Starch grain and phytolith evidence for early ninth millennium B.P. maize from the Central Balsas River Valley, Mexico

Starch grains were recovered from 19 of the grinding stones and 3 of the chipped stone tools. Maize was the dominant starch type on every tool, accounting for 90% of all grains recovered (Table 1; Fig. 1). Previous research has demonstrated that starch grain size and morphology are of significant utility for differentiating maize from wild grasses native to North, Central, and South America (6–8, 10, 13) (see also SI Materials and Methods and Table S1). Our work in Mexico required a good understanding of starch grain characteristics in wild Zea, represented by 4 species and 3 subspecies of teosinte occurring from Mexico to Nicaragua (3). Recent research by I.H. and D.R.P. has demonstrated that size and morphological criteria can be effectively used to separate the teosintes, including ssp. parviglumis, from maize (9); see SI Materials and Methods and Table S2 for a summary. These data, combined with the results for non-Zea wild grasses, provide a secure method for identifying maize in archaeological starch grain records from the Central Balsas region.

Tables 1 and 2 summarize size and morphological characteristics of the grass starch grains isolated from the artifacts. For both the preceramic- and ceramic-phase artifacts, average grain size was commonly 12–17 μm, which is characteristic of maize and outside the mean for any species or subspecies of teosinte (9; Table S2). The largest average size recorded in Balsas teosinte, the wild progenitor of maize, is 9.5 μm (9). The maximum size of individual grains in the Xihuatoxtla samples is commonly 20–26 μm, which also indicates maize, because it greatly exceeds that recorded in all types of teosinte except Race Chalco (Zea mays ssp. mexicana), which is known to frequently hybridize with maize. Starch grain morphological characteristics also indicate the presence of maize (Table 2; Fig. 1); for comparison, Fig. S1A–C shows modern teosinte and maize starch grains. For example, the grains isolated from the preceramic- and ceramic-period artifacts are predominantly irregularly shaped, and most have defined compression facets, unlike those of teosinte. Conversely, oval and bell-shaped grains, which are common in teosinte, were rare to absent on the tools, as they are in maize (9; Table S2).

The starch grain assemblages also contain higher frequencies of a type of fissure called a “transverse” fissure (a natural crack on the grain formed at the grain hilum or center) than are seen in modern teosinte samples (Fig. 1A); for example, the highest percentage of transverse fissures recorded in teosinte is 20% in the race Central Plateau. The archaeological frequencies are characteristic of Mexican popcorns (9; Table S2). Four of the preceramic grinding stones (316d, 318d, 318e, and 365a) have particularly good sample sizes, clearly demonstrating that every aspect of the starch grain assemblages indicates the presence of maize throughout the Xihuatoxtla preceramic sequence. Starch from few other taxa was present, indicating that maize was a focus of early food processing strategies. Grain morphology suggests the presence of popcorns or other hard-endosperm maize types (6, 9).

Eight of the grinding stones from which maize starch was recovered were securely stratified deep in preceramic levels (layers D and E), originating from 60–90 cm of units 1 and 2, well below a date of 4730 ± 40 B.P. (5590–5320 cal. B.P.) on charcoal from 49 cm b.s. (layer C) of unit 2 (Table 1) (1). A large piece of charcoal from 65 cm b.s. of unit 1 (layer D) yielded an age of 7920 ± 40 B.P. (8990–8610 cal. B.P.; intercept, 8710 cal. B.P.) (1). Four of the grinding stones and 2 of the chipped stone tools with maize starch occurred below this dated sample.

The sequence of steps used to analyze the artifacts showed that starch survival was very poor in sediments that occurred immediately around and below the stones; most samples had no grains (Table S3). This finding conforms to the expected rapid degradation of starch after deposition into soil (10). On all but one tool in which needle probe analysis was carried out, grains were retrieved from deep inside small surface cracks and crevices, and nearly all of them originated from tool facets showing signs of use wear, such as smoothing or polish (1). The remainder of the grains were recovered from analysis of sediments that were firmly adhered to the tools when they were removed from the ground (called the “first wash” step in Table S3) and during the final extraction step when washed stones were shaken in an ultrasound unit. Patterns of grain occurrence thus indicate that the starch represents plants that were processed with the artifacts, which, as other studies have shown, provided protected environments enabling preservation (6–8, 10, 13).

The phytolith record provides independent and mutually supportive evidence for maize and amplifies how it was exploited during the early Archaic Period and later. Short-cell phytoliths diagnostic of the glumes and cupules of maize cobs, called wavy-top rondels and ruffle-top rondels, are present in a number of different contexts: (i) in the same residues from preceramic- and ceramic-phase grinding stones that yielded starch grains, (ii) in sediments closely associated with these artifacts, and (iii) in column samples spanning the site's occupation (Table 1; Fig. S2). The types of long-cell phytoliths that always occur in high numbers in (and are diagnostic of) teosinte fruitcases (i.e., the hard structures that enclose kernels; see SI Materials and Methods) are not present in any sample. Other rondel phytoliths, designated “maize-type” in Table 1, cannot be unequivocally assigned to maize, because they occur in some non-Zea grasses: however, they are the most common rondel types found in maize cobs, and because they co-occur with ruffle-top and wavy-top forms, they are likely from maize. Furthermore, these phytoliths lack edge ornamentations and thus are not characteristic of rondels from teosinte (Fig. S3).

These results corroborate the starch grain findings in indicating that the ears of teosinte were not exploited at Xihuatoxtla and that the Zea remains are exclusively from maize. The rondel phytoliths retrieved along with maize starch grains from the surfaces of grinding stones during needle probe analysis likely represent chaff that adhered to the kernels when they were removed from cobs and processed. Thus, all of the data indicate that maize was being grown during the early ninth millennium cal. B.P.

Some investigators have proposed that Zea was initially collected and cultivated to consume the sugar in its stalk and/or to make an alcoholic beverage from it (15, 16). This hypothesis is testable using the idiosyncratic short-cell phytoliths that maize and teosinte stalks produce in significant quantities and that would be expected to occur in the phytolith record (see SI Materials and Methods). These phytoliths were searched for through extensive scanning of the microscope slides containing the phytolith preparations and also through examination of the phytoliths occurring in the stone tool residues. They were not seen in any context at Xihutoxtla. Thus, it appears that the major focus of maize utilization was directed toward the cob of the plant.

A second major domesticated plant identified through phytolith analysis is a squash (Cucurbita sp.). Spherical “scalloped” phytoliths diagnostic of the fruit rinds of Cucurbita (11) are common in preceramic levels and rare in ceramic levels (Table 1; Fig. 2). This difference is most likely attributable to the frequent cultivation of human-selected, soft-rinded, and thus phytolith-poor fruits during the ceramic period, rather than to the near cessation of Cucurbita growing by the site's ceramic-phase occupants. Both phytolith and lignin production in Cucurbita fruits, which combine to make the fruit exterior hard and protect plants from herbivores, are known to be under the control of the hard rind (Hr) domestication gene (11, 14). Wild fruits (except rare mutants that would be deleterious in the wild) are homozygous for the dominant Hr allele and have very hard rinds with abundant phytoliths, whereas fruits that have been selected for soft rinds have very few or no phytoliths. The rarity of Cucurbita fruit phytoliths in a ceramic-period, pre-Columbian occupation following earlier occupations at the same site containing numerous such phytoliths follows a trend observed in a number of other Neotropical regions (11), suggesting that human selection for soft rinds was widespread by the middle and later Holocene.

Both phytolith size and morphology indicate that a domesticated Cucurbita was present along with maize during the earliest preceramic occupations of the site (Table 1; Fig. 2). Studies of fruits from numerous populations of C. sororia and other wild species native to Mexico [e.g., C. pepo ssp. fraterna (L. H. Bailey) Andres; C. lundelliana Bailey] indicate a maximum phytolith length of 100 μm (and average length no greater than 75 μm) and a maximum thickness of 68 μm in wild plants. In modern domesticated fruits, phytolith size often is considerably larger (11, 17). Three sediment samples from 3 different preceramic levels contain Cucurbita phytoliths with lengths of 106–120 μm and 2 have Cucurbita phytoliths with a thickness of 84 μm, all exceeding the firmly established baseline values for wild plants. Two of these samples, 319d and 318e, are sediments closely associated with grinding stones showing evidence of maize processing, and the other 2 samples are from 60–75 cm b.s. of the column sample. The mean Cucurbita length of 80 μm in the column sample from 60–65 cm b.s. also substantially exceeds the mean value recorded in wild species.

In addition, the known effects of the Hr gene on the morphology of these phytoliths point to their domesticated status. In the preceramic samples, > 73% of the phytoliths exhibit surface features characteristic of incomplete silicification that are typical of phytoliths in modern domesticated fruits and hybrids between wild and domesticated species heterozygous for Hr that have rinds of intermediate hardness (Fig. 2 and Fig. S4; SI Materials and Methods) (11, 14). These types of phytoliths are absent to rare in wild fruits that are homozygous for Hr and have very hard rinds (Fig. S5; SI Materials and Methods). The data imply that human selection for reduced lignification and silicification of fruits was underway by 8700 cal. B.P.*

We cannot say with certainty which domesticated taxon was used, because the types of phytoliths present occur in both of the domesticated squashes native to Mexico, C. pepo and C. argyrosperma (11, 14). The former occurs at 10,000 cal. B.P. in the central Mexican highlands (18), and it conceivably could have been dispersed at an early date to the study region. Because the wild ancestor of C. argyrosperma is native to the central Balsas watershed (4), and its early domesticated product would have been better adapted to the region's climate than C. pepo, the preceramic squash may more likely be from C. argyrosperma.

The sampling of stone tools involved a series of steps designed to carefully evaluate whether grains recovered from the stones represent plants that were processed with the artifacts (6). Unwashed grinding stones were first placed under a stereoscopic microscope at a power of ×100. The point of a fine needle was inserted deep into cracks and crevices of the surfaces to loosen and remove any residue. The residue was then transferred directly to microscope slides, mounted in water, and examined at a power of ×400. Between 8 and 21 locations covering the used and nonused facets of 9 grinding stones were examined in this manner. Because this step is time-consuming, a focus was put on preceramic-phase artifacts for the needle point studies; those analyzed by this method were 319d, 318d, 318e, 316d, 322c, 315c, 361a, 365a, and 367a (see Table 1 for the proveniences of these stones). For stones not analyzed in this way, starch analysis began with the next step, described below.

The stones were then placed underneath running water and washed with a brush to remove any sediment that was strongly adhered to them when they were removed from the ground. The sediment was saved, and starch was removed from it through the addition of a heavy liquid metal at a density of 1.8 made from cesium chloride (CsCl). Next, the washed stones were shaken in an ultrasound unit for 5–10 min to further dislodge starch grains, which were then isolated from the solution using CsCl as in the previous step. Between 10 and 20 cm³ of sediment sampled from immediately beneath and around (within 10–15 cm) the stones while the excavation was in progress also was analyzed for starch content using this heavy liquid treatment.

Chipped stone tools were analyzed by placing them directly in the ultrasound unit and continuing the analysis as described above.

Phytolith extraction from sediments followed standard techniques involving sediment dispersion, removal of carbonates and organic materials, and separation of phytoliths using a heavy liquid solution at a density of 2.3 (11).