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1 Supporting Information Piperno et al /pnas SI Materials and Methods Modern Reference Collections and Microfossil Identification. Our reference collections of phytoliths and starch grains include more than 2,000 and about 500 species, respectively, and include many wild taxa of economic importance, most of the known domesticated plants native to Central and South America, and wild progenitors and other close wild relatives of the crop plants. Investigating the history of maize and squash in the study region was one of our priorities; therefore, our reference collections include all known species and subspecies of teosinte; 24 maize races from Central and South America, including 10 traditional Mexican land races; and all domesticated and most known wild species of Cucurbita, including all those found in Mesoamerica, such as C. argyrosperma ssp. sororia, which is native to the study region and is the wild ancestor of C. argyrosperma (the silverseeded squash or cushaw pumpkin) (1). With regard to starch grain identification, previous research has demonstrated that starch grains in maize commonly range from about 8 to 26 m in maximum length and from 12 to 16 m in mean length (2 8). In non-zea grasses, grain size typically ranges from about 2 to 18 m in maximum length and from 3 to 11 m in mean length. In many wild species, the maximum grain size is only 6 9 m (2, 4 8). Often these grains are too small to allow confident discernment of surface features, but species with larger grains have been found to have dissimilar morphological characteristics to maize (4 7). In this study, we examined additional species of non-zea grasses common in the Mexican flora (Table S1), including a putative early cultivar from highland Mexico, Setaria parvifolia (Poiert) (formerly S. geniculata) (9). As in other non-zea grasses, starch grain size is considerably smaller than in maize, and morphological characteristics also serve as distinguishing criteria. With regard to the differentiation of maize and teosinte on the basis of starch grain size (see Table S2), mean grain length in teosinte ranges from 9.5 m (Race Balsas) to 11.9 m (Z. luxurians, endemic to Guatemala). Maximum grain length varies from 2 mto28 m; the latter was represented by a single grain observed in a specimen of Chalco teosinte (Z. mexicana), a race that commonly hybridizes with maize (7). Maximum grain size does not exceed 20 m in non-chalco teosintes and 18 m in Balsas teosinte. In contrast, in maize, mean length varies from 11.4 to 15.8 m, and in most races, mean length is 12.5 m and maximum length is 20 m, reaching 26 m in some cases (7). Differences in such features as grain shape and surface sculptoring also provide clear morphological contrasts between teosinte and maize (7) (Table S2). For example, nearly every race of maize studied has dominant proportions of irregular grains (those without a clearly describable shape), and many have defined (deeply impressed) compression facets, which develop when the grains are packed together during their formation in the cellular organelles called amyloplasts. In contrast, teosinte exhibits significant percentages of oval and bell-shaped grains, which are nearly absent in maize, and has far fewer irregular grains. Most teosinte grains also lack defined compression facets and have different types of fissures (i.e, cracks at the hilum, the botanical center of the grain). With regard to phytolith identification, criteria for the identification of maize and teosinte phytoliths developed by ourselves and other investigators, including with the use of large blind studies, are well described elsewhere (10 16). Importantly, the considerable differences in morphological attributes of phytoliths formed in the fruitcases of teosinte and cobs of maize, structures which are homologous to each other, have been shown to be controlled primarily by tga1, a major domestication gene with significant effects (10, 17, 18). tga1 underwrites the degree of silicification of the glumes and rachids (cupules) of the fruitcases and cobs. In teosinte, the entire epidermis, consisting of both the long and short cells, is silicified, whereas in maize (which requires less natural protection from its herbivores), silicification is greatly reduced, and only the short epidermal cells (which produce the phytoliths called rondels) are filled with silica. In addition, the rondels produced in teosinte are more highly decorated than those in maize (a result also of more extensive lignification in teosinte), and the rondel phytoliths in maize cobs have a more diverse morphology and are in forms not found in teosinte. These differences result in the formation of distinct and identifiable phytoliths in maize and teosinte that allow them to be distinguished from each other and from non-zea wild grasses, including the genus Tripsacum (10 16). Other (Non-Maize) Starch Grains Present on the Stone Tools. Four yam grains (Dioscorea sp.), 3 legume grains, and 1 Marantaceae grain occurred on tool 316d, a large, preceramic grinding stone base recovered from cm b.s. of unit 1. The yam grains cannot be identified as belonging to either a wild or domesticated species, because considerable work is needed on wild Dioscorea species native to Mexico to rule out possible confusion with cultivated/domesticated taxa. This is the first empirical indication of yam usage in tropical Mexico during the pre-columbian era, however. The legume grains are similar to some that occur in Phaseolus, but they lack some attributes common in P. vulgaris and P. lunatus, such as the presence of lamellae and fissures; thus, we cannot unequivocally assign them to a specific legume taxon at this time. Similarly, the Marantaceae grain cannot be assigned to a specific genus. One unknown grain occurred on tool 365a. Other Types of Phytoliths Present in the Sediments. Marantaceae seed phytoliths, probably from either Maranta or Stromanthe, were well-represented throughout the sedimentary sequence. These phytoliths are not like those from arrowroot (M. arundinacea L.). A type of phytolith produced in the foliage and sometimes the wood of various tree species also was common. Also persistently present in lower frequencies were phytoliths from palms, Cyperaceae, and Asteraceae. Discriminating Phytoliths from Maize and Teosinte Culms (Stalks). Culms or stalks of grasses produce various idiosyncratic forms that do not occur in the leaves and inflorescences of the plants (19). Culm phytoliths in maize often are thick and irregularly cross or bilobate in shape with unusual, deeply notched bases. These are distinct from phytoliths produced in leaves and cobs. A stalk of Z. mays ssp. parviglumis from Guerrero state sampled from the herbarium folders at the U.S. National Museum of Natural History (NMNH ) produced the same types of phytoliths, as well as other phytoliths not seen in the maize stalks studied. Although further work is needed to more robustly assess whether the maize and teosinte phytoliths are diagnostic to Zea or to the subspecies level, these phytoliths can be used to identify stalk deposition. To be confident that young maize stalks, which presumably would have been used because they contain the highest quantity of sugar, produce phytoliths, we grew maize from seed at the Smithsonian Tropical Research Institute in Panama. Stalks were harvested 53 days after they were planted and investigated for silica content and phytolith morphological 1of10

2 attributes. The phytolith content was high, and the diagnostic phytoliths produced by mature stalks were commonly present in the young stalks. We restudied phytolith samples from important sites in Panama containing starch grain and phytolith evidence for preceramic maize (4, 10). No Zea-type stalk phytoliths were observed. Presence of Preceramic Phytoliths Indicative of Human Selection at the Hr Genetic Locus. In modern domesticated species and the F 1 and F 2 progeny of hybrids made between C. sororia and C. argyrosperma and between C. sororia and other domesticated species, many phytoliths from plants that are heterozygous at the Hr locus (Hr hr), and thus exhibit softer rinds than typically occur in wild plants, acquire characteristic surface features, such as incompletely formed and fainter scalloped impressions and even holes at the surface (Fig. S4B). These patterns may result from the influence of modifier genes or incomplete dominance of the Hr locus (20). In any case, the types of phytoliths produced significantly outnumber the completely silicified forms that dominate wild fruits (Fig. S5). The incompletely silicified phytoliths also commonly form as half-spheres (10, 20). All of these features are linked to the suppression of lignification and silicification under artificial selection for softer rinds. We explored this issue in greater detail by examining 100 phytoliths from each of 4 different fruits representing 3 different populations of C. argyrosperma ssp. sororia, the wild ancestor of C. argyrosperma. The fruits are homozygous at the Hr locus. In 2 of these fruits, only 3 phytoliths with surface features (e.g., marks or holes) characteristic of incomplete silicification were recorded. In the other 2 fruits, 1 and 0 phytoliths of this type occurred. Scans of phytolith preparations made from other fruits of C. sororia and other wild species further indicate that these characteristics are rare in wild Cucurbita. In preceramic samples 319d, 325 h, 316c, 318d, and 318e, in which numerous squash phytoliths occurred, 73% of the phytoliths (a far greater amount than in any wild species) exhibited surface features like those in modern specimens with domesticated germ plasm heterozygous for Hr. Half-spheres also were routinely recorded. 1. Sanjur OI, Piperno DR, Andres TC, Wessel-Beaver L (2001) Phylogenetic relationships among domesticated and wild species of Cucurbita (Cucurbitaceae) inferred from a mitochondrial gene: Implications for crop plant evolution and areas of origin. Proc Natl Acad Sci USA 99: Reichert ET (1913) The Differentiation and Specificity of Starches in Relation to Genera, Species, etc. (Carnegie Institution of Washington, Washington, DC). 3. Seidemann J (1966) Stärke-Atlas (Paul Parey, Berlin). 4. Piperno DR, Ranere AJ, Holst I, Hansell P (2000) Starch grains reveal early root crop horticulture in the Panamanian tropical forest. Nature 407: Pearsall DM, Chandler-Ezell K, Zeidler JA (2004) Maize in ancient Ecuador: Results of residue analysis of stone tools from the Real Alto site. J Archaeol Sci 31: Zarillo S, Kooyman B (2006) Evidence for berry and maize processing on the Canadian plains from starch grain analysis. Am Antiq 71: Holst I, Moreno JE, Piperno DR (2007) Identification of teosinte, maize, and Tripsacum in Mesoamerica by using pollen, starch grains, and phytoliths. Proc Natl Acad Sci USA 104: Zarillo S, Pearsall DM, Raymond JS, Tisdale MA, Quon DJ (2008) Directly dated starch residues document Early Formative maize (Zea mays L.) in tropical Ecuador. Proc Natl Acad Sci USA 105: Austin DR (2006) Fox-tail millets (Setaria:Poaceae) abandoned food in two hemispheres. Econ Bot 60: Piperno DR (2006) Phytoliths: A Comprehensive Guide for Archaeologists and Paleoecologists (AltaMira, Lanham, MD). 11. Piperno DR, Pearsall DM (1993) Phytoliths in the reproductive structures of maize and Teosinte: Implications for the study of maize evolution. J Archaeol Sci 20: Bozarth SR (1993) Maize (Zea mays) cob phytoliths from a central Kansas Great Bend Aspect archaeological site. Plains Anthropol 38: Mulholland SC (1993) in Current Research in Phytolith Analysis: Applications in Archaeology and Paleoecology, eds Pearsall DM, Piperno DR (MASCA, University Museum of Archaeology and Anthropology, Univ. of Pennsylvania, Philadelphia), pp Pearsall DM, Chandler-Ezell K, Chandler-Ezell A (2003) Identifying maize in Neotropical sediments and soils using cob phytoliths. J Archaeol Sci 30: Piperno DR, et al. (2007) Late Pleistocene and Holocene environmental history of the Iguala Valley, Central Balsas watershed of Mexico. Proc Natl Acad Sci USA 104: Iriarte J (2003) Assessing the feasibility of identifying maize through the analysis of cross-shape size and three-dimensional morphology of phytoliths in the grasslands of southeastern South America. J Archaeol Sci 29: Dorweiler JE, Stec A, Kermicle J, Doebley J (1993) Teosinte glume architecture 1: A major locus controlling a key step in maize evolution. Science 262: Dorweiler JE, Doebley J (1997) Developmental analysis of teosinte glume architecture 1: A key locus in the evolution of maize (Poaceae). Am J Bot 84: Piperno DR, Pearsall DM (1998) The silica bodies of tropical American grasses: Morphology, taxonomy, and implications for grass systematics and fossil phytolith identification. Smith Cont Bot 85: Piperno DR, Holst I, Wessel-Beaver L, Andres TC (2002) Evidence for the control of phytolith formation in Cucurbita fruits by the hard rind (Hr) genetic locus: Archaeological and ecological implications. Proc Natl Acad Sci USA 99: Piperno DR, Weiss E, Holst I, Nadel D (2004) Processing of wild cereal grains in the Upper Paleolithic revealed by starch grain analysis. Nature 430: Henry A, Piperno DR (2008) Using plant microfossils from dental calculus to recover human diet: A case study from Tell al-raqí, Syria. J Archaeol Sci 35: of10

3 Fig. S1. Starch grains from modern kernels of Z. mays ssp. parviglumis (A) and maize races Pepetilla (B) and Harinoso de Ocho (C). Maize starch is larger and mostly irregularly shaped, with well-defined compression facets and various types of fissures. In contrast, grains in ssp. parviglumis are predominantly oval to round, with fewer compression facets and other surface features typical of maize. 3of10

4 Fig. S2. Rondel phytoliths diagnostic of maize cobs. (A and B) Ruffle-top rondels from sediments directly associated with grinding stone 318d and from the needle probe analysis of a used facet of 318d, respectively. (C and D) Wavy-top rondels from the cm b.s. level of the unit 1 column sample. 4of10

5 Fig. S3. Rondels typical of maize cobs from the needle probe analysis of a used facet of grinding stone 318d. 5of10

6 Fig. S4. Cucurbita phytoliths from sediments associated with grinding stone 318 (A), from a modern hybrid of C. sororia and C. argyrosperma that is heterozygous at the Hr locus (Hr hr)(b), and from the cm b.s. level from the unit 1 column sample (C and D). The phytoliths exhibit surface features (surface cavities and marks, faint scalloped impressions) typical of fruits that have undergone human manipulation at the Hr locus. 6of10

7 Fig. S5. Phytoliths from C. argyrosperma ssp. sororia. Unlike in domesticated fruits and hybrids with domesticated germ plasm that are heterozygous at the Hr locus, these phytoliths exhibit no signs of incomplete silicification, such as surface cavities and faint scalloped impressions. 7of10

8 Table S1. Starch grain size in panicoid grasses common in the Mexican flora Length, m Range, m SD n Setaria geniculata (NH ) (NH ) Heteropogon melanocarpus (NH ) Hackelochloa granularis (NH ) (NH ) Schizachyrium condensatus (NH ) Aristida schiediana (NH ) Our starch research focused primarily on the Panicoideae, the subfamily to which maize belongs. Various studies have shown that other subfamilies of grasses are characterized by different types of starch grains that would not be confused with maize; for example, the Pooideae have simple round lenticular grains, sometimes with lamellae, or compound (and thus more highly angled) grains (2, 21, 22), and the Chloridoideae have mostly compound grains, whereas maize has simple grains (2). 8of10

9 Table S2. Starch grain characteristics in a representative sample of maize and teosinte Teosinte Maize race Balsas Chalco Central Plateau Bolita Reventador Naltel Tabloncillo Pepetilla Shape Round 44 (32 55) 33 (16 58) 11 (2 20) Oval 15 (11 19) 6 (2 10) 12 (12 13) Bell 10 (2 16) 3 (0 5) 7 (5 8) Irregular 30 (27 33) 58 (40 73) 70 (61 78) Hilum Cavity 24 (15 33) 12 (6 18) 9 (9 10) Compression facets Slight 58 (54 63) 47 (35 64) 44 (42 45) Defined 29 (21 35) 54 (36 63) 56 (55 58) Fissures Transverse 16 (12 19) 14 (12 18) 20 (11 30) Total with fissure 38 (37 40) 47 (41 54) 37 (25 48) Range of mean size, m Range of individual grain sizes, m Numbers are percentages of the different types of grains and surface features. Numbers in parentheses are the ranges for percentages of each attribute found in different populations studied. The range of mean size in teosinte represents the mean size in the different populations studied. (Data from ref. 7.) 9of10

10 Table S3. Number and distribution of starch grains recovered from each step of the stone tool analyses Tool catalogue number Sediment Needle probe First wash Ultrasound Total number Ground stone tools 310a unit 1, layer B, cm b.s. NA NNP a unit 1, layer B, cm b.s. NA NNP c unit 1, layer C, cm b.s. 1 NNP c unit 1, layer C, 57 cm b.s c unit1, layer D, cm b.s. 0 NNP d unit 1, layer D, cm b.s d unit 1, layer E, 77 cm b.s e unit 1, layer E, cm b.s d unit 1, layer E, 78 cm b.s c unit 1, layer E, cm b.s a unit 2, layer B, cm b.s. NA NNP a unit 2, layer B, cm b.s. NA NNP a unit 2, layer B, 45 cm b.s. 0 NNP unit 2, layer B, cm b.s. NA NNP a unit 2, layer C, 49 cm b.s c unit 2, layer C, 51 cm b.s. NA NNP b unit 2, layer C, 54 cm b.s. 0 NNP a unit 2, layer C, 57 cm b.s. 3 NNP unit 2, layer C, cm b.s. NA NNP a unit 2, layer D, 63 cm b.s a unit 2, layer D, 63 cm b.s. 1 NNP Chipped stone tools 308a unit 1, layer B, cm b.s. NA NNP a unit 1, layer B, cm b.s. NA NNP unit 2, layer B, cm b.s. NA NNP unit 2, layer E, cm b.s. NA NNP a unit 1, layer E, cm b.s. NA NNP NA, sediment was not analyzed; NNP, no needle probes were carried out. On tool 316d, the distribution of different types of grains was as follows: 68 from maize, 4 from Dioscorea, 3 from the Fabaceae, and 1 from the Marantaceae. On tool 365a, one unknown grain occurred. On tool 365a, 24 of the starch grains were from maize. Sediment is that sampled from immediately below and around the tools. 10 of 10