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From the modern to the archaeological: starch grains from millets and their wild relatives in China
Journal of Archaeological Science 39 (2012) 247e254
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From the modern to the archaeological: starch grains from millets and their wild relatives in China Xiaoyan Yanga, *, Jianping Zhangb, Linda Perryc, d, Zhikun Maa, e, Zhiwei Wana, e, Mingqi Lia, Xianmin Diaof, Houyuan Lub a
Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China The Foundation for Archaeobotanical Research in Microfossils, PO Box 37, Fairfax, VA 22038, USA d Department of Geography and Geoinformation Science, Center for Earth Observing and Space Research, George Mason University, 4400 University Drive MS6C3, Fairfax, VA 22030, USA e Graduate University of Chinese Academy of Sciences, Beijing 100049, China f Institute of Crop Science, Chinese Academy of Agriculture Sciences, Beijing 100081, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2011 Received in revised form 31 August 2011 Accepted 2 September 2011
Starch grains from 31 modern samples of millets derived from the seeds of 7 species within the genus Setaria and 2 species within the genus Panicum were analyzed to determine diagnostic morphological characteristics. Ancient starch grains recovered from a sample of broomcorn millet (Panicum miliaceum) excavated from the Cishan site (10.0e7.6 cal yr BP) in the North China Plain were then subjected to the same analyses to determine the differences in morphologies, if any, between modern and ancient samples. The data indicate that morphological features, and particularly surface and fissure features, will allow for solid identifications of ancient millet starches, while size classes will be helpful, but will not be dependable taxonomic indicators. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Starch grain analysis Millets and their wild relatives Modern starches Archaeological starches
1. Introduction Foxtail millet (Setaria italica) and broomcorn millet (Panicum miliaceum) were initially cultivated and eventually domesticated in North China. These two species of millets were the dominant traditional crops in Northern China where people have cultivated these plants for at least eight thousand years (Lu et al., 2009; Chen, 2002). The origins and subsequent dispersals of millets, therefore, have been subjects of great interest to and study by archaeobotanists in the region (Huang, 1982; Liu and Kong, 2004; Zhao, 2006; Lee et al., 2007; Lu et al., 2009; Zhang et al., 2011). Previous studies that targeted ancient millets focused on the identification of both macrofossil and microfossil remains of foxtail and broomcorn millets and their wild relatives. Green foxtail millet (Setaria viridis) is believed to be the wild ancestor of foxtail millet (de Wet et al., 1979), but the ancestor of broomcorn millet has not yet been determined.
* Corresponding author. Tel.: þ86 10 64889443; fax: þ86 10 64872274. E-mail address: [email protected] (X. Yang). 0305-4403/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2011.09.001
Experiments with the charring of millet seeds have been completed for comparative purposes (Liu and Kong, 2004), and diagnostic characteristics in phytolith morphology have been studied and used to distinguish between foxtail millet and broomcorn millet (Lu et al., 2009), as well as wild foxtail grass and domesticated foxtail millet (Zhang et al., 2011). Since the 1980s, starch grain analysis has been employed in archaeobotanical studies to understand plant use history, ancient diet, tool function, and agricultural origins and dispersals (e.g. Ugent et al., 1984; Piperno and Holst, 1998; Piperno et al., 2000; Perry et al., 2006, 2007; Barton, 2007; Zarrillo et al., 2008; Macader, 2009; Henry et al., 2011). The basic diagnostic morphological characteristics of the starches derived from both foxtail and broomcorn millet were documented in the first half of the 19th century by Payen (Reichert, 1913), and have since been studied by others including researchers in the food sciences (e.g., Fujita et al., 1996; Zarnkow et al., 2007). The detailed diagnostic characteristics of starch grains from foxtail and broomcorn millets and their wild relatives, and their application in archaeobotanical investigations, however, have not yet been reported. Here we discuss the diagnostic morphological characteristics of starch grains from a suite of
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70o
modern millets, then compare these specimens to starches derived from archaeological millet excavated from the Cishan site in the North China Plain.
80o
90o
100o
110o
120o
130o 50o
2. Materials and methods 40o
40o
2.1. Modern samples Thirty-one modern samples from the two genera of millets domesticated in North China were collected for modern starch grain analysis. The collections included 21 samples from 7 species within the genus Setaria and 10 samples from 2 species within the genus Panicum (Table 1). The modern samples were collected from 14 provinces ranging from north to south in eastern and central China including Inner Mongolia, Guangdong, Gansu, and Jiangsu (Fig. 1). Before each sample was analyzed, all implements such as knives, test tubes, glass stirring rods, etc. were both sonicated and boiled for 15 min to prevent possible cross contamination between the samples. The protocol for extraction of starch grains from modern seeds is as follows: 1) Tweezers were used to remove the glumes surrounding the seeds. One to three seeds were sampled based upon seed size. 2) Seeds were placed into clean test tubes with ultrapure water and soaked for an hour. 3) A clean, glass stirring rod was used to crush the soaked seeds and release the starch grains from the endosperm into the water. Table 1 Modern samples for starch grain analysis. Lab Source Samples No.
Location
Range/mm Mean 1/mm Mean 2/mm
F1 F2 F3 F4 F5 F6 F7 Fa Fb Fc Fd Fe M1 M2 M3 M4 M5 M6 M7 M8 M9 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
Ningxia Shanxi Henan Inner Mongolia Hebei Hebei Liaoning Anhui Guangdong Shandong Jiangsu ? Hebei Gansu Jilin Liaoning Inner Mongolia Hebei Henan Hebei Hebei Hebei Hebei Shanxi Shanxi Shaanxi Shaanxi Hebei Gansu Hebei Beijing
3.2e11.2 3.0e11.0 2.5e11.7 2.1e12.3 2.0e14.0 2.3e10.0 3.1e12.3 3.2e7.9 3.9e11.1 3.8e8.2 3.8e7.3 2.8e7.7 3.2e15.4 3.4e15.6 2.1e17.3 2.8e15.1 2.1e17.3 2.5e16.0 2.2e19.3 2.4e19.5 2.5e17.2 2.3e10.6 2.0e10.9 2.3e11.5 2.5e10.9 2.3e10.5 2.1e9.6 2.1e11.9 2.3e10.0 2.0e11.5 2.2e9.3
IM IM IM IM IM IM IM HUH HUH HUH HUH HUH IM IM IM IM IM IM IB IB IB IGG IGG IGG IGG IGG IGG IGG IGG IGG IGG
S. viridis S. viridis S. viridis S. viridis S. viridis S. viridis S. viridis S. faberil S. plicata S. pumila S. chondrachne S. parviflora S. italica S. italica S. italica S. italica S. italica S. italica S. italica S. italica S. italica P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. bisulcatum
6.9 7.8 9.6 8.0 8.2 7.4 8.0 6.3 7.4 6.6 6.0 5.7 9.7 11.2 11.4 9.7 11.7 10.3 9.8 10.0 9.3 7.6 7.3 6.8 7.4 7.0 7.0 7.8 6.7 7.9 6.9
1.1 1.6 1.6 1.6 1.6 1.1 1.6 0.8 1.2 0.7 0.6 0.8 1.6 1.5 2.2 2.2 1.7 2.2 2.5 2.6 2.2 1.4 1.3 1.1 1.3 1.3 1.2 1.4 1.1 1.5 1.2
6.6 5.4 7.2 6.8 5.9 6.4 6.7 5.4 7.3 6.2 5.4 4.4 8.0 10.1 8.4 9.0 8.8 7.7 e e e 6.6 5.9 5.8 6.3 e e e 6.8 e e
1.4 2.5 2.9 2.3 2.0 2.4 1.9 1.2 1.4 1.1 1.0 0.9 2.5 2.1 1.7 2.6 2.3 3.1
1.8 1.4 1.5 1.5
30o
30o
20o
90o
100o
110o
120o
Fig. 1. Geographic locations from which samples were collected. : S. viridis; : S. italica; : P. miliaceum; : S. plicata; : S. chondrachne : S. pumila : S. faberil : Cishan site. The sample S. parviflora was collected in 1972, the location is unknown.
4) A drop of water/starch slurry was placed onto a clean glass slide, was mounted in 10% glycerine and 90% ultrapure water, and was sealed with acrylic nail polish. 5) Starch grains were examined using compound light microscopy in both white and cross-polarized light at 400 magnification. 6) 50 to 150 starch grains were measured for each sample. 7) Morphological features of the starch grains including basic shape and surface microscopic morphology were examined.
2.2. Archaeological samples The Cishan site (36 34.511N, 114 06.720E) is located between the Taihang Mountain range and the North China Plain at an elevation of 260e270 m above sea level. Between 1976 and 1978, excavations uncovered numerous storage pits that contained the charred remains of grain crops that, unfortunately, were oxidized to ashes after being exposed to the air (Tong, 1984). In a previous study (Lu et al., 2009), we analyzed the phytoliths from the ashes of 46 seed samples recovered from five newly excavated storage pits (CS-I to CS-V) at the Cishan site, and one sample (CS-BWG) preserved in a storage bottle from the Culture Museum of Cishan. The sample CS-BWG was collected during the excavation when the crop remains were still intact grains, and 99.6% (n ¼ 1000) of the phytoliths from this sample were diagnostic of broomcorn millet. For this study, we subsampled from CS-BWG to recover archaeological starch grains from broomcorn millet. The sample dates to 5684 37 cal yr BC by AMS14C (Lu et al., 2009). One tenth gram (0.1 g) of the sample was subsampled for analysis. The protocol for the recovery of starch grains from the sample CS-BWG follows:
1.8
Notes: IM: Crop Gene Bank of Institute of Millets, Hebei Provincial Academy of Agricultural Sciences; HUH: Harvard University Herbaria; IB: Institute of Botany, Chinese Academy of Sciences; IGG: Institute of Geology and Geophysics, Chinese Academy of Sciences. Column of Mean 1 results from starch grains in size larger than 5 mm and column Mean 2 results from all starch grains in the range. Sample Fe was collected in 1972, the location is unknown.
1) A solution of 6% H2O2 was used to break down some of the larger charred particles by oxidation, releasing starch grains possibly trapped within them or adhering to them, as well as to oxidize some of the extraneous organic material from the residues. 2) A heavy liquid solution of CsCl at a density of 1.3 was added to the residue to remove any material with a specific gravity of less than 1.3.
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Fig. 2. Modern and archaeological starch grains. aec, S. viridis from Shanxi, Hebei and Inner Mongolia. d, S. pumila from Shandong Province; e, S faberil from Anhui Province; f, S. plicata from Guangdong; g, S. chondrachne from Jiangsu Province; h, S. parviflora from unknown region in South China; iel, S. italica from Gansu, Hebei (j and k) and Inner Mongolia; m-o, P. miliaceum from Hebei, Gansu and Shaanxi Provinces. p, P. bisulcatum from Beijing. qet, ancient starch grains from P. miliaceum recovered from the Cishan site. Scale bar: 20 mm.
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3) Starch grains were floated using a CsCl solution at a density of 1.8, after which the heavy liquid was rinsed from the samples 4) The material recovered from the second extraction was mounted in 10% glycerine and 90% water on a clean glass slide. For a detailed protocol see (Zarrillo et al., 2008; Yang and Jiang, 2010). Steps 5e7 were completed as for the modern samples above so that the archaeological residues and modern samples would be directly comparable. Our starch keys and classifications for modern starches emphasize attributes demonstrated by previous studies to be useful in identification: overall grain shape; contour and surface features; position and form of the hilum and fissures; number and characteristics of pressure facets; presence or absence of demonstrable lamellae and mean maximum length averaged from the measurement of 50e150 grains. Attributes of the extinction cross, an optical interference pattern created by the layered semi-crystalline structure of the starch grains, was observed using cross-polarized light.
Because both the modern and archaeological starch grain component smaller than 5 mm is typically somewhat featureless, and, therefore, non-diagnostic, these starches were discarded from the study. Small, non-diagnostic starch grains occur in nearly every taxonomic group, and, thus, they are not useful for archaeobotanical analysis. For reasons that are not understood, starch grains of this small size do not typically occur in archaeological assemblages from North China. Nonetheless, because this component was discarded from both modern and archaeological samples, the assemblage analyses to be presented in the remainder of the manuscript remain unaffected for any region where these plants may occur. Subsamples from the studied millets were deposited in the modern reference collection of more than 170 Asian species housed at the Institute of Geographic Sciences and Natural Resources Research at the Chinese Academy of Sciences. This reference collection includes members of plant families known to have been important economically in China including Poaceae, Fagaceae,
Fig. 3. Starch grains from some economically significant plant families in China. a, Vicia Faba, Family Leguminosae, range, 7.4e55.8 mm, mean, 29.3 11.54 mm; b, Quercus dentata, Family Fagaceae, range, 6.0e24.8 mm, mean, 13.2 3.8 mm; c, Fallopia multiflora, Family Polygonaceae, range, 4.2e20.3 mm, mean, 10.9 3.5 mm; d, Fritillaria cirrhosa, Family Liliaceae, range, 8.2e49.0 mm, mean, 33.2 8.2 mm; e, Dioscorea opposite, Family Dioscoreaceae, range, 5.9e43.6 mm, mean, 22.9 7.3 mm; f, Nelumbo nucifera, Family Nymphaeaceae, range, 8.0e72.4 mm, mean, 36.5 15.5 mm; g, Ginkgo biloba, Family Ginkgoaceae, range, 5.9e30.6 mm, mean, 15.3 4.2 mm; h, Stahlianthus involucratus, Family Zingiberaceae, range, 7.1e21.8 mm, mean, 15.4 2.8 mm; i, Typhonium divaricatum, Family Areceae, range, 6.3e19.8 mm, mean, 12.1 2.8 mm. Scale bar: 20 mm.
X. Yang et al. / Journal of Archaeological Science 39 (2012) 247e254
Nymphaeaceae, Alismataceae, Araceae, Cyperaceae, Polygonaceae, Juglandaceae, and others. Laboratory processing of the modern samples was carried out in the Laboratory of Environmental Archeology, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, the Archaeobiology Lab of the Smithsonian National Museum of Natural History and the Archaeobotanical Lab at the Foundation for Archaeobotanical Research in Microfossils. Examination of the archaeological sample was carried out in the Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences.
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3. Results 3.1. Foxtail grasses All starch grains from wild millets within the genus Setaria have wrinkled surfaces and coarse edges (Fig. 2aeh). Starch grains from green foxtail grass (S. viridis) are the largest in maximum length followed by those of S. pumila. Starch grains from S. pumila and S. parviflora are mainly spherical in shape with short lines that radiate from the center to the edges of the grains. Grains from S. viridis often have fissures through the hila which vary in form, while those from other species seldom have strongly-defined fissures.
Fig. 4. Starch grains from various genera within the Family Poaceae. a, Roegneria kamoji. Range, 3.6e16.3 mm, mean 8.2 2.5 mm; b, Elymus dahuricu. Range, 3.8e18.2 mm, mean 11.1 3.2 mm; c, Leymus chinensis. Range, 6.5e26.2 mm, mean, 14.0 4.6 mm; d, Agropyron criststum. Range, 3.5e43.0 mm, mean, 24.8 8.5 mm e, Secale cereale. Range, 4.7e45.2 mm, mean, 28.9 13.3 mm f, Aegilops tauschi. Range, 5.9e42.9 mm, mean, 17.3 8.0 mm; g, Triticum aestivum. Range, 5.7e39.4 mm, mean, 18.7 8.8 mm; h, Hordeum vulgare var. nudum. Range, 5.2e22.4 mm, mean, 15.4 3.6 mm; i, Coix lacryma-jobi. Range, 5.4e20.4 mm, mean, 12.4 3.0 mm; j, Sorghum bicolor, Range, 6.8e22.2 mm, mean, 14.1 3.7 mm; k, Zizania caduciflora. Range, 3.2e8.2 mm, mean, 5.0 0.7 mm; l, Oryza sativa. Range, 5.0e11.27 mm, mean 6.5 1.2 mm (starch grains less than 5 mm are excluded); m, Eriochloa villosa. Range, 3.2e6.8 mm, mean, 4.8 0.8 mm; n, Avena nuda. Range, 3.1e7.7 mm, mean, 5.1 1.0 mm; o, Eleusine coracana. Range, 3.3e11.8 mm, mean, 6.8 1.6 mm. Scale bar: k and m, 10 mm; others, 20 mm.
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The largest single grain from the wild grasses measured 14.0 mm in maximum length and is derived from the Hebei sample. The mean length measurements of all 7 samples of S. viridis are between 7 and 8 mm, with the largest mean value in the Henan sample. The mean of 700 grains from 7 samples is 7.7 1.4 mm, and the confidence interval (CI) is 7.6e7.9 mm.
Table 3 Percentage content of different surface-fissured starch grains of millets and their relatives. Samples
Fissure type (%) None
Linear
Transverse
Y-shaped
Stellate
75 65.9 34.5 30.2
10.8 14.7 21.5 14.0
2.2 16.6 6.6 8.8
11.8 3.8 32 34.7
0 0 5.4 12.2
3.2. Foxtail millet Starch grains from foxtail millets have a basic polyhedral shape with occasional spherical or oval grains, and the surfaces are smooth (Fig. 2iel). These observations are in line with those reported previously (Reichert, 1913). The hila of starch grains from foxtail millets are centric, and the majority of grains have hila that are traversed by deep, linear, transverse, Y-shaped or stellate fissures. Radiating lines occur commonly from the center to the edge on the surface of grains. 987 starch grains from 9 samples were measured. The mean maximum length measurements of 9 samples are 9e12 mm with the largest starch grains occurring in the sample collected in Inner Mongolia, and the smallest from the sample collected in Hebei Province. The mean of 987 starch grains is 9.9 2.3 mm, 95% CI is 9.7e10.0 mm.
P. milliaceum P. bisulcatum S. viridis S. italica
grains have deep Y-shaped and transverse fissures, and the extinction crosses are quite weak (Fig. 3q). The remaining 26 grains range from 4.7 to 15.1 mm, and the mean is 10.5 2.8 mm 16 of 26 grains are larger than 10 mm. The grains are polyhedral with centric hila (Fig. 2ret). In a population of 26 intact, undamaged starch grains, only 3 grains (11.5%) have a thin linear fissure transecting the hilum, and the remaining starch grains are unfissured. 4. Discussion
3.3. Broomcorn millet 4.1. Family-level identification Polyhedral starch grains dominate the sample (Fig. 2meo), an observation that is in line with previous research (Reichert, 1913). The hila of starch grains from broomcorn millets are centric, and almost 70% of the grains are unfissured. The remaining 30% grains have slight fissures through the hila, and a very few starch grains have transverse or Y-shaped fissures. The mean maximum length measurements of 9 samples are 6.5e7.5 mm, and, notably, starch grains that are larger than 10 mm in length make up less than 2% of the population. Only 3 of 923 grains measured more than 10 mm and the largest size is 11.9 mm. The mean maximum length of the 923 starch grains from broomcorn millets is 7.3 1.4 mm and the 95% CI is 7.2e7.4 mm. 3.4. Wild relative of broomcorn millet The wild ancestor of broomcorn millet is still unknown. Panicum bisulcatum, a wild grass and congeneric species with broomcorn millet, has similar morphological features in its starch grains in both size and surface features. The differences lie in the size classes and fissure patterns. The ration of length to width of wild grass starches measure 1.0e1.7 um larger than those of broomcorn millet (w1.0) (Fig. 2p), and starch grains from P. bisulcatum have fissures even less frequently than the starches of broomcorn millet. 3.5. Ancient starch grains We recovered 37 starch grains from sample CS-BWG, 11 of which appear to be damaged by some processing method. These damaged
The vast botanical literature addressing the subject of starch grains is in agreement that these structures have diagnostic morphological features that can be used to determine genera, species, and, in some cases, even varieties of plants (Reichert, 1913; Carlquist, 1961; Perez et al., 2009). The starch analyst must carefully study the modern reference collections so that these features can be recognized and used to separate taxa within the archaeobotanical assemblage. Both macrofossil and microfossil plant remains from members of the Fagaceae, Nymphaeaceae, Alismataceae, Araceae, Cyperaceae, Polygonaceae, and Juglandaceae have been recovered from Neolithic and historic sites in China (Zhao, 2010). Starch grains derived from the Poaceae are easily distinguished from those of these other economically significant families (Figs. 3) (Yang et al., 2009b; Wan et al., 2011). The genera Setaria and Panicum are classified within the family Poaceae. 4.2. Genus-level identification The tribes and genera within the Poaceae are easily distinguishable from one another using very basic morphological traits and assemblage characteristics (Reichert, 1913; Tateoka, 1962). Fig. 4 shows the starch grains extracted from the seeds of plants within the genera Oryza, Zizania, Sorghum, Coix, Eriochloa, Avena as well as 8 genera from the Tribe Triticeae. Starch grains from the Tribe Triticeae are lenticular, a morphology that is very different from others which are typically dominated by polyhedral shapes.
Table 2 Percentage content of different sized starch grains of millets and their relatives. Samples
10e11 mm
11e12 mm
12e13 mm
13e14 mm
14e15 mm
>15 mm
Total
P. milliaceum P. bisulcatum S. viridis S. italica
13.3% 0 7.3% 17.7%
0 0 2.6% 17.7%
0 0 1.1% 11.8%
0 0 0.4% 6.5%
0 0 0 2.5%
0 0 0 1.5%
13.3% 0 11.4% 55.0%
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The starch grains from the genera Avena, Oryza and Zizania are the smallest, are typically characterized by compound grains, and the single component granules are very angular. The largest sized grains are from Sorghum, and the starch grains from Job’s-tear (Coix) are the second largest among these economically significant Old World plants.
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Table 4 Percentage content of different shapes of starch grains from foxtail millet (S. italica). Samples
Spherical (%)
Polyhedral (%)
M1, M2, M3, M5, M6, M7,
15 4 10 35 30 12
85 96 90 65 70 88
Hebei-1 Gansu Jilin IM Hebei-2 Henan
4.3. Species-level identification In the case of the genera Setaria and Panicum, both the sizes and the morphological features of the starch grains from foxtail millet, broomcorn millet and their relatives overlap somewhat. Thus, a method using a basic statistical assemblage analysis of a population of starch grains was devised to separate these important crop plants from one another. While the millets recovered from the middle and late Neolithic sites in China will be representative of fully domesticated crops, at the onset of the Neolithic, when millets were first being exploited, it is possible that the wild relatives of these plants were also collected and used. Thus, the combined features of related wild millet species were studied as well as the domesticated crop plants. The size of starch grains from foxtail millet (S. italica) is larger than those of others. The wild relatives of foxtail millet, with the exception of S. viridis, are much smaller with very few grains larger than 10 mm. Table 2 and Table 3 show the percentage content of different sized starch grains and different surface-fissured starch grains of millets and their relatives, respectively. When both size classes and fissuring patterns are taken into consideration, starch assemblages with characteristics typical of millets can be partitioned into taxonomic groups using the following dichotomous key.
4.4. Environmental context and gene control The basic morphology of starch grains is under genetic control, however, size and shape can be modified by both the internal and external environments of the plant (Nikuni, 1978; Oliverira et al., 1994; Haase and Plate, 1996). Some variability in size has been noted in barley and wheat (Lindeboom et al., 2004), but unfortunately, to our knowledge there are no studies published on environmental effects and the size classes of millets. We did note that the percentages of spherical grains are greater in samples from the north (Table 4), a finding that could indicate that some seeds were not completely mature when collected (Reichert, 1913). Even though the starches may be from immature seeds, the size of starch grains from foxtail millet still increases from south to north, which may indicate that higher rainfall would not result in a larger grain size. Southern Hebei and Henan Provinces are located in the warm temperate zone, while Inner Mongolia and Gansu Provinces are situated in the semi-arid area. The annual temperature and precipitation in the southern Hebei and Henan Provinces are higher than those in the northern Inner Mongolia and Gansu Provinces. Further study on the relationship between size change and environment is needed to refine our understanding of these issues.
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4.5. Comparison between ancient and modern starch grains Gong et al. (2011) analyzed starch grains from ancient noodles and cakes made of P. miliaceum unearthed at the Subeixi site (500300 cal yr BP) in Xinjiang, northwestern China. Their research indicated that the size of ancient starch grains was identical to that of the modern comparative specimens. In contrast, the mean size of starch grains from the CS-BWG sample averages about 3 mm larger than the modern starches, a difference of 40%. The range of sizes of the ancient starch grains is also very different from the modern with 61.5% of the grains measuring more than 10 mm in size. Despite the differences in size, the other morphological features of the modern and ancient samples of broomcorn millet are identical. Previous work also found that ancient foxtail millet starch grains recovered from stone tools excavated from the Shangzhai site (w7000 cal yr BP) (Yang et al., 2009a) were also larger than modern starch grains. Here we cannot explain the reason why the ancient starch grains from P. miliaceum at the Subeixi site have the same size as the modern starches. Both the Cishan site and Shangzhai site are located in the North China Plain where annual rainfall is 700e800 mm, but the Subeixi Site is located in an area characterized by the typical continental desert climate: the annual rainfall is 25.2 mm. Again, experimental work may help clarify the effects of environmental conditions on millet starch grain size. 5. Conclusion Phytoliths derived from glumes, lemmas, and paleas of millets are positive indicators of millets in archaeological contexts (Lu et al., 2009; Zhang et al., 2011). Starches, however, are generally extracted from processing tools, thus providing evidence of the plants’ use in a secure, food related context. The combination of analyses may provide a more solid approach to the identification of millets in archaeological contexts. The comparative studies demonstrate that grass starches of millets can be identified using an assemblage approach and basic statistical methods. If only a few starch grains of millets are present in a given context, it is more difficult to make a species-level determination because there is some inter-species overlap in morphological features. When the parameters deriving from modern starches are employed to identify ancient starch grains, the range and mean size are not absolutely comparable, but do assist in initial divisions of the archaeobotanical assemblage. The best approach to secure identification of millets includes consideration of multiple factors including the geographic area, the age of the archaeological site, the local flora, and, finally, the detailed morphological features of the starch grains in the archaeobotanical assemblage. Acknowledgments Dr. Xiaoyan Yang worked in the Archaeobiology Lab of Smithsonian National Museum of Natural History as a postdoctoral fellow during 2007e2008. She would like to thank the Smithsonian Institution for financial support and Prof. D. R. Piperno, A.G. Henry, S. Simms, and other people for their helpful advice in research. Xiaoyan Yang gratefully acknowledges the support of the National Natural Science Foundation of China (Grant No. 40771205 and 41072140) and the CAS Strategic Priority Research Program (Grant No. XDA05130603 and XDA05130402). References Barton, H., 2007. Starch residues on museum artefacts: implications for determining tool use. Journal of Archaeological Science 34, 1752e1762. Carlquist, S., 1961. Comparative Plant Anatomy: a Guide to Taxonomic and Evolutionary Application of Anatomical Data in Angiosperms. Holt, Reinhart, and Winston, New York, pp. 12e13.
Chen, W., 2002. Agricultural Archaeology. Cultural Relics Press, Beijing, pp. 42e48 (in Chinese). de Wet, J.M.J., Oestry-Stidd, L.L., Cubero, J.I., 1979. Origins and evolution of foxtail millets (Setaria italica). Journal of Agricultural and Traditional Botany Application 26, 53e64. Fujita, S., Sugimoto, Y., Yamashita, Y., Fuwa, H., 1996. Physicochemical studies of starch from foxtail millet (Setaria italica Beauv). Food Chemistry 55, 209e213. Gong, Y., Yang, Y., Ferguson, D.K., Tao, D., Li, W., Wang, C., Lu, E., Jiang, H., 2011. Investigation of ancient noodles, cakes, and millet at the Subeixi Site, Xinjiang, China. Journal of Archaeological Science 38, 470e479. Haase, N.U., Plate, J., 1996. Properties of potato starch in relation to varieties and environmental factors. Starch/Stärke 48, 167e171. Henry, A.G., Brooks, A.S., Piperno, D.R., 2011. Microfossils in calculus demonstrates consumption of plants and cooked food in Neanderthal diets (Shanidar III, Iraq; Spy I and II, Belgium). Proceedings of the National Academy of Sciences 108, 486e491. Huang, Q., 1982. Spodograms study and its application in archaeology. Kaogu (Archaeology) 4, 418e420 (in Chinese). Lee, G.A., Crawford, G.W., Liu, L., Chen, X., 2007. Plants and people from the early neolithic to shang periods in North China. Proceedings of the National Academy of Sciences 104, 1087e1092. Lindeboom, N., Chang, P.R., Tyler, R.P., 2004. Analytical, biochemical and physicochemical aspects of starch granule size, with emphasis on small granule starches: a review. Starch/Stärke 56, 89e99. Liu, C., Kong, Z., 2004. The morphological comparison of grains between foxtail and broomcorn millet and its application for identification in archaeology remains. Kaogu (Archaeology) 3, 76e83 (in Chinese). Lu, H., Zhang, J., Liu, K., Wu, N., Li, Y., Zhou, K., Ye, M., Zhang, T., Zhang, H., Yang, X., Shen, L., Xu, D., Li, Q., 2009. Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago. Proceedings of the National Academy of Sciences 106, 7367e7372. Macader, J., 2009. Mozambican grass seed consumption during middle stone age. Science 326, 1680e1682. Nikuni, Z., 1978. Studies on starch granules. Starch/Stärke 30, 105e111. Oliverira, A.B., Rasnusson, D.C., Fulcher, R.G., 1994. Genetic aspects of starch granule traits in barley. Crop Science 34, 1176e1180. Perry, L., Dickau, R., Zarrillo, S., Holst, I., Pearsall, D.M., Piperno, D.R., Berman, M.J., Cooke, R.G., Rademaker, K., Ranere, A.J., Raymond, J.S., Sandweiss, D.H., Scaramelli, F., Tarble, K., Zeidler, J.A., 2007. Starch fossils and the domestication and dispersal of chili peppers (Capsicum spp. L.) in the Americas. Science 315, 986e988. Perry, L., Sandweiss, D., Piperno, D., Rademaker, K., Malpass, M., Umire, A., de la Vera, P., 2006. Early maize agriculture and interzonal interaction in southern Peru. Nature 440, 76e79. Piperno, D.R., Holst, I., 1998. The presence of starch grains on prehistoric stone tools from the humid neotripics: indications of early tuber use and agriculture in Panama. Journal of Archaeological Science 25, 765e776. Piperno, D.R., Ranere, A.J., Holst, I., Hansell, P., 2000. Starch grains reveal early root crop horticulture in the Panamanian tropical forest. Nature 407, 894e897. Perez, S., Baldwin, P.M., Gallant, D.J., 2009. Structural features of starch granules i. In: BeMiller, J., Whistler, R. (Eds.), Starch: Chemistry and Technology, third ed. Academic Press, Amsterdam, pp. 149e192. Reichert, E.T.,1913. The Differentiation and Specificity of Starches in Relation to Genera, Species, Etc. Carnegie Institution of Washington, Washington DC. 199e200. Tateoka, T., 1962. Starch Grains of Endosperm in Grass Systematics. Botanical Magazine, Tokyo 75. 377e383. Tong, W.H., 1984. Original agricultural relics of Cishan sites and related problems. Agricutural Archaeology 1, 194e207 (in Chinese). Ugent, D., Pozorski, S., Pozorski, T., 1984. New evidence cultivation for ancient cultivation of Canna edulis in Peru. Economic Botany 38, 417e432. Wan, Z., Yang, X., Ge, Q., Jiang, M., 2011. Morphological characteristics of starch grains of root and tuber plants in South China. Quaternary Sciences 31, 736e744 (in Chinese with English abstract). Yang, X., Jiang, L., 2010. Starch grain analysis reveals ancient diet at Kuahuqiao site, Zhejiang Province. Chinese Science Bulletin 55, 1150e1156. Yang, X., Yu, J., Lu, H., Cui, T., Guo, J., Ge, Q., 2009a. Starch grain analysis reveals function of grinding stone tools at Shangzhai site, Beijing. Earth Sciences 52, 1164e1171. Science in China Series D. Yang, X., Kong, Z., Liu, C., Zhang, Y., Ge, Q., 2009b. Characteristics of starch grains from nuts category in North China. Quaternary Sciences 29, 153e158 (in Chinese with English abstract). Zarrillo, S., Pearsall, D.M., Raymond, J.C., et al., 2008. Directly dated starch residues document early formative maize (Zea mays L.) in tropical Ecuador. Proceedings of the National Academy of Sciences 105, 5006e5011. Zarnkow, M., Mauch, A., Back, W., Arendt, E.K., Kreisz, S., 2007. Proso millet (Panicum miliaceum L.): an evaluation of the microstructural changes in the endosperm during the malting process by using scanning-electron and confocal laser microscopy. Journal of Institute of Brewing 113, 355e364. Zhao, Z., 2006. Domestication of millet: paleoethnobotanic data and ecological perspective. Archaeology in China and Sweden. In: Institute of Archaeology Chinese Academy of Social Sciences and the Institute of Archaeology Swedish National Heritage Board. Science Press, Beijing, pp. 97e104. Zhao, Z., 2010. Paleoethnobotany: Theories, Methods and Practice. Science Press, Beijing, pp. 71e222 (in Chinese). Zhang, J., Lu, H., Wu, Q., Yang, X., Diao, X., 2011. Phytolith analysis for differentiating between foxtail millet (Setaria italica) and green foxtail (Setaria viridis). PLoS ONE 6, 19726. doi:10.1371/journal.pone.0019726.
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Journal of Archaeological Science journal homepage: http://www.elsevier.com/locate/jas
From the modern to the archaeological: starch grains from millets and their wild relatives in China Xiaoyan Yanga, *, Jianping Zhangb, Linda Perryc, d, Zhikun Maa, e, Zhiwei Wana, e, Mingqi Lia, Xianmin Diaof, Houyuan Lub a
Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China The Foundation for Archaeobotanical Research in Microfossils, PO Box 37, Fairfax, VA 22038, USA d Department of Geography and Geoinformation Science, Center for Earth Observing and Space Research, George Mason University, 4400 University Drive MS6C3, Fairfax, VA 22030, USA e Graduate University of Chinese Academy of Sciences, Beijing 100049, China f Institute of Crop Science, Chinese Academy of Agriculture Sciences, Beijing 100081, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2011 Received in revised form 31 August 2011 Accepted 2 September 2011
Starch grains from 31 modern samples of millets derived from the seeds of 7 species within the genus Setaria and 2 species within the genus Panicum were analyzed to determine diagnostic morphological characteristics. Ancient starch grains recovered from a sample of broomcorn millet (Panicum miliaceum) excavated from the Cishan site (10.0e7.6 cal yr BP) in the North China Plain were then subjected to the same analyses to determine the differences in morphologies, if any, between modern and ancient samples. The data indicate that morphological features, and particularly surface and fissure features, will allow for solid identifications of ancient millet starches, while size classes will be helpful, but will not be dependable taxonomic indicators. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Starch grain analysis Millets and their wild relatives Modern starches Archaeological starches
1. Introduction Foxtail millet (Setaria italica) and broomcorn millet (Panicum miliaceum) were initially cultivated and eventually domesticated in North China. These two species of millets were the dominant traditional crops in Northern China where people have cultivated these plants for at least eight thousand years (Lu et al., 2009; Chen, 2002). The origins and subsequent dispersals of millets, therefore, have been subjects of great interest to and study by archaeobotanists in the region (Huang, 1982; Liu and Kong, 2004; Zhao, 2006; Lee et al., 2007; Lu et al., 2009; Zhang et al., 2011). Previous studies that targeted ancient millets focused on the identification of both macrofossil and microfossil remains of foxtail and broomcorn millets and their wild relatives. Green foxtail millet (Setaria viridis) is believed to be the wild ancestor of foxtail millet (de Wet et al., 1979), but the ancestor of broomcorn millet has not yet been determined.
* Corresponding author. Tel.: þ86 10 64889443; fax: þ86 10 64872274. E-mail address: [email protected] (X. Yang). 0305-4403/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2011.09.001
Experiments with the charring of millet seeds have been completed for comparative purposes (Liu and Kong, 2004), and diagnostic characteristics in phytolith morphology have been studied and used to distinguish between foxtail millet and broomcorn millet (Lu et al., 2009), as well as wild foxtail grass and domesticated foxtail millet (Zhang et al., 2011). Since the 1980s, starch grain analysis has been employed in archaeobotanical studies to understand plant use history, ancient diet, tool function, and agricultural origins and dispersals (e.g. Ugent et al., 1984; Piperno and Holst, 1998; Piperno et al., 2000; Perry et al., 2006, 2007; Barton, 2007; Zarrillo et al., 2008; Macader, 2009; Henry et al., 2011). The basic diagnostic morphological characteristics of the starches derived from both foxtail and broomcorn millet were documented in the first half of the 19th century by Payen (Reichert, 1913), and have since been studied by others including researchers in the food sciences (e.g., Fujita et al., 1996; Zarnkow et al., 2007). The detailed diagnostic characteristics of starch grains from foxtail and broomcorn millets and their wild relatives, and their application in archaeobotanical investigations, however, have not yet been reported. Here we discuss the diagnostic morphological characteristics of starch grains from a suite of
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70o
modern millets, then compare these specimens to starches derived from archaeological millet excavated from the Cishan site in the North China Plain.
80o
90o
100o
110o
120o
130o 50o
2. Materials and methods 40o
40o
2.1. Modern samples Thirty-one modern samples from the two genera of millets domesticated in North China were collected for modern starch grain analysis. The collections included 21 samples from 7 species within the genus Setaria and 10 samples from 2 species within the genus Panicum (Table 1). The modern samples were collected from 14 provinces ranging from north to south in eastern and central China including Inner Mongolia, Guangdong, Gansu, and Jiangsu (Fig. 1). Before each sample was analyzed, all implements such as knives, test tubes, glass stirring rods, etc. were both sonicated and boiled for 15 min to prevent possible cross contamination between the samples. The protocol for extraction of starch grains from modern seeds is as follows: 1) Tweezers were used to remove the glumes surrounding the seeds. One to three seeds were sampled based upon seed size. 2) Seeds were placed into clean test tubes with ultrapure water and soaked for an hour. 3) A clean, glass stirring rod was used to crush the soaked seeds and release the starch grains from the endosperm into the water. Table 1 Modern samples for starch grain analysis. Lab Source Samples No.
Location
Range/mm Mean 1/mm Mean 2/mm
F1 F2 F3 F4 F5 F6 F7 Fa Fb Fc Fd Fe M1 M2 M3 M4 M5 M6 M7 M8 M9 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
Ningxia Shanxi Henan Inner Mongolia Hebei Hebei Liaoning Anhui Guangdong Shandong Jiangsu ? Hebei Gansu Jilin Liaoning Inner Mongolia Hebei Henan Hebei Hebei Hebei Hebei Shanxi Shanxi Shaanxi Shaanxi Hebei Gansu Hebei Beijing
3.2e11.2 3.0e11.0 2.5e11.7 2.1e12.3 2.0e14.0 2.3e10.0 3.1e12.3 3.2e7.9 3.9e11.1 3.8e8.2 3.8e7.3 2.8e7.7 3.2e15.4 3.4e15.6 2.1e17.3 2.8e15.1 2.1e17.3 2.5e16.0 2.2e19.3 2.4e19.5 2.5e17.2 2.3e10.6 2.0e10.9 2.3e11.5 2.5e10.9 2.3e10.5 2.1e9.6 2.1e11.9 2.3e10.0 2.0e11.5 2.2e9.3
IM IM IM IM IM IM IM HUH HUH HUH HUH HUH IM IM IM IM IM IM IB IB IB IGG IGG IGG IGG IGG IGG IGG IGG IGG IGG
S. viridis S. viridis S. viridis S. viridis S. viridis S. viridis S. viridis S. faberil S. plicata S. pumila S. chondrachne S. parviflora S. italica S. italica S. italica S. italica S. italica S. italica S. italica S. italica S. italica P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. miliaceum P. bisulcatum
6.9 7.8 9.6 8.0 8.2 7.4 8.0 6.3 7.4 6.6 6.0 5.7 9.7 11.2 11.4 9.7 11.7 10.3 9.8 10.0 9.3 7.6 7.3 6.8 7.4 7.0 7.0 7.8 6.7 7.9 6.9
1.1 1.6 1.6 1.6 1.6 1.1 1.6 0.8 1.2 0.7 0.6 0.8 1.6 1.5 2.2 2.2 1.7 2.2 2.5 2.6 2.2 1.4 1.3 1.1 1.3 1.3 1.2 1.4 1.1 1.5 1.2
6.6 5.4 7.2 6.8 5.9 6.4 6.7 5.4 7.3 6.2 5.4 4.4 8.0 10.1 8.4 9.0 8.8 7.7 e e e 6.6 5.9 5.8 6.3 e e e 6.8 e e
1.4 2.5 2.9 2.3 2.0 2.4 1.9 1.2 1.4 1.1 1.0 0.9 2.5 2.1 1.7 2.6 2.3 3.1
1.8 1.4 1.5 1.5
30o
30o
20o
90o
100o
110o
120o
Fig. 1. Geographic locations from which samples were collected. : S. viridis; : S. italica; : P. miliaceum; : S. plicata; : S. chondrachne : S. pumila : S. faberil : Cishan site. The sample S. parviflora was collected in 1972, the location is unknown.
4) A drop of water/starch slurry was placed onto a clean glass slide, was mounted in 10% glycerine and 90% ultrapure water, and was sealed with acrylic nail polish. 5) Starch grains were examined using compound light microscopy in both white and cross-polarized light at 400 magnification. 6) 50 to 150 starch grains were measured for each sample. 7) Morphological features of the starch grains including basic shape and surface microscopic morphology were examined.
2.2. Archaeological samples The Cishan site (36 34.511N, 114 06.720E) is located between the Taihang Mountain range and the North China Plain at an elevation of 260e270 m above sea level. Between 1976 and 1978, excavations uncovered numerous storage pits that contained the charred remains of grain crops that, unfortunately, were oxidized to ashes after being exposed to the air (Tong, 1984). In a previous study (Lu et al., 2009), we analyzed the phytoliths from the ashes of 46 seed samples recovered from five newly excavated storage pits (CS-I to CS-V) at the Cishan site, and one sample (CS-BWG) preserved in a storage bottle from the Culture Museum of Cishan. The sample CS-BWG was collected during the excavation when the crop remains were still intact grains, and 99.6% (n ¼ 1000) of the phytoliths from this sample were diagnostic of broomcorn millet. For this study, we subsampled from CS-BWG to recover archaeological starch grains from broomcorn millet. The sample dates to 5684 37 cal yr BC by AMS14C (Lu et al., 2009). One tenth gram (0.1 g) of the sample was subsampled for analysis. The protocol for the recovery of starch grains from the sample CS-BWG follows:
1.8
Notes: IM: Crop Gene Bank of Institute of Millets, Hebei Provincial Academy of Agricultural Sciences; HUH: Harvard University Herbaria; IB: Institute of Botany, Chinese Academy of Sciences; IGG: Institute of Geology and Geophysics, Chinese Academy of Sciences. Column of Mean 1 results from starch grains in size larger than 5 mm and column Mean 2 results from all starch grains in the range. Sample Fe was collected in 1972, the location is unknown.
1) A solution of 6% H2O2 was used to break down some of the larger charred particles by oxidation, releasing starch grains possibly trapped within them or adhering to them, as well as to oxidize some of the extraneous organic material from the residues. 2) A heavy liquid solution of CsCl at a density of 1.3 was added to the residue to remove any material with a specific gravity of less than 1.3.
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Fig. 2. Modern and archaeological starch grains. aec, S. viridis from Shanxi, Hebei and Inner Mongolia. d, S. pumila from Shandong Province; e, S faberil from Anhui Province; f, S. plicata from Guangdong; g, S. chondrachne from Jiangsu Province; h, S. parviflora from unknown region in South China; iel, S. italica from Gansu, Hebei (j and k) and Inner Mongolia; m-o, P. miliaceum from Hebei, Gansu and Shaanxi Provinces. p, P. bisulcatum from Beijing. qet, ancient starch grains from P. miliaceum recovered from the Cishan site. Scale bar: 20 mm.
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3) Starch grains were floated using a CsCl solution at a density of 1.8, after which the heavy liquid was rinsed from the samples 4) The material recovered from the second extraction was mounted in 10% glycerine and 90% water on a clean glass slide. For a detailed protocol see (Zarrillo et al., 2008; Yang and Jiang, 2010). Steps 5e7 were completed as for the modern samples above so that the archaeological residues and modern samples would be directly comparable. Our starch keys and classifications for modern starches emphasize attributes demonstrated by previous studies to be useful in identification: overall grain shape; contour and surface features; position and form of the hilum and fissures; number and characteristics of pressure facets; presence or absence of demonstrable lamellae and mean maximum length averaged from the measurement of 50e150 grains. Attributes of the extinction cross, an optical interference pattern created by the layered semi-crystalline structure of the starch grains, was observed using cross-polarized light.
Because both the modern and archaeological starch grain component smaller than 5 mm is typically somewhat featureless, and, therefore, non-diagnostic, these starches were discarded from the study. Small, non-diagnostic starch grains occur in nearly every taxonomic group, and, thus, they are not useful for archaeobotanical analysis. For reasons that are not understood, starch grains of this small size do not typically occur in archaeological assemblages from North China. Nonetheless, because this component was discarded from both modern and archaeological samples, the assemblage analyses to be presented in the remainder of the manuscript remain unaffected for any region where these plants may occur. Subsamples from the studied millets were deposited in the modern reference collection of more than 170 Asian species housed at the Institute of Geographic Sciences and Natural Resources Research at the Chinese Academy of Sciences. This reference collection includes members of plant families known to have been important economically in China including Poaceae, Fagaceae,
Fig. 3. Starch grains from some economically significant plant families in China. a, Vicia Faba, Family Leguminosae, range, 7.4e55.8 mm, mean, 29.3 11.54 mm; b, Quercus dentata, Family Fagaceae, range, 6.0e24.8 mm, mean, 13.2 3.8 mm; c, Fallopia multiflora, Family Polygonaceae, range, 4.2e20.3 mm, mean, 10.9 3.5 mm; d, Fritillaria cirrhosa, Family Liliaceae, range, 8.2e49.0 mm, mean, 33.2 8.2 mm; e, Dioscorea opposite, Family Dioscoreaceae, range, 5.9e43.6 mm, mean, 22.9 7.3 mm; f, Nelumbo nucifera, Family Nymphaeaceae, range, 8.0e72.4 mm, mean, 36.5 15.5 mm; g, Ginkgo biloba, Family Ginkgoaceae, range, 5.9e30.6 mm, mean, 15.3 4.2 mm; h, Stahlianthus involucratus, Family Zingiberaceae, range, 7.1e21.8 mm, mean, 15.4 2.8 mm; i, Typhonium divaricatum, Family Areceae, range, 6.3e19.8 mm, mean, 12.1 2.8 mm. Scale bar: 20 mm.
X. Yang et al. / Journal of Archaeological Science 39 (2012) 247e254
Nymphaeaceae, Alismataceae, Araceae, Cyperaceae, Polygonaceae, Juglandaceae, and others. Laboratory processing of the modern samples was carried out in the Laboratory of Environmental Archeology, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, the Archaeobiology Lab of the Smithsonian National Museum of Natural History and the Archaeobotanical Lab at the Foundation for Archaeobotanical Research in Microfossils. Examination of the archaeological sample was carried out in the Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences.
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3. Results 3.1. Foxtail grasses All starch grains from wild millets within the genus Setaria have wrinkled surfaces and coarse edges (Fig. 2aeh). Starch grains from green foxtail grass (S. viridis) are the largest in maximum length followed by those of S. pumila. Starch grains from S. pumila and S. parviflora are mainly spherical in shape with short lines that radiate from the center to the edges of the grains. Grains from S. viridis often have fissures through the hila which vary in form, while those from other species seldom have strongly-defined fissures.
Fig. 4. Starch grains from various genera within the Family Poaceae. a, Roegneria kamoji. Range, 3.6e16.3 mm, mean 8.2 2.5 mm; b, Elymus dahuricu. Range, 3.8e18.2 mm, mean 11.1 3.2 mm; c, Leymus chinensis. Range, 6.5e26.2 mm, mean, 14.0 4.6 mm; d, Agropyron criststum. Range, 3.5e43.0 mm, mean, 24.8 8.5 mm e, Secale cereale. Range, 4.7e45.2 mm, mean, 28.9 13.3 mm f, Aegilops tauschi. Range, 5.9e42.9 mm, mean, 17.3 8.0 mm; g, Triticum aestivum. Range, 5.7e39.4 mm, mean, 18.7 8.8 mm; h, Hordeum vulgare var. nudum. Range, 5.2e22.4 mm, mean, 15.4 3.6 mm; i, Coix lacryma-jobi. Range, 5.4e20.4 mm, mean, 12.4 3.0 mm; j, Sorghum bicolor, Range, 6.8e22.2 mm, mean, 14.1 3.7 mm; k, Zizania caduciflora. Range, 3.2e8.2 mm, mean, 5.0 0.7 mm; l, Oryza sativa. Range, 5.0e11.27 mm, mean 6.5 1.2 mm (starch grains less than 5 mm are excluded); m, Eriochloa villosa. Range, 3.2e6.8 mm, mean, 4.8 0.8 mm; n, Avena nuda. Range, 3.1e7.7 mm, mean, 5.1 1.0 mm; o, Eleusine coracana. Range, 3.3e11.8 mm, mean, 6.8 1.6 mm. Scale bar: k and m, 10 mm; others, 20 mm.
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The largest single grain from the wild grasses measured 14.0 mm in maximum length and is derived from the Hebei sample. The mean length measurements of all 7 samples of S. viridis are between 7 and 8 mm, with the largest mean value in the Henan sample. The mean of 700 grains from 7 samples is 7.7 1.4 mm, and the confidence interval (CI) is 7.6e7.9 mm.
Table 3 Percentage content of different surface-fissured starch grains of millets and their relatives. Samples
Fissure type (%) None
Linear
Transverse
Y-shaped
Stellate
75 65.9 34.5 30.2
10.8 14.7 21.5 14.0
2.2 16.6 6.6 8.8
11.8 3.8 32 34.7
0 0 5.4 12.2
3.2. Foxtail millet Starch grains from foxtail millets have a basic polyhedral shape with occasional spherical or oval grains, and the surfaces are smooth (Fig. 2iel). These observations are in line with those reported previously (Reichert, 1913). The hila of starch grains from foxtail millets are centric, and the majority of grains have hila that are traversed by deep, linear, transverse, Y-shaped or stellate fissures. Radiating lines occur commonly from the center to the edge on the surface of grains. 987 starch grains from 9 samples were measured. The mean maximum length measurements of 9 samples are 9e12 mm with the largest starch grains occurring in the sample collected in Inner Mongolia, and the smallest from the sample collected in Hebei Province. The mean of 987 starch grains is 9.9 2.3 mm, 95% CI is 9.7e10.0 mm.
P. milliaceum P. bisulcatum S. viridis S. italica
grains have deep Y-shaped and transverse fissures, and the extinction crosses are quite weak (Fig. 3q). The remaining 26 grains range from 4.7 to 15.1 mm, and the mean is 10.5 2.8 mm 16 of 26 grains are larger than 10 mm. The grains are polyhedral with centric hila (Fig. 2ret). In a population of 26 intact, undamaged starch grains, only 3 grains (11.5%) have a thin linear fissure transecting the hilum, and the remaining starch grains are unfissured. 4. Discussion
3.3. Broomcorn millet 4.1. Family-level identification Polyhedral starch grains dominate the sample (Fig. 2meo), an observation that is in line with previous research (Reichert, 1913). The hila of starch grains from broomcorn millets are centric, and almost 70% of the grains are unfissured. The remaining 30% grains have slight fissures through the hila, and a very few starch grains have transverse or Y-shaped fissures. The mean maximum length measurements of 9 samples are 6.5e7.5 mm, and, notably, starch grains that are larger than 10 mm in length make up less than 2% of the population. Only 3 of 923 grains measured more than 10 mm and the largest size is 11.9 mm. The mean maximum length of the 923 starch grains from broomcorn millets is 7.3 1.4 mm and the 95% CI is 7.2e7.4 mm. 3.4. Wild relative of broomcorn millet The wild ancestor of broomcorn millet is still unknown. Panicum bisulcatum, a wild grass and congeneric species with broomcorn millet, has similar morphological features in its starch grains in both size and surface features. The differences lie in the size classes and fissure patterns. The ration of length to width of wild grass starches measure 1.0e1.7 um larger than those of broomcorn millet (w1.0) (Fig. 2p), and starch grains from P. bisulcatum have fissures even less frequently than the starches of broomcorn millet. 3.5. Ancient starch grains We recovered 37 starch grains from sample CS-BWG, 11 of which appear to be damaged by some processing method. These damaged
The vast botanical literature addressing the subject of starch grains is in agreement that these structures have diagnostic morphological features that can be used to determine genera, species, and, in some cases, even varieties of plants (Reichert, 1913; Carlquist, 1961; Perez et al., 2009). The starch analyst must carefully study the modern reference collections so that these features can be recognized and used to separate taxa within the archaeobotanical assemblage. Both macrofossil and microfossil plant remains from members of the Fagaceae, Nymphaeaceae, Alismataceae, Araceae, Cyperaceae, Polygonaceae, and Juglandaceae have been recovered from Neolithic and historic sites in China (Zhao, 2010). Starch grains derived from the Poaceae are easily distinguished from those of these other economically significant families (Figs. 3) (Yang et al., 2009b; Wan et al., 2011). The genera Setaria and Panicum are classified within the family Poaceae. 4.2. Genus-level identification The tribes and genera within the Poaceae are easily distinguishable from one another using very basic morphological traits and assemblage characteristics (Reichert, 1913; Tateoka, 1962). Fig. 4 shows the starch grains extracted from the seeds of plants within the genera Oryza, Zizania, Sorghum, Coix, Eriochloa, Avena as well as 8 genera from the Tribe Triticeae. Starch grains from the Tribe Triticeae are lenticular, a morphology that is very different from others which are typically dominated by polyhedral shapes.
Table 2 Percentage content of different sized starch grains of millets and their relatives. Samples
10e11 mm
11e12 mm
12e13 mm
13e14 mm
14e15 mm
>15 mm
Total
P. milliaceum P. bisulcatum S. viridis S. italica
13.3% 0 7.3% 17.7%
0 0 2.6% 17.7%
0 0 1.1% 11.8%
0 0 0.4% 6.5%
0 0 0 2.5%
0 0 0 1.5%
13.3% 0 11.4% 55.0%
X. Yang et al. / Journal of Archaeological Science 39 (2012) 247e254
The starch grains from the genera Avena, Oryza and Zizania are the smallest, are typically characterized by compound grains, and the single component granules are very angular. The largest sized grains are from Sorghum, and the starch grains from Job’s-tear (Coix) are the second largest among these economically significant Old World plants.
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Table 4 Percentage content of different shapes of starch grains from foxtail millet (S. italica). Samples
Spherical (%)
Polyhedral (%)
M1, M2, M3, M5, M6, M7,
15 4 10 35 30 12
85 96 90 65 70 88
Hebei-1 Gansu Jilin IM Hebei-2 Henan
4.3. Species-level identification In the case of the genera Setaria and Panicum, both the sizes and the morphological features of the starch grains from foxtail millet, broomcorn millet and their relatives overlap somewhat. Thus, a method using a basic statistical assemblage analysis of a population of starch grains was devised to separate these important crop plants from one another. While the millets recovered from the middle and late Neolithic sites in China will be representative of fully domesticated crops, at the onset of the Neolithic, when millets were first being exploited, it is possible that the wild relatives of these plants were also collected and used. Thus, the combined features of related wild millet species were studied as well as the domesticated crop plants. The size of starch grains from foxtail millet (S. italica) is larger than those of others. The wild relatives of foxtail millet, with the exception of S. viridis, are much smaller with very few grains larger than 10 mm. Table 2 and Table 3 show the percentage content of different sized starch grains and different surface-fissured starch grains of millets and their relatives, respectively. When both size classes and fissuring patterns are taken into consideration, starch assemblages with characteristics typical of millets can be partitioned into taxonomic groups using the following dichotomous key.
4.4. Environmental context and gene control The basic morphology of starch grains is under genetic control, however, size and shape can be modified by both the internal and external environments of the plant (Nikuni, 1978; Oliverira et al., 1994; Haase and Plate, 1996). Some variability in size has been noted in barley and wheat (Lindeboom et al., 2004), but unfortunately, to our knowledge there are no studies published on environmental effects and the size classes of millets. We did note that the percentages of spherical grains are greater in samples from the north (Table 4), a finding that could indicate that some seeds were not completely mature when collected (Reichert, 1913). Even though the starches may be from immature seeds, the size of starch grains from foxtail millet still increases from south to north, which may indicate that higher rainfall would not result in a larger grain size. Southern Hebei and Henan Provinces are located in the warm temperate zone, while Inner Mongolia and Gansu Provinces are situated in the semi-arid area. The annual temperature and precipitation in the southern Hebei and Henan Provinces are higher than those in the northern Inner Mongolia and Gansu Provinces. Further study on the relationship between size change and environment is needed to refine our understanding of these issues.
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4.5. Comparison between ancient and modern starch grains Gong et al. (2011) analyzed starch grains from ancient noodles and cakes made of P. miliaceum unearthed at the Subeixi site (500300 cal yr BP) in Xinjiang, northwestern China. Their research indicated that the size of ancient starch grains was identical to that of the modern comparative specimens. In contrast, the mean size of starch grains from the CS-BWG sample averages about 3 mm larger than the modern starches, a difference of 40%. The range of sizes of the ancient starch grains is also very different from the modern with 61.5% of the grains measuring more than 10 mm in size. Despite the differences in size, the other morphological features of the modern and ancient samples of broomcorn millet are identical. Previous work also found that ancient foxtail millet starch grains recovered from stone tools excavated from the Shangzhai site (w7000 cal yr BP) (Yang et al., 2009a) were also larger than modern starch grains. Here we cannot explain the reason why the ancient starch grains from P. miliaceum at the Subeixi site have the same size as the modern starches. Both the Cishan site and Shangzhai site are located in the North China Plain where annual rainfall is 700e800 mm, but the Subeixi Site is located in an area characterized by the typical continental desert climate: the annual rainfall is 25.2 mm. Again, experimental work may help clarify the effects of environmental conditions on millet starch grain size. 5. Conclusion Phytoliths derived from glumes, lemmas, and paleas of millets are positive indicators of millets in archaeological contexts (Lu et al., 2009; Zhang et al., 2011). Starches, however, are generally extracted from processing tools, thus providing evidence of the plants’ use in a secure, food related context. The combination of analyses may provide a more solid approach to the identification of millets in archaeological contexts. The comparative studies demonstrate that grass starches of millets can be identified using an assemblage approach and basic statistical methods. If only a few starch grains of millets are present in a given context, it is more difficult to make a species-level determination because there is some inter-species overlap in morphological features. When the parameters deriving from modern starches are employed to identify ancient starch grains, the range and mean size are not absolutely comparable, but do assist in initial divisions of the archaeobotanical assemblage. The best approach to secure identification of millets includes consideration of multiple factors including the geographic area, the age of the archaeological site, the local flora, and, finally, the detailed morphological features of the starch grains in the archaeobotanical assemblage. Acknowledgments Dr. Xiaoyan Yang worked in the Archaeobiology Lab of Smithsonian National Museum of Natural History as a postdoctoral fellow during 2007e2008. She would like to thank the Smithsonian Institution for financial support and Prof. D. R. Piperno, A.G. Henry, S. Simms, and other people for their helpful advice in research. Xiaoyan Yang gratefully acknowledges the support of the National Natural Science Foundation of China (Grant No. 40771205 and 41072140) and the CAS Strategic Priority Research Program (Grant No. XDA05130603 and XDA05130402). References Barton, H., 2007. Starch residues on museum artefacts: implications for determining tool use. Journal of Archaeological Science 34, 1752e1762. Carlquist, S., 1961. Comparative Plant Anatomy: a Guide to Taxonomic and Evolutionary Application of Anatomical Data in Angiosperms. Holt, Reinhart, and Winston, New York, pp. 12e13.
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