Morphometric distinction between Acute bulbosus phytoliths (silicified epidermal hair cells) from Panicum miliaceum and Setaria italica leaves

Abstract

The taxonomic differentiation of Panicum miliaceu (broomcorn millet) and Setaria italica (foxtail millet) is of high relevance for archaeology and archaeobotany. The identification of these millets heavily relies on the morphology of the phytoliths in the inflorescence bracts (husks), while other plant parts have been less researched. This study offers a meticulous examination of a distinctive variety of phytoliths in millet leaves, Acute bulbosus. It carries out a comparative analysis of examples of these from modern P. miliaceum and S. italica leaves. To support the robustness of our findings, a case study was done using pieces of burnt clay daub excavated from the Shuanghuaishu site, central China (5,290–4,527 cal bp), which showed clear impressions of millet. The results agree with parallel investigations of diagnostic phytoliths extracted from millet husks, in addition to ethnographic observations of the uses of millet. This research establishes the possibility of differentiating between the leaves of Panicum miliaceum and Setaria italica, from the dimensions and shapes of the Acute bulbosus phytoliths there. These distinguishing features are quantified using a range of criteria and show the potential for identifying the remains of leaves of S. italica and P. miliaceum from archaeological contexts by using morphometric distinctions between the Acute bulbosus phytoliths.

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Vol.:(0123456789)
Vegetation History and Archaeobotany
https://doi.org/10.1007/s00334-024-00999-5
ORIGINAL ARTICLE
Morphometric distinction betweenAcute bulbosus phytoliths (silicified
epidermal hair cells) fromPanicum miliaceum andSetaria italica leaves
TingAn1 · JiaxinChen1· YunfeiZheng2· ShudeHuo1· WanfaGu3
Received: 17 August 2023 / Accepted: 19 February 2024
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024
Abstract
The taxonomic differentiation of Panicum miliaceu (broomcorn millet) and Setaria italica (foxtail millet) is of high relevance
for archaeology and archaeobotany. The identification of these millets heavily relies on the morphology of the phytoliths in
the inflorescence bracts (husks), while other plant parts have been less researched. This study offers a meticulous examina-
tion of a distinctive variety of phytoliths in millet leaves, Acute bulbosus. It carries out a comparative analysis of examples
of these from modern P. miliaceum and S. italica leaves. To support the robustness of our findings, a case study was done
using pieces of burnt clay daub excavated from the Shuanghuaishu site, central China (5,290–4,527cal bp), which showed
clear impressions of millet. The results agree with parallel investigations of diagnostic phytoliths extracted from millet husks,
in addition to ethnographic observations of the uses of millet. This research establishes the possibility of differentiating
between the leaves of Panicum miliaceum and Setaria italica, from the dimensions and shapes of the Acute bulbosus phy-
toliths there. These distinguishing features are quantified using a range of criteria and show the potential for identifying the
remains of leaves of S. italica and P. miliaceum from archaeological contexts by using morphometric distinctions between
the Acute bulbosus phytoliths.
Keywords Leaf phytolith· Acute bulbosus· Millet agriculture· Dry farming
Introduction
Setaria italica (foxtail millet) and Panicum miliaceum
(broomcorn millet) are some of the most ancient culti-
vated foods in human history (Lu etal. 2009; Zhao 2020).
Today, they remain important crops in northern China and
in many other parts of the world. The analysis of botanical
macroremains from many Neolithic sites in northern China
has indicated that P. miliaceum was common during the
Early Neolithic period (Qin 2012; Zhao 2014). This domi-
nance gradually gave way to S. italica around 6,000years
bp. However, a comparative assessment of phytoliths from
archaeological sites in the same area has yielded divergent
results (Zhang etal. 2010; Wang etal. 2018), thereby chal-
lenging the conclusions drawn from identifying the macrore-
mains. This inconsistency emphasizes the need to explore
alternative approaches for identifying millets to study the
diverse patterns of millet growing and use at different places
and dates.
Previous research has mostly concentrated on distin-
guishing the grains of P. miliaceum and S. italica (such
as Liu and Kong 2004; Zhao and Fang 2007). However,
millet leaves, which comprise a larger portion of the plant,
are much less detected. Leaves represent by-products of
cereal harvests, and they had much economic significance.
They were used in various ways by different communities,
for example as livestock fodder, temper for building mate-
rials such as daub, crafts and weaving, medicinal uses,
food ingredients and more (for example, Grubben and
Partohardjono 1996; di Lernia etal. 2012; Li etal. 2020;
Teira-Brión 2022). Simultaneously, carbon and nitrogen
isotope analyses have found the use of millet as fodder for
livestock (Chen etal. 2012; Yang etal. 2023). However,
Communicated by K. Neumann.
* Ting An
jiezian@zju.edu.cn
1 School ofArt andArchaeology, Zhejiang University,
Hangzhou310000, China
2 Zhejiang Institute ofCultural Relics andArchaeology,
Hangzhou310014, China
3 Zhengzhou Institute ofCultural Relics andArchaeology,
Zhengzhou450007, China

Vegetation History and Archaeobotany
the varied ways in which broomcorn and foxtail millets
were used have scarcely been touched. The exploration of
taxonomic identification methods for millet leaves holds
great relevance for archaeology and archaeobotany.
Phytoliths, composed of biogenic opal, offer valuable
applications in the identification of plant parts (such as
culms, leaves and inflorescences) and in understanding
their use through the study of the remains of food prod-
ucts, building material, tempering agents and crop process-
ing residues (Out and Madella 2016). Previous phytolith
identifications of P. miliaceum and S. italica are to a great
extent based on the morphology of the phytoliths in husks
(for example, Lu etal. 2009; Shang etal. 2016; Weisskopf
and Lee 2016), with other plant parts receiving much less
attention. In particular, Lu etal. (2009) described crucial
diagnostic morphological characters of phytoliths from
husks of Panicum miliaceum and Setaria italica, which
have found widespread application in the identification of
these taxa from archaeological microremains (for exam-
ple, Zhang etal. 2010; Yang etal. 2020; An etal. 2022;
Laugier etal. 2022; Wang etal. 2022).
There are a few studies on the systematics of Pani-
coideae based on phytoliths from millet leaves (such as
Renvoize 1987; Ellis 1988; Zucol 1998; Krishnan etal.
2000; Lu and Liu 2003; Fahmy 2008; Shaheen etal. 2011,
2012). However, these have mainly concentrated on wild
taxa, with limited attention devoted to the analysis of P.
miliaceum and S. italica. In contrast, considerable work
has been done on bilobate phytoliths found in millet leaves,
alongside other distinctive phytolith types, as in studies by
Renvoize (1987), Lu and Liu (2003), Fahmy (2008) and
Out and Madella (2016). Notably, Out and Madella (2016,
2019) used morphometric analysis to investigate bilobate
phytoliths and short cell assemblages from P. miliaceum
and S. italica leaves. Nevertheless, the application of
their findings to differentiate between these two taxa from
archaeological remains is largely unexplored. Addition-
ally, systematic research on other characteristic phytoliths
present in millet leaves has been limited.
This study aims to investigate a specific type of phyto-
lith called Acute bulbo sus, which are formed as entirely
silicified epidermal hair cells. It aims to determine whether
measurements can differentiate between these phytoliths
in P. miliaceum and S. italica leaves. The study is mainly
on these phytoliths due to their common occurrence and
abundance in archaeological assemblages. The main ques-
tions addressed in this study are as follows:
Can morphometry effectively distinguish Acute bul -
bos us phytoliths from P. miliaceum and from S. italica
leaves?
Are the diagnostic features of these phytoliths applicable
for distinguishing between the two taxa in an archaeological
context?
The findings will contribute to understanding the culti-
vation and use of these two millets in ancient agriculture,
providing valuable insights into the versatile roles of millet
leaves in past societies.
Materials andmethods
In this study, we investigate the morphology of distinct
leaf phytoliths found in modern P. miliaceum and S. ital-
ica leaves in order to establish criteria for differentiation
between the two species. We then applied these criteria to
archaeological samples to determine the viability of using
our diagnostic features for identification. Specifically, we
made a case study using pieces of burnt clay daub from the
remains of wattle and daub structures excavated from the
site of Shuanghuishu (5,290–4,527cal bp) in Henan prov-
ince, China. The site is associated with millet growing, as
indicated by both macroremains and husk phytolith analyses
(Sun etal. 2023; Zhong and Zhao 2023).
Acute bulb osus phytoliths have been found not only
in millet leaves but also in other plants belonging to the
Poaceae and Cyperaceae families (for example, Brown 1984;
Piperno 1988, 2006; Strömberg 2003), including Equisetum,
Selaginella, certain dicotyledons and Arecaceae (Piperno
1988; Strömberg 2003). However, in cases where there is
independent evidence of the range of taxa present, as there
was for the Shuanghuaishu daub, any variations in Acute
bulbo sus are likely to represent the differences between
those in P. miliaceum and in S. italica leaves.
This study used four modern populations of P. miliaceum
and S. italica, provided by farmers from Dalian in Liaoning
Province, Chaoyang in Liaoning Province, Yulin in Shaanxi
Province and Chifeng in Inner Mongolia. To ensure consist-
ency and minimize potential variations in leaf development
and silicification, a systematic approach was adopted for leaf
sampling, in which 10–15 leaves from the upper part of the
plant, just below the highest leaf, were randomly selected
from two plants per population. The lowest and highest
leaves were avoided as they might have significant differ-
ences in leaf development and silicification. Leaf blades
were mainly sampled, as they are more taxonomically
relevant according to Metcalfe (1960). Additionally, they
may have higher levels of silica deposition due to increased
evapo-transpiration, as indicated by Prychid etal. (2003) and
Chauhan etal. (2011). In contrast, leaf sheaths, which are
tougher, are mainly found on the lower part of the plant and
were not included in our sampling procedure.
The archaeological samples which we investigated were
obtained from the pieces of daub found at the site of Shuan-
ghuaishu in Henan province, which is dated to the middle
and late Yangshao Culture and Longshan Culture, ranging
from approximately 5,290–4,527cal bp (Fig.1). Previous

Vegetation History and Archaeobotany
archaeobotanical research at the same site by Zhong and
Zhao (2023) has revealed a significant presence of charred
P. miliaceum and S. italica grains. Additionally, there are
impressions of millet grains and stems both on and within
the daub (Fig.2, images with better resolution provided in
ESM1). For this investigation, a total of ten samples were
cut from two fragments of daub, a and b, which show clearly
visible millet grains and impressions. Fragment a came from
an ash pit while fragment b came from Level 4 (Fig.2), both
dated to the transitional period from the late Yangshao to
Longshan period by the typology of the finds. Essentially,
these pieces of daub are remnants of ancient building mate-
rials, offering valuable clues to the building techniques and
material culture of the prehistoric community that inhabited
the site.
We studied the Acute bulbosus phytoliths in several
ways, first by surface views on the leaves, secondly by
extracting phytoliths from leaf samples and thirdly from
archaeological daub samples.
The ash patterns left by burnt plant parts (spodograms),
which provide surface views of the phytoliths insitu, were
prepared following this specific method:
1. Labelling and rinsing: the leaf samples were first
labelled for identification and then rinsed with distilled
water to remove any surface impurities or contaminants.
2. Air drying and clamping: two glass slides were used
to hold each sample in place between them and then
clamped securely with clips. This setup allowed for
proper air drying of the samples.
3. Baking in a muffle furnace: the clamped glass slides
with the samples were then placed in a muffle furnace
and heated until the tissues reached a whitish colour,
indicating successful preparation.
4. Cooling and slide preparation: after the heating process,
the samples were allowed to cool down. Then one of
the glass slides was cautiously removed, revealing the
phytoliths in place on the exposed surface.
5. Making microscope slides: the burnt leaf tissue prepara-
tion, showing the phytoliths in position, was then used to
make microscope slides. Neutral balsam was applied to
fix the specimen on the slide, ensuring its stability and
preservation for study with a microscope.
Secondly, leaf samples for morphometric analysis of the
phytoliths were prepared using a sedimentation method. This
was efficient and allowed us to observe and measure the
Acute bu lbosus phytoliths of P. miliaceum and S. italica
accurately. A step-by-step description of the preparation
process is as follows:
1. Cleaning and drying: the labelled leaf samples were
first cleaned using distilled water and were put in an
ultrasound bath to remove any surface particles and then
thoroughly dried.
2. Cutting and weighing: the dried samples were carefully
cut into manageable pieces and weighed to ensure accu-
rate measurements during the subsequent steps.
3. Oxidation and removal of organic inclusions: to elimi-
nate organic inclusions and achieve complete oxidation,
the samples were placed in a crucible and heated to a
high temperature in a muffle furnace.
4. Sedimentation and particle removal: after the heating
process, 1g of each sample was randomly selected. The
remaining particles were removed through a sedimenta-
tion process. The samples were then dried and weighed
again.
5. Mixing with glass beads: to facilitate even distribu-
tion during microscopic observation and morphologi-
cal measurement, the samples were evenly mixed with
0.0225g of glass beads, roughly 30–40μm, similar in
size to the phytoliths.
6. Microscopic slide preparation: finally, microscope slides
were prepared using neutral balsam.
The morphometric analysis of the Acute bulbosus phy-
toliths (epidermal hairs) in this study involved qualitative
descriptions and identifications at 10 × and 40 × magnifi-
cation using the Motic Panthera i biological microscope.
There are two types of Acute bulbosus phytoliths, those
with a single protrusion and others with two (Fig.3). For
each phytolith, two sets of variables were measured using
the MotiConnect software app: the length and width, and the
protrusion length and length of the whole Acute bul bosus
phytolith. These measurements were used to quantify the
overall shape and dimensions of each phytolith under inves-
tigation, and then a comparative analysis was done with the
data from the P. miliaceum and the S. italica leaves.
Fig. 1 Location of the Shuanghuaishu site in Henan Province, central
China

Vegetation History and Archaeobotany
A total of 50 Acute bulbosus, comprising both intact and
fragmented specimens, were selected at random from leaves
of P. miliaceum and S. italica for the analysis. It is note-
worthy that broken pointed ends are evident in numerous
instances of Acute bulbosus with single (long) protrusions
from both P. miliaceum and S. italica leaves. This suggests
that both complete and incomplete Acute bulbosus phyto-
liths, such as those with broken pointed ends, are likely to
be encountered in archaeological samples as well. Therefore,
as well as the intact examples, these incomplete ones were
included and analysed.
To quantify the yield of Acute bulbosus phytoliths pro-
duced per gram of dried leaves, the following formula was
used:
Number of Acute bulbosus per 1g sample = (number of
Acute bulbos us counted × number of glass beads added) /
(number of glass beads counted × weight of sample).
This calculation provided the concentration of Acute
bulbosus in the samples, thereby enabling a comparative
assessment of their abundance in either P. miliaceum or S.
italica leaves.
Thirdly, for the archaeological samples, instead of using
the sedimentation method used for the modern material, a
Fig. 2 The two pieces of burnt clay daub with millet impres-
sions investigated in the current study. a Daub fragment (code
2020HGSIT3448H1045), from an ash pit and dated to the transitional
period from late Yangshao to Longshan period; b Daub fragment
2020HGSIT1453, from Level 4 and the same date as (a); c–g Photos
of millet impressions from daub fragment 2020HGSIT3448H1045; h
SEM image of a silicone cast made from a grain impression of Pani-
cum miliaceum from daub fragment 2020HGSIT1453; i MicroCT
image of a Setaria italica grain from daub fragment 2020HGSIT1453

Vegetation History and Archaeobotany
heavy liquid separation method was used to extract phy-
toliths from the daub. This ensured the efficient extraction
and purification of phytoliths. The detailed procedure is as
follows:
1. Cleaning and pretreatment: the daub sample was
cleaned, crushed and finely ground using a pestle and
mortar to ensure proper preparation for phytolith extrac-
tion.
2. Weighing and mixing: a 1g subsample of the pretreated
material was accurately measured and placed in a test
tube. Approximately 0.0225g of glass beads was added
to the sample and mixed thoroughly.
3. Removal of iron oxide: this was done by adding 4ml
of 1mol/l dilute hydrochloric acid to the sample. After
ultrasonic treatment and centrifugation at 3000rpm for
15min, the supernatant was discarded, and the sample
was left to stand overnight.
4. Removal of organic matter: hydrogen peroxide was
added to the sample to remove any residual organic mat-
ter from the daub. The sample was allowed to stand for
3–4h after sonication.
5. Hexametaphosphate treatment: 4ml of 5% sodium
hexametaphosphate solution was added to the sample
and it was left standing for 2–5h to disperse clay parti-
cles. Then the sample was centrifuged at 3000rpm for
15min, the supernatant was discarded, and the sam-
ple was washed three times by adding 4ml of purified
water, shaking, washing and centrifuging at 3000rpm
to remove any remaining impurities.
6. Heavy liquid separation: the sample was mixed with
6ml of 2.30 specific gravity heavy solution (cadmium
polytungstate), then shaken and centrifuged at 1500rpm
for 30min. The top layer of heavy liquid was pipetted
out and 14ml of pure water was added for further wash-
ing. The sample was then centrifuged at 3000rpm and
the top layer of liquid was discarded.
7. Drying and slide preparation: the washed and separated
sample was allowed to dry. Once dry, the phytolith sam-
ples were transferred to slides, fixed with neutral balsam,
and a cover slip added for subsequent analysis with a
microscope.
The analysis of Acute bulbosus from the archaeological
samples followed a similar approach to that used for the
modern examples. Given the small numbers of these phyto-
liths in the archaeological samples, all examples, including
both intact and fragmented specimens, were photographed
for analysis later. The following procedure was used:
1. Qualitative description and classification: The Acute
bulbosus from the archaeological samples were quali-
tatively described and classified at 10 × and 40 × magni-
fication using a Motic Panthera biological microscope.
This step involved observing and recording the morpho-
logical characteristics and features of the phytoliths.
2. Concentration assessment: the concentration of Acute
bulbo sus was determined by counting the number of
phytoliths per 600 glass beads. This count provided a
quantitative measurement of the amount of these phy-
toliths in the archaeological samples.
3. Photography: the phytoliths were photographed using a
stereo microscope at both 10 × and 40 × . These images
served as visual documentation and reference for further
analysis.
4. Morphological measurements: these were taken using
the MotiConnect app, which ensured accurate and stand-
ardized measurements, contributing to reliable data anal-
ysis.
Results
Acute bulbosus phytoliths in modern material, according
to our analysis of leaves reduced to ash, derive from the
epidermal hair cells of leaves (Fig.4). There were notable
differences in the numbers of these phytoliths in either Pani-
cum miliaceum or Setaria italica leaves (Table1).
The dried leaves of S. italica from Dalian, Chaoyang,
Yulin and Chifeng yielded 1.86 × 105, 3.68 × 105, 3.82 × 105
and 1.06 × 105 Acu te bul bosus phytoliths from each 1g
sample, whereas the same amount of P. miliaceum produced
1.50 × 104, 7.48 × 103, 2.59 × 104 and 6.88 × 102. In all cases,
S. italica produced 12 times more Acute bulbosus than P.
miliaceum per gram of leaf material.
Moreover, the shapes of the Acute bulbosus phytoliths
from S. italica and P. miliaceum leaves were also noticeably
Fig. 3 Morphometric measurement method used for the analysis of
Acute bulbosus phytoliths which are divided into two types: a With a
single protrusion; b A double protrusion

Vegetation History and Archaeobotany
different (ESM2 and 3). Among the samples investigated,
the majority of Acute bulbosus from S. italica leaves in
all four populations had two protrusions (ESM2), whereas
the majority of those from P. miliaceum leaves had just one
(ESM3; Fig.5).
The two species can also be effectively distinguished
by their size differences (Fig.6). The length range of
Acute bulbos us from S. italica is mostly between 25 and
60μm, while in P. miliaceum it is longer, ranging from
50 to 120μm. Moreover, the length of the protrusion in
these phytoliths from S. italica ranges from 2 to 10μm,
whereas in P. miliaceum, it is mostly between 8 and 40μm.
In simple terms, these phytoliths from P. miliaceum are
typically longer and with larger protrusions than those
of S. italica. There is a significant overlap in the width
of these phytoliths between S. italica and P. miliaceum.
However, the width range of the Acute bulbosus from
P. miliaceum leaves (4–50μm) is wider than that from S.
italica (15–35μm).
Our analyses of the Shuanghuaishu archaeological sam-
ples have revealed the presence of Acute bulbosus in all ten
samples (A-J, Table2). Among the 154 of these phytoliths
investigated from the Shuanghuaishu samples (ESM4), the
majority (60%, N = 92) have only one protrusion (Fig.7),
resembling the pattern observed in modern P. miliaceum
leaves (Fig.5).
Fig. 4 Location of Acute bul-
bosus phytoliths in epidermal
hair cells of the millet leaves
under study. The phytoliths may
be distributed along the surface
of the hair cell or concentrated
at specific points, such as the
tips or bases of the trichomes.
The boundary between the
Acute bulbosus phytolith
and the hair cell is not always
sharply defined. a,b Phytoliths
with two protrusions from
Setaria italica leaves; c,d With
one protrusion from Panicum
miliaceum leaves
Table 1 Acute bulbosus
phytoliths from modern Setaria
italica and Panicum miliaceum
leaves in the current study. Dry
weight here refers to air dried
but not ashed tissue
Sampling location Millet species Dry weight (g) Ash weight (g) Glass bead
weight (g)
Glass bead
number
(pcs)
Acute
bulbosus
phytoliths,
protru-
sions
One Two
Dalian S. italica 0.68 0.08 0.0225 302 23 104
P. miliaceum 0.98 0.1 0.0224 305 14 1
Chaoyang S. italica 0.93 0.0815 0.0226 302 78 265
P. miliaceum 0.86 0.0522 0.0209 305 7 0
Chifeng S. italica 0.69 0.0693 0.0236 314 2 71
P. miliaceum 0.15 0.0109 0.0024 310 1 0
Yulin S. italica 0.71 0.0798 0.0210 312 30 272
P. miliaceum 0.65 0.0570 0.0122 309 29 3

Vegetation History and Archaeobotany
The distributions of length and width data of the Shuang-
huaishu samples overlap considerably with those of P. mili-
aceum (Fig.8a). The length of Acu te bulb osus from the
Shuanghuaishu samples mainly ranged from 50 to 100μm,
bringing them closer to those of P. miliaceum (50–120μm).
The width of Acute bulbosus from the Shuanghuaishu sam-
ples, ranging from 14 to 60μm, is closer to P. miliaceum
(4–50μm) than S. italica (15–35μm). Furthermore, the pro-
trusion length of the Shuanghuaishu samples (2–35μm) is
closer to those of P. miliaceum (8–40μm), despite a notable
overlap with S. italica (2–10μm) as shown in Fig.8b.
Discussion
Our analysis of phytoliths from four modern populations of
the two millets establishes the feasibility of distinguishing
leaves of Panicum miliaceum from those of Setaria italica
based on the shape, dimensions and number of their Acute
bulbosus phytoliths per gram of leaf material. Morphometric
identification criteria include the length, width, protrusion
length and the number of protrusions.
The primary distinction between these phytoliths from
P. miliaceum and S. italica lies in their shape, specifically
the presence of one or two protrusions. There is also a
notable difference in the dimensions of the phytoliths,
with those from P. miliaceum generally being longer and
larger than those from S. italica, so that the length range
of Ac ute b ulbo sus from S. italica is mainly between 25
and 60μm, while in P. miliaceum it ranges from 50 to
120μm. The protrusion length of Acute bu lbosus from S.
italica ranges from 2 to 10μm, whereas in P. miliaceum,
it is mostly from 8 to 40μm. There is a notable overlap
between the width of Acute b ulbosu s from S. italica and
P. miliaceum leaves. However, the width range of those
from P. miliaceum (4–50μm) leaves is wider than from S.
italica (15–35μm).
The contrast between the dimensions of P. miliaceum
and S. italica phytoliths agrees with a recent ethnological
investigation by the authors in West Henan province (pers.
comm.). Through an interview with Li Fengxue, a local
farmer whose family has grown both millet species for gen-
erations, it was revealed that P. miliaceum is preferred as
temper for the traditional building technique in which stalks
are mixed with mud or clay to make daub. In contrast, S.
italica stalks are usually used as animal fodder. According to
Li, livestock show a preference for foxtail millet stalks over
broomcorn millet due to the latter’s bitterness. However, it
should be noted that this “bitterness” may be more accu-
rately described as toughness (ge-zui咯嘴 in Chinese). The
longer and larger Acute bulbosus phytoliths in P. miliaceum
leaves, as revealed in the current study, give them a coarser
texture compared to those of S. italica, presumably making
them less palatable to the livestock.
Furthermore, an equivalent quantity of leaves from S. ital-
ica yielded a significantly larger amount of Acute bulbosus
compared to P. miliaceum (Table1). Although the numbers
of these phytoliths per gram leaf from each species varied
slightly between different populations, a clear distinction
between the two species was consistently observed. In our
study, S. italica produced more than 12 times more Acute
bulbosus phytoliths than P. miliaceum.
The dimensions and shapes of the Acut e b ul bos us
from the Shuanghuaishu archaeological samples closely
Fig. 5 Number of protrusions
in Acute bulbosus phytoliths in
the modern Setaria italica and
Panicum miliaceum leaves from
the sites under study

Vegetation History and Archaeobotany
Fig. 6 Scatter plot of Acute
bulbosus phytolith measure-
ments from Setaria italica and
Panicum miliaceum leaves; a
Phytolith length and width; b
Phytolith length and protrusion
length

Vegetation History and Archaeobotany
resembled those found in modern P. miliaceum leaves,
the majority of which feature a single protrusion and are
longer and larger than those from S. italica leaves. The
noted overlap in protrusion length between the Shuang-
huaishu samples (2–40μm) and S. italica ones (2–10μm)
could be attributed to the inclusion of incomplete phy-
toliths in the analysis. This inclusion involves phytoliths
with a single long protrusion and a broken pointed end,
which, according to our observations, are numerous in the
Shuanghuaishu samples.
Moreover, a 1g sample of dried leaves from S. italica
yielded more than 12 times the quantity of Acute bulbosus
compared to P. miliaceum (Table1). Consequently, if these
phytoliths in an archaeological assemblage show a closer
resemblance to those of P. miliaceum, which typically pro-
duces fewer phytoliths than S. italica, it is reasonable to infer
that a significantly larger amount of broomcorn millet leaves
would have been incorporated into the Shuanghuaishu daub.
If the observed pattern holds true across a broader con-
text, our results agree with the findings from other analyses
of Shuanghuaishu samples, from the phytoliths of millet
husks (Sun etal. 2023). However, they diverge from mac-
roremains analyses of the same site, which report a higher
abundance of Setaria italica grains compared to Panicum
miliaceum (Zhong and Zhao 2023). In other words, the mac-
rofossil results from flotation and charred grain identification
show foxtail millet as the dominant crop in the late Yangshao
period, whereas phytolith analyses, either of millet husks or
leaves as in our case, suggest broomcorn millet as the main
crop instead. This discrepancy, which has also been noticed
from various sites in northern China (Qin 2012; Zhao 2014),
calls for a more thorough investigation.
On the one hand, the agreement between our results and
parallel investigations of diagnostic phytoliths from millet
husks shows the potential underestimation of the dominance
of broomcorn millet when relying exclusively on macrobot-
anical results. Wang and Lu (2020) attribute this difference
in results to variations in carbonization temperature, which
may mean that P. miliaceum grains are less easily charred
and thus preserved compared to S. italica. In contrast, phyto-
lith identifications of both millet leaves and husks may more
accurately reflect the actual proportions of broomcorn and
foxtail millet which were grown during the late Yangshao
period.
On the other hand, the discrepancy between the results
from charred grain identification and those from phytolith
analysis of daub may be attributed to the different archaeo-
logical contexts from which the respective samples origi-
nated. Macro remains are typically extracted from soil
samples and usually relate to subsistence activities, while
daub is linked to building materials, representing a differ-
ent aspect of human-plant interaction. As previously men-
tioned, the ethnological research in Henan suggested that
Table 2 Presence of Acute
bulbosus phytoliths in burnt
clay daub fragments from
Shuanghuaishu
Daub sample code Sample letter Sample dry
weight (g)
Glass bead
weight (g)
Glass bead
number (pcs)
Acute
bulbosus
phytoliths,
protrusions
One Two
2020HGSIT3448
H1045
A 0.99 0.0223 603 5 2
B 1.00 0.0232 601 10 8
C 0.98 0.0216 612 17 13
D 1.03 0.0220 601 5 3
E 0.98 0.0224 605 15 9
2020HGSIT1453 F 0.97 0.0236 611 9 6
G 0.98 0.0241 602 3 2
H 1.01 0.0225 603 11 8
I 1.01 0.0223 612 9 6
J 1.00 0.0221 606 8 5
Fig. 7 Protrusion number in Acute bulbosus phytoliths from Shuan-
ghuaishu

Vegetation History and Archaeobotany
S. italica leaves and stalks are preferred for animal fodder,
whereas P. miliaceum leaves and stalks are frequently mixed
with mud or clay to make building material such as daub.
Consequently, it would be wise to consider the possibility
that P. miliaceum leaves may have been mainly used in daub,
Fig. 8 Scatter plot of Acute
bulbosus phytolith measure-
ments from archaeological
samples and modern Setaria
italica and Panicum miliaceum
leaves; a Length and width; b
Phytolith length and protrusion
length and length

Vegetation History and Archaeobotany
even though S. italica may have been more important in the
diets of both humans and animals.
Additionally, this study suggests two potential distinct
pathways for Acu te bulbosus phytoliths from P. miliaceum
or S. italica leaves to end up in daub (Fig.9), and a simi-
lar scheme has been well discussed in existing literature
(for example, Willcox and Tengberg 1995; Newton 2004;
Homsher 2012; Dzhanfezova 2020). Provided that Panicum
miliaceum and Setaria italica were the main crops grown at
Shuanghuaishu, the deliberate use of P. miliaceum leaves
and stalks for daub seems possible. This is supported by
the notable presence of Acute bulbosus in the assemblage,
resembling those found in modern P. miliaceum leaves. Con-
versely, Setaria italica leaves and stems were probably used
for fodder, suggesting the potential presence of these phy-
toliths in animal dung. Insituations where dung is added to
daub, a practice documented in various contexts (Jordanova
etal. 2018; Ray etal. 2023), the distinct Acute bul bosus
from Setaria italica leaves, accompanied by visible undi-
gested grains as shown in Fig.2, may also persist, albeit
at a smaller frequency compared to those associated with
Panicum milicaeum. Future studies require comprehensive
research to shed light on the prehistoric use of dung in build-
ing material during the Yangshao period.
Conclusions
This project carried out a quantitative analysis of the mor-
phology of Acute bulbosus phytoliths from modern Pani-
cum miliaceum and Setaria italica leaves. Several criteria
for separating and identifying them, including numbers per
g leaf and the width, length and number of protrusions may
be used to differentiate between the two species. This study
shows the potential for identifying P. miliaceum and S. ital-
ica leaf material from archaeological contexts.
The examination of the archaeological daub fragments
from Shuanghuaishu in this case study gave results that are
consistent with similar investigations of diagnostic phyto-
liths from millet husks and ethnographic observations on the
uses of the two millets. Future research will be dedicated to a
more rigorous analysis to quantify morphometric variations
in Acute b ulbosus phytoliths in various cultivated millets
and their wild counterparts, as well as in other taxa.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00334- 024- 00999-5.
Author contributions TA, YZ and WG planned the project. WG pro-
vided archaeological samples. TA, JC and SH performed experimen-
tal work and data analysis. TA and JC wrote the paper. All authors
participated in the discussions of the results and commented on the
manuscript.
Funding This study was supported by the National Social Science
Fund of China grant No. 20CKG024 (TA) and grant No. 19ZDA227
(WG).
Data availability All data needed to evaluate the conclusions in the
paper are present in the paper and/or the ESM. Additional data related
to this paper may be requested from the authors.
Declarations
Competing interests The authors declare that they have no competing
interests.
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