Results of the use wear analysis. Scars, use-wear marks, and perforation marks in varying sizes were observed on all the twelve perforated shells from the Xianrendong and Diaotonghuan sites (Fig. 1B①-⑫, Fig. 2a, b). Further, transverse and diagonal striations were detected on the used edges, the thinner side (which also means the used edge) with white luster, of all the perforated shells (Fig. 2c-h).
Results of starch grain analysis. More than 83 starch grains were recovered from 24 subsamples of the twelve perforated shells, and they can be divided into 5 categories (Table 1).
Type A - the Fagaceae: The common features of type A grains (n=4) were their various ovate shapes. Some type A grains were aggregated together in a cluster. When the grains were rotated, they still retained their ovate shapes. The properties of type A starches were consistent with the morphological characteristics of Fagaceae, therefore, we identified the type A grains as a type of Fagaceae. Based on the differences in the size of their diameters, surface morphologies, and fissure types, the type A grains could be divided into two subtypes: type A1 grains (Fig. 2i, j) with lengths 15.4μm and 18.7μm, which are consistent with the features of the grains of Cyclobalanopsis aggregated together; type A2 grains (Fig. 2k, l) with a smooth surface, regular oval shape, and smaller particle sizes (diameter lengths of 11.0 μm and 14.7 μm), which are consistent with the features of the disarticulated grains of Cyclobalanopsis.
Type B - the Poaceae: Thirty-nine starch grains were polyhedral in shape with a centric hilum and lacked the presence of lamellae; most grains also had deeply crossed or winged fissures. Their size range and mean diameter were 7.3–25.9 μm and 15.8 ± 2.4 μm, respectively. Modern reference collections show that starch grains from the pith of Sorghum, Zea, Setaria, Panicum, and Coix are similar to the type B starch grains, as characterized by their pressure facets. In general, it is understood that the genera Sorghum and Zea were initially domesticated in Africa and America about 9,000 years ago, respectively30,31 whereas the genera Setaria and Panicum were utilized and domesticated in China about 23,000 and 10,000 years ago, respectively32,33. The morphological characteristics and size range (the length of diameter, 5–20 μm) of starches from the genera Setaria and Panicum were consistent with the ancient starch grains extracted from the shell artifacts. In our previous study, after the starch grains derived from the seeds of 31 modern millet samples were analyzed, a system of classification was established to define the diagnostic morphological characteristics of the starch grains, including those of wild millets, which are small particles (the length is usually 10–14 μm) with wrinkled surfaces and rough edges. Only domesticated foxtail millets (Setaria italica) have starch grains measuring >14.0 μm34. Further, polyhedral and spherical starches (range, 20–32.3 μm) in millet stems and lenticular starches (range, 9.7–38.4 μm) in millet leaves were found in experimental harvesting20,22. Starches from Coix such as Job's tears (Coixchinensis) are polyhedral in shape and exhibit linear-, V-, Y-shaped or radiated fissures (range, 5.3–29.2 μm). In previous studies, a zig-zag arm on the extinction cross and an eccentric hilum have been observed as diagnostic features in the seed starch of Job's tears35. Some ancient starches in type B are similar to those from the seeds of Coix. Therefore, we divided thirty-nine type B starch grains into three groups: type B1 starch grains, those of wild millets from genera of Setaria and Panicum, which are small particles (n=19; range, 7.3–9.7 μm; mean size, 8.7±1.8 μm) with wrinkled surfaces and rough edges (Fig. 2m, n); type B2 starch grains, those from Coix, which are polyhedral in shape with a smooth surface, a zig-zag arm on the extinction cross, and an eccentric hilum (n=13, 20–25.9 μm, 24.5±3.1 μm; Fig. 2o, p); the remaining 7 starch grains (type B3 starch grains, n=7, 8.5~19.9 μm, 18.9±2.7 μm; Fig. 2q, r), which may contain grains from ordinary seeds, stems of millets, and Coix plants (Fig. 2s, t). In summary, the type B starch grains are derived from grasses in the Panicoideae, probably genera from Setaria, Panicum and Coix plants, including some starches from stems of the three genera.
Type C - geophytes: nineteen starches were characterized by a larger granule size, an eccentric hilum, irregular triangular or oval shape, and bent extinction crosses (range, 9.0–26.5 μm; mean size, 16.2±4.8 μm). These starches show strong similarities in morphology and size with those of the roots and tubers in our modern samples36. Based on the morphological characteristics of type C starch grains, they were categorized as polyhedral type C1 starch grains (Fig. 2u, v) with a slightly eccentric hilum, vertical or slightly bent extinction crosses, lamellae, and fissuring, which occasionally appears on the large grains (n=11; range, 9.0–22.5 μm; mean size, 15.7±5.2 μm), oval type C2 starch grains (Fig. 2w, x) with an irregular oval shape, an extremely eccentric hilum, and craters on the surface (n=2, size 24.8 μm and 25.7 μm); and the common hemispherical type C3 starch grains (Fig. 2y, z) with a slightly eccentric hilum, slightly bent extinction crosses, and a few compound grains (n=6; range, 17.8–26.5 μm; mean size, 14.1±4.2 μm). Type C1 and a portion of type C3 starch grains are similar to those derived from taro corms, which have slightly eccentric hila, vertical or slightly bent extinction crosses, some compound grains, and large grains occasionally have lamellae and fissuring. Other root and tuber plants, however, also contain a certain number of hemispherical starch grains, so type C3 starch grains may also be derived from some other root and tuber plants. Type C2 starch grains show strong similarities in morphology and size with those of Dioscorea in the modern database. In summary, Type C starch grains are probably derived from root and tuber plants, including taros and Dioscorea.
Type D - the Poaceae: Seventeen starch grains had an irregular lenticular shape (such as an ellipse or a semicircle) with a centric hilum and craters on the surface (range, 14.3–38.0 μm; mean size, 24.1 ± 5.6 μm; Fig. 2A, B).The extinction crosses appeared either as “X” or “＋” in shape. The starch grains were olivary and had an equatorial groove when rotated (Fig. 2C, D).The type D grains were similar to the starch grains of the tribe Triticeae studied by Yang and Perry37, and thus we inferred the type D grains were from the tribe Triticeae. In addition, three starches (Fig. 2E, F) were similar to the starch grains collected from the leaves of Panicoideae, Pooideae, and Phragmites22. Therefore, we inferred that the type D grains were from the tribe Triticeae and may also contain a certain number of starches from the stems and leaves of Panicoideae, Pooideae, and Phragmites.
Type E (Fig. 2G, H) - unidentifiable: The remaining 4 starches that could not be identified because of the damage or an absence of identifiable characteristics are grouped into Type E.
Results of phytolith analysis. According to the references for phytolith taxa in China38,39, specific phytolith types are predominantly, although not restricted to, those produced by certain plants. The most commonly occurring forms include bilobate, saddle, rondel, acicular hair cells, smooth elongate, square and trapeziform sinuate phytoliths from stems and leaves of the Poaceae40; bilobates from Panicoideae41; and long-saddle forms from Bambusoideae42.
Only six phytoliths were retrieved from the study samples (Table 1), and they were all from stems and leaves of the Poaceae. One smooth elongate (Fig. 2I) and one acicular hair cell (Fig. 2J) were recovered from residues on the shell 99WX-1746 E10N12 Z:②A③from the Xianrendong site. Four phytoliths were recovered from three perforated shells in the Diaotonghuan site, including one bilobate of the Panicoideae from shell 93WW-2265 W3S1 Z:L (Fig. 2K), one long-saddle of the Bambusoideae from 95WW-3452 W3N0 Z:H (Fig. 2L), and two squares from 95WW-3353 W2S3 Z:G and 95WW-3452 W3N0 Z:H (Fig. 2M).
No starches and phytoliths were extracted from the dust samples in the storeroom where the twelve shells were stored.