in the diet.
Research of starch remains was also successful in
the central European Neolithic context. In Tiszasziget-
Agyagbánya in southeastern Hungary, a site belonging to the
Late Neolithic Tisza culture (ca. 5000–4500 BC cal.) was
investigated. Here in a pit associated with postholes – the
remains of a longhouse – three intact vessels and one grinding
stone were unearthed. The layout of objects in the pit evokes
its ritual character. The unearthed vessels were situated in
the hole of the former central post of the building’s structure.
The subject of interest by microscopic and biochemical
analysis was organic matter from the bottom of the vessels.
The methods used were complex, including the study of
phytoliths, starch and biochemical signals (Pető
et al.
, 2013).
Phytolithic morphologies present in the samples have their
anatomical origin from the leaf and inforescence of
Poaceae
(as well as cereals). Among the phytoliths, which indicate
the predominance of grasses, fve partly-damaged starches
were observed. Based on their identifcation, the starches
from the sample most likely represent wheat (
triticum
sp.).
Starch analysis has been carried out in the case of a set
of grinding stones from the Neolithic site of Hrdlovka
(Northwest Bohemia, Czech Republic). The samples
were taken from grindstones deposited in feature 838 and
dated to 4620–4458 BC cal. and were situated in the area
of house 8 from the Late SBK period. The deposition of
millstones could have a ritual or social meaning connected
with the foundation of the house. Altogether 8 grinding
stones were analysed for the presence of starch grains and
their surfaces revealed 49 starch grains (Beneš
et al.
, 2015).
Overall, it was possible to determine 12 circular starch
grains as belonging to plants of the family
Poaceae
. Starch
grains of an oval shape with the characteristic extinction
cross belonged to the family
Fabaceae
. Determination of
starch grains from the Neolithic Hrdlovka site represents
the frst such positive analysis of micro-remains in the
Czech Republic.
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The last above-mentioned examples from central Europe
represent cases of a similar archaeological situation, where
artefacts from sunken features dating to the Neolithic period
played a central role, as well as a specifc, possibly ritual,
meaning for both artefact deposits. In both cases starch analysis
contributed to specifying their role in the Neolithic community.
4. Conclusion
Starch grain analysis is a useful tool for evaluating
archeobotanical specimens from soil samples, artefacts, and
from human teeth. Using starch grain analysis, it is possible
to identify plants that are in a non-fowering growth phase
and would not be identifed by pollen analysis. A further
great advantage is that humans preferred plants with a large
amount of starch. Those plants, and their individual organs
with a high starch content, were used as energy sources from
human nutrition. People used and processed a lot of material
that can be identifed in archaeological contexts. However,
for the same reason, there is a risk of contamination of the
sample from present-day materials containing starch grains.
Unfortunately, starch grains are relatively susceptible to
environmental conditions, such as those not of optimal
temperature and neutral pH. Another disadvantage is the
inability to capture plants from the family Asteraceae, as
they do not form starch but inulin. Starch grain analysis is
more efective in combination with other bioarchaeological
methods, such as pollen, phytolith determinations and
analysis of botanical macroremains. Hence the method of
starch analysis is also notably useful in combination with
biochemical analysis targeted at a specifc group such as
amino acids or lipids.
In general, starch analysis constitutes a useful tool in
bioarchaeological investigations. Despite its relatively
limited taxonomical power, it is an efective supporting
method in the functional determination of artefacts such as
millstones and grinders, and a key source of information in
the bioarchaeological research of food remains on pottery.
It is also possible to point towards the propagation, use and
processing of plant species. The anthropological application
of this method targeted on dental calculus is also particularly
important. Current studies of these biological traces have
shed new light on the ratios between meat- and plant-based
diets. Some results clearly indicate a higher ratio of plant
diets among Palaeolithic human populations than previously
thought. In future research, starch analysis – together with
pollen and phytolith analyses – will form a crucial basis for
functional bioarchaeological interpretation.
Acknowledgements
This article was supported by the OPVK project PAPAVER
– Centre for human and plant studies in Europe and
Northern Africa in the post-glacial period, registration no.
CZ.1.07/2.3.00/20.0289.
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