Supporting Information Piperno et al. 10.1073/pnas.0812525106 SI Materials and Methods structures which are homologous to each other, have been shown Modern Reference Collections and Microfossil Identification. Our to be controlled primarily by tga1, a major domestication gene reference collections of phytoliths and starch grains include with significant effects (10, 17, 18). tga1 underwrites the degree more than 2,000 and about 500 species, respectively, and include of silicification of the glumes and rachids (cupules) of the many wild taxa of economic importance, most of the known fruitcases and cobs. In teosinte, the entire epidermis, consisting domesticated plants native to Central and South America, and of both the long and short cells, is silicified, whereas in maize wild progenitors and other close wild relatives of the crop plants. (which requires less natural protection from its herbivores), Investigating the history of maize and squash in the study region silicification is greatly reduced, and only the short epidermal was one of our priorities; therefore, our reference collections cells (which produce the phytoliths called rondels) are filled with include all known species and subspecies of teosinte; 24 maize silica. In addition, the rondels produced in teosinte are more races from Central and South America, including 10 traditional highly decorated than those in maize (a result also of more Mexican land races; and all domesticated and most known wild extensive lignification in teosinte), and the rondel phytoliths in species of Cucurbita, including all those found in Mesoamerica, maize cobs have a more diverse morphology and are in forms not such as C. argyrosperma ssp. sororia, which is native to the study found in teosinte. These differences result in the formation of region and is the wild ancestor of C. argyrosperma (the silver- distinct and identifiable phytoliths in maize and teosinte that seeded squash or cushaw pumpkin) (1). allow them to be distinguished from each other and from With regard to starch grain identification, previous research non-Zea wild grasses, including the genus Tripsacum (10–16). has demonstrated that starch grains in maize commonly range from about 8 to 26 ␮m in maximum length and from 12 to 16 ␮m Other (Non-Maize) Starch Grains Present on the Stone Tools. Four in mean length (2–8). In non-Zea grasses, grain size typically yam grains (Dioscorea sp.), 3 legume grains, and 1 Marantaceae ranges from about 2 to 18 ␮m in maximum length and from 3 to grain occurred on tool 316d, a large, preceramic grinding stone 11 ␮m in mean length. In many wild species, the maximum grain base recovered from 60–67 cm b.s. of unit 1. The yam grains size is only 6–9 ␮m (2, 4–8). Often these grains are too small to cannot be identified as belonging to either a wild or domesticated allow confident discernment of surface features, but species with species, because considerable work is needed on wild Dioscorea larger grains have been found to have dissimilar morphological species native to Mexico to rule out possible confusion with characteristics to maize (4–7). In this study, we examined cultivated/domesticated taxa. This is the first empirical indica- additional species of non-Zea grasses common in the Mexican tion of yam usage in tropical Mexico during the pre-Columbian flora (Table S1), including a putative early cultivar from high- era, however. The legume grains are similar to some that occur land Mexico, Setaria parvifolia (Poiert) (formerly S. geniculata) in Phaseolus, but they lack some attributes common in P. vulgaris (9). As in other non-Zea grasses, starch grain size is considerably and P. lunatus, such as the presence of lamellae and fissures; thus, smaller than in maize, and morphological characteristics also we cannot unequivocally assign them to a specific legume taxon serve as distinguishing criteria. at this time. Similarly, the Marantaceae grain cannot be assigned With regard to the differentiation of maize and teosinte on the to a specific genus. One unknown grain occurred on tool 365a. 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. Other Types of Phytoliths Present in the Sediments. Marantaceae luxurians, endemic to Guatemala). Maximum grain length varies seed phytoliths, probably from either Maranta or Stromanthe, from 2 ␮mto28␮m; the latter was represented by a single grain were well-represented throughout the sedimentary sequence. observed in a specimen of Chalco teosinte (Z. mexicana), a race These phytoliths are not like those from arrowroot (M. arundi- that commonly hybridizes with maize (7). Maximum grain size nacea L.). A type of phytolith produced in the foliage and does not exceed 20 ␮m in non-Chalco teosintes and 18 ␮min sometimes the wood of various tree species also was common. Balsas teosinte. In contrast, in maize, mean length varies from Also persistently present in lower frequencies were phytoliths 11.4 to 15.8 ␮m, and in most races, mean length is 12.5 ␮m and from palms, Cyperaceae, and Asteraceae. maximum length is 20 ␮m, reaching 26 ␮m in some cases (7). Differences in such features as grain shape and surface Discriminating Phytoliths from Maize and Teosinte Culms (Stalks). sculptoring also provide clear morphological contrasts between Culms or stalks of grasses produce various idiosyncratic forms teosinte and maize (7) (Table S2). For example, nearly every race that do not occur in the leaves and inflorescences of the plants of maize studied has dominant proportions of ‘‘irregular’’ grains (19). Culm phytoliths in maize often are thick and irregularly (those without a clearly describable shape), and many have cross or bilobate in shape with unusual, deeply notched bases. defined (deeply impressed) compression facets, which develop These are distinct from phytoliths produced in leaves and cobs. when the grains are packed together during their formation in A stalk of Z. mays ssp. parviglumis from Guerrero state sampled the cellular organelles called amyloplasts. In contrast, teosinte from the herbarium folders at the U.S. National Museum of exhibits significant percentages of oval and bell-shaped grains, Natural History (NMNH 3123148) produced the same types of which are nearly absent in maize, and has far fewer irregular phytoliths, as well as other phytoliths not seen in the maize stalks grains. Most teosinte grains also lack defined compression facets studied. Although further work is needed to more robustly assess and have different types of fissures (i.e, cracks at the hilum, the whether the maize and teosinte phytoliths are diagnostic to Zea botanical center of the grain). or to the subspecies level, these phytoliths can be used to identify With regard to phytolith identification, criteria for the iden- stalk deposition. To be confident that young maize stalks, which tification of maize and teosinte phytoliths developed by ourselves presumably would have been used because they contain the and other investigators, including with the use of large blind highest quantity of sugar, produce phytoliths, we grew maize studies, are well described elsewhere (10–16). Importantly, the from seed at the Smithsonian Tropical Research Institute in considerable differences in morphological attributes of phyto- Panama. Stalks were harvested 53 days after they were planted liths formed in the fruitcases of teosinte and cobs of maize, and investigated for silica content and phytolith morphological Piperno et al. www.pnas.org/cgi/content/short/0812525106 1of10 attributes. The phytolith content was high, and the diagnostic dominate wild fruits (Fig. S5). The incompletely silicified phy- phytoliths produced by mature stalks were commonly present in toliths also commonly form as half-spheres (10, 20). All of these the young stalks. features are linked to the suppression of lignification and We restudied phytolith samples from important sites in Pan- silicification under artificial selection for softer rinds. ama containing starch grain and phytolith evidence for prece- We explored this issue in greater detail by examining 100 ramic maize (4, 10). No Zea-type stalk phytoliths were observed. phytoliths from each of 4 different fruits representing 3 different populations of C. argyrosperma ssp. sororia, the wild ancestor of Presence of Preceramic Phytoliths Indicative of Human Selection at C. argyrosperma. The fruits are homozygous at the Hr locus. In the Hr Genetic Locus. In modern domesticated species and the F 2 of these fruits, only 3 phytoliths with surface features (e.g., 1 marks or holes) characteristic of incomplete silicification were and F progeny of hybrids made between C. sororia and C. 2 recorded. In the other 2 fruits, 1 and 0 phytoliths of this type argyrosperma and between C. sororia and other domesticated occurred. Scans of phytolith preparations made from other fruits species, many phytoliths from plants that are heterozygous at the of C. sororia and other wild species further indicate that these Hr locus (Hr hr), and thus exhibit softer rinds than typically occur characteristics are rare in wild Cucurbita. in wild plants, acquire characteristic surface features, such as In preceramic samples 319d, 325 h, 316c, 318d, and 318e, in incompletely formed and fainter scalloped impressions and even which numerous squash phytoliths occurred, Ͼ 73% of the holes at the surface (Fig. S4B). These patterns may result from phytoliths (a far greater amount than in any wild species) the influence of modifier genes or incomplete dominance of the exhibited surface features like those in modern specimens with Hr locus (20). In any case, the types of phytoliths produced domesticated germ plasm heterozygous for Hr. Half-spheres also significantly outnumber the completely silicified forms that were routinely recorded. 1. Sanjur OI, Piperno DR, Andres TC, Wessel-Beaver L (2001) Phylogenetic relationships 13. Mulholland SC (1993) in Current Research in Phytolith Analysis: Applications in among domesticated and wild species of Cucurbita (Cucurbitaceae) inferred from a Archaeology and Paleoecology, eds Pearsall DM, Piperno DR (MASCA, University mitochondrial gene: Implications for crop plant evolution and areas of origin.