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Journal of Archaeological Science 40 (2013) 868e873

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Journal of Archaeological Science

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Maize, beans and the floral isotopic diversity of highland Oaxaca, Mexico

Christina Warinner a, Nelly Robles Garcia b, Noreen Tuross c,*aDepartment of Anthropology, Harvard University, United Statesb Instituto Nacional de Antropología e Historia de México, MexicocDepartment of Human Evolutionary Biology, Harvard University, Cambridge, MA 2138, United States

a r t i c l e i n f o

Article history:Received 30 October 2011Received in revised form2 July 2012Accepted 3 July 2012

Keywords:MesoamericaOaxacaPaleodietStable isotopesCarbonNitrogenTrophic levelAgriculture

* Corresponding author.E-mail address: tuross@fas.harvard.edu (N. Tuross

0305-4403/$ e see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.jas.2012.07.003

a b s t r a c t

Oaxaca, Mexico has been inhabited by humans for over 10,000 years. From the time of earliest habitation,this environment provided a wide floral subsistence to its human inhabitants, notably documented at thecave site of Guila Naquitz. Light stable isotopes, primarily carbon and nitrogen, have been used in dietaryand environmental reconstructions throughout Mexico and Central America. We report a large isotopicstudy of wild and market plants from Oaxaca that demonstrates 1) overlapping d13C values of C4 plants,including maize, and plants that utilize the Crassulacean Acid Metabolism (CAM) pathway for biosyn-thesis, 2) the existence of a significant C4 grass biomass, 3) the lack of isotopic separation in the d15N oflegumes and non-leguminous plants and 4) the increase in the nitrogen isotopic composition of cropplants relative to wild plant averages. These four observations are potential complicating factors ininterpretations involving the origins and spread of maize agriculture, the relative amount of maize in thediet and assessments of trophic level or meat contributions to the human diet.

� 2012 Published by Elsevier Ltd.

1. Introduction

Over the last thirty years, light stable isotope analysis hasbecome a standard tool of archaeological inquiry, and it hasexpanded our understanding of shifting global subsistence strate-gies and dietary practices over time. During subsistence recon-struction, idealized plant types and isotopic values are typicallyused as input parameters in paleodietary models; however, asarchaeologists increasingly employ isotopic evidence in high-resolution paleodietary studies, a more detailed understanding ofthe underlying foodweb is required.

In Mesoamerica human light isotopic data derived fromarchaeological contexts have been primarily employed to investi-gate three main paleodietary questions: 1) the origin and spread ofmaize agriculture, 2) the relative proportion of dietary maizeconsumed by farming populations, and 3) the proportion of plantand animal proteins in human diets (i.e., trophic level). However,efforts to address these questions have been impaired by thelimited amount of data available on the isotopic diversity of Mes-oamerican plants, especially from highland contexts. For example,there is little consensus regarding basic isotopic information, such

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as the average d13C of common C3 and C4Mesoamerican foods, andthe presumed d15N of legumes and non-leguminous plants varieswidely from study to study. Additionally, there is a growing body ofevidence that agricultural practices can dramatically impact thed15N of cultivated plants (e.g., Bateman et al., 2005; Bogaard et al.,2007), but the isotopic differences between wild and cultivatedplants inMesoamerica have not yet been systematically explored ordescribed.

In this study, we report new carbon and nitrogen isotopic valuesfor more than 300 plants collected from traditional markets, a fieldtransect, and archaeological deposits in the Valley of Oaxaca, aswell as comparative floral data from a lowland, coastal market andMesoamerican herbarium collections. This enlarged data set, rep-resenting a more than three-fold increase over all previously pub-lished Mesoamerican plant isotopic data combined, allowsassumptions made in Mesoamerican paleodietary modeling to bedirectly tested within a regional context.

Although this study focuses on plant isotopic diversity withinMesoamerica, the patterns observed have larger implications forpaleodietary modeling around the world. There is an increasingawareness that many basic assumptions about plant isotopicecology are in urgent need of reevaluation and refinement (Lee-Thorp, 2008). Although a discussion of plant physiology, ecologyand isotopic composition is beyond the scope of this paper,multiple reviews highlight the complexity of influences on

C. Warinner et al. / Journal of Archaeological Science 40 (2013) 868e873 869

observed isotopic composition in plants (e.g., Craine et al., 2009;Dawson et al., 2002; Kohn, 2010; Yoneyama et al., 2003).

One important component needed to interpret paleodiets andpaleoenvironments based on isotopic studies are empiricalstudies of diverse ecosystems. This study demonstrates the valueof an empirical approach and illustrates how local and regionalenvironments differ from theoretical, idealized or nonlocalaverages.

2. Materials and methods

Plants obtained for this study were analyzed for d13C and d15N atthe Harvard University Biogeochemistry Laboratory using estab-lished isotopic methods (Warinner and Tuross, 2009). Carbon andnitrogen isotopic values are expressed relative to VPDB and AIR,respectively. Statistical analysis was performed using IBM SPSSStatistics 19 software for Mac. Original data is available inWarinner(2010) and from the corresponding author upon request. Sampleswere obtained from both highland and lowland locations inMexico, and include modern, historical, and archaeological plantsfrom wild and cultivated contexts (Fig. 1). Highland market plantswere purchased during the winter and summer seasons from threemarkets in the Valley of Oaxaca (elevation c. 1500 m). In addition toplants, fermented pulque was also acquired. A total of 196 samplesfrom more than 40 taxa were collected from the Valley of Oaxacamarkets. Comparative lowland market plants were collected fromthe coastal city of Villahermosa, located approximately 30 km fromthe Gulf of Mexico at an elevation of 12 m. A total of 45 plantsamples from more than 30 taxa were collected from the Villa-hermosa market.

Wild vegetation was sampled in the winter along a 185-m ele-vational transect in the vicinity of the Guilá Naquitz rockshelter,located in the UNESCO protected prehistoric caves area betweenMitla and Yagul in the Valley of Oaxaca. Plant collection zones wereestablished at intervals from the rockshelter to the valley floor andconsisted of complete sampling of all plants within a 5-m radius ofeach collection point. A total of 96 samples were collected from fivecollection zones, and an additional 54 samples were collected non-systematically between collection zones. Plants were classified into

-40

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Fig. 1. Box plot comparison of d13C values for (left to right) C4, CAM, and C3 plantscollected from the caves area between Yagul and Mitla (black), and modern market(white). Among modern plants, both wild- and market-collected C4 and CAM plantshave similar d13C values, except for maize, which is on average 1e2& higher. Both wildand market C3 plants exhibit a wide range of d13C values and mean d13C valuesof �28.1& and -27.3&, respectively. Historic and archaeological plant samples exhibitelevated d13C values across all plant types (see Appendix). Error bars represent 95%confidence intervals; stars represent extreme outliers.

three broad categories: 1) grasses and sedges, 2) cacti and succu-lents, and 3) other plants.

Carbonized plant remains were obtained from archaeologicaldeposits at the Zapotec site of Atzompa, a satellite community ofMonte Alban located in the Valley of Oaxaca. A total of fivesamples from different contexts were analyzed. Calibratedradiocarbon dates for the plant remains range from the 5th to 16thcenturies AD.

Eight plant samples were obtained for isotopic analysis from theHarvard University Herbaria collections. Samples were originallycollected between 1894 and 1906 fromOaxaca, Mexico. All sampleswere desiccated before mounting and exhibit good structuralpreservation.

3. Results

3.1. Carbon isotopes

A total of 296 plant d13C values were measured from Oaxacancollections. One hundred sixty modern plant d13C values weremeasured from the Oaxacan market collection. No significantdifference in d13C was observed between summer- and winter-collected specimens and the data were combined for subsequentanalysis. One hundred fifty modern wild plant d13C values weremeasured from a transect collection in the caves area betweenMitla and Yagul in Oaxaca, and d13C values were also measured foreight historic (1894e1906) and five archaeological Oaxacan plantspecimens. In all collections, C3 and C4 plants exhibit good isotopicseparation, although carbon isotopic averages differ from commonestimates reported in paleodietary models (Fig. 1). Among C3plants, the mean d13C of modern wild (�28.0 � 2.3&; n ¼ 88) andmarket (�27.3� 2.0&; n¼ 133) plants is similar to that reported inother empirical studies (O’Leary,1988; Smith and Epstein,1971; vander Merwe, 1982), but 1e3& lower than the estimates commonlycited for modern plant means in Mesoamerican studies (Table 1)and paleodietary reviews (e.g., Lee-Thorp, 2008; Tykot, 2004,2006). More important than the mean d13C of C3 plants utilized fordietary reconstruction is the large range (>10&) and standarddeviation (>2.0&) observed in this study. The range and standarddeviation are similar for both wild and market plants, indicatingthat a high degree of C3 isotopic diversity is characteristic of Oax-acan plants in general and is not the product of particular

Table 1Modern plant d13C and d15N values assumed in Mesoamerican paleodietary studies.

Location d13C d15N Referencesa

C4 C3 Legume Non-legume

Central Mexico �9.0 �25.0 0.0 6.2 2Belize �9.0 �26.5 1 2e4 1Belize �9.5 �27.0 e e 5Belize �9.6 �26.4 e e 8Belize �11.0 �25.0 0 2e6 9Belize �9.0 �26.0 e e 10Belize �9.5 �26.5 e e 11Belize �11.0 �25.0 0 2e6 12Belize/Guatemala/

Honduras�12.5 �26.5 e e 4

Guatemala �9.6 �26.0 e e 3Guatemala �12.5 �27.0 e e 7Guatemala �10 �26 e e 14Honduras �12.5 �27.0 1 9 6Honduras �12.5 �27.0 1 9 13

a 1 ¼ Coyston et al. (1999); 2 ¼ DeNiro and Epstein (1981); 3 ¼ Emery et al.(2000); 4 ¼ Gerry and Krueger (1997); 5 ¼ Powis et al. (1999); 6 ¼ Reed (1994);7¼ Reed (1999); 8¼ van derMerwe et al. (2000); 9¼White (2005); 10¼White andSchwarcz (1989); 11 ¼ White et al. (1993); 12 ¼ White et al. (2001a,b);13 ¼ Whittington and Reed (1997); 14 ¼ Wright (1997).

C. Warinner et al. / Journal of Archaeological Science 40 (2013) 868e873870

agricultural practices or selective sampling. Although sample sizesfor historic (�23.2& � 2.3; n ¼ 5) and archaeological(�23.9&� 1.3; n¼ 4) C3 plants were too small to characterize pastisotopic variation, their mean d13C is higher than for modern plants(see Appendix 4 and 5 of supplemental data). The differences inmodern vs. historic and archaeological d13C values are consistentwith the addition of industrial CO2 to modern plant material (theSuess effect), although it is interesting to note in this small samplethat the Oaxacan historic plants collected early in the industrialrevolution do not differ in their carbon isotopic composition(Revelle and Suess, 1957).

Modern cultivated d13C values (�13.1& � 0.7; n ¼ 6) and wild(�14.3& � 3.4; n ¼ 30) for CAM plants are on average lower thanfor market maize (�11.8& � 1.0; n ¼ 5), but not wild C4 grasses(�13.3& � 0.5; n ¼ 32) or market amaranth (�12.8&; n ¼ 2). Thed13C values for historic and archaeological maize (�9.4; n ¼ 1),amaranth (�10.1; n ¼ 1), and CAM specimens (�10.5; n ¼ 2) arehigher than those of their modern counterparts. Importantly, thecultivated CAM plants measured in this study, which include agaveand Opuntia cacti, do not vary widely in d13C, as has been found inother environments (Griffiths, 1992; Winter and Holtum, 2002;O’Leary, 1988). Within the wild caves area transect collection it isalso noteworthy that 90% of the grasses sampled are C4, and that80% of CAM plants have C4-like d13C values (>�15.0&). The rela-tively enriched CAM d13C values coupled with a significant C4 grassbackground in Oaxaca, suggests that, absent other information,determining the introduction or relative contribution of maize tothe human diet on the basis carbon isotopic data alone isproblematic.

3.2. Nitrogen

The mean d15N of Oaxacan market plants (3.6& � 3.6; n ¼ 143)is significantly higher than that for wild transect plants (1.6&� 2.3;n ¼ 119) by 2& (Fig. 2; One way ANOVA, F ¼ 30.23, df ¼ 1,p < 0.001), suggesting that the agricultural management practicesin contemporary Oaxaca result in crop plants with enrichednitrogen isotopic composition. Although contemporary cultivation

Fig. 2. Box plot comparison of d15N values from modern wild- and market-collectedOaxacan plants. The significantly higher mean d15N of market plants (One wayANOVA; F ¼ 30.23; df ¼ 1, p < 0.001) may be related to cultivation practices. Error barsrepresent 95% confidence intervals. Hollow circles represent outliers; stars representextreme outliers.

methods certainly differ from those in the past, there is a growingbody of archaeological evidence for complex pre-Columbian agri-cultural management and soil conservation practices (Beach et al.,2009; Bloom et al., 1983; Jacob 1995; Kunen, 2001). Raised fieldagricultural technology, in particular, has been shown to substan-tially alter local nitrogen cycles, resulting in sustainable, high-yieldagriculture (Biesboer et al., 1999; Erickson, 1988). Thus, an agri-culturally based increase in plant d15N could significantlycompromise the interpretations of human trophic placement orestimations of dietary meat consumption.

Little isotopic discrimination could be found between legumesand non-legumes of the Oaxacan market plants (Fig. 3). Althoughsimilar results have been found in ecological studies of South Africaand the Sonoran Desert (Heaton, 1987; Shearer et al., 1983), muchof the Mesoamerican archaeological literature assumes that thed15N distribution between legumes and non-legumes are distinctand largely non-overlapping (Table 1). In the Oaxaca marketcollection, we find that the d15N values for 22 legumes and 121 non-legumes range from �3.1& to 16.8& with complete isotopicoverlap of the two plant types.

Interestingly, legumes as a whole do not represent a homoge-nous group. Using discriminant function analysis (DFA) we foundthat d15N values could be used to distinguish the common beanfrom leguminous trees and shrubs (Wilk’s l ¼ 0.65, c21 ¼ 7:94,p ¼ 0.005), with a correct classification rate of c. 85% and cross-validation rates of 71e84%. The mean d15N of the leguminoustrees and shrubs (0.4& � 1.9; n ¼ 9), including mesquite (Prosopisspp.), sicima (Pachyrhizus erosus) and guaje (Leucaena spp.), issignificantly lower (Fig. 3; One way ANOVA, F ¼ 13.12, df ¼ 1,p ¼ 0.002) than that of the common bean, Phaseolus vulgaris(4.0& � 2.6; n ¼ 13). This difference likely results from differentcultivation techniques and further emphasizes the role of agri-culture in shaping plant d15N values. Thus, the d15N of consumertissue in Oaxaca and likely elsewhere in the region cannot be usedto assess the amount of legume consumption in agriculturalsettings.

Fig. 3. Box plot comparison of d15N values from modern, market-collected legumesand non-legumes from Oaxaca, Mexico. Discriminant function analysis supports thedivision of legumes into two categories: the common bean (Phaseolus vulgaris) andother legumes (Prosopis spp. and Leucaena spp.). The mean d15N of Phaseolus vulgaris,an intensively cultivated crop, is similar to that for non-legumes and is significantlyhigher than that of other legumes (One way ANOVA, F ¼ 10.33, df ¼ 1, p ¼ 0.005). Errorbars represent 95% confidence intervals. Hollow circles represent outliers; starsrepresent extreme outliers.

C. Warinner et al. / Journal of Archaeological Science 40 (2013) 868e873 871

3.3. Highland vs. lowland

The d13C and d15N of highland and lowland market sampleswere compared to determine if elevation and environmentaldifferences result in regional isotopic differences. Forty-five d13Cvalues and 44 d15N values were measured for lowland marketplants. No significant difference in d13C was observed among C3plants, CAM plants, or maize between the two regions. Withrespect to nitrogen, the lowland market non-legumes (5.6& � 3.3;n ¼ 39) and legumes (5.4& � 2.4; n ¼ 5) are more enriched thanhighland crops. The difference is significant for non-legumes (Oneway ANOVA; F ¼ 8.93; df ¼ 1; p ¼ 0.003), but only marginally so forlegumes (One way ANOVA; F ¼ 4.24; df ¼ 1; p ¼ 0.051), for whichthe small sample size is likely a factor. Previous studies have indi-cated that aridity may lead to plant nitrogen isotopic enrichment(Ambrose, 1991), but in this study it is the wetter environment thatyielded the higher plant nitrogen isotopic values. The differences inmean d15N between these two regions may instead be due todiffering soil fertility or cultivation practices.

4. Discussion

Establishing the isotopic baseline of a local foodweb is essentialbefore effective stable isotope-based paleodietary modeling can beperformed. The Valley of Oaxaca has strong C4/CAM representationin the wild flora, and we find that Mesoamerican C4 and CAMplants from both wild and cultivated contexts exhibit similar andoverlapping d13C values. Pulque, a fermented agave beverage withpre-Columbian origins, is also enriched in the heavy isotope ofcarbon, consistent with its CAM plant source, agave. In accordancewith previous studies (e.g., Schwarcz et al., 1985; Tieszen and Fagre,1993), maize is found to be 1e2& heavier on average than other C4and CAM taxa, including amaranth, another domesticated C4 grain.

Among C3 plants, we find that the commonly assumed isotopicmean of modern C3 plants, �26.5&, is 1e2& more enriched thanthe measured mean d13C of wild and cultivated plants in Oaxaca, aswell in our comparative lowland market collection. A review of theliterature reveals that in large-scale studies, a mean d13C of�26.5&has only been observed among African savannah C3 grasses (Vogel,1978). Traditional Mesoamerican diets do not include C3 grasses; infact, of the 72 market-collected C3 specimens measured in thisstudy, none are grasses, demonstrating that grasses are a poorisotopic proxy for the C3 component of Mesoamerican diets.Although modern Mesoamerican C3 plants are more isotopicallydepleted than commonly assumed, ancient C3 plants may havebeen far more enriched than current models of the Suess effect(Suess, 1955) posit. In this study, a small sample of historic(1894e1906) and archaeological (AD 400e1600) Oaxacan C3 plantsare approximately 4& more enriched than modern Oaxacan C3plants. If the enriched carbon isotopic values of historic andarchaeological C3 plants in this study are authentic and not theresult of unknown taphonomic processes, current paleodietarymodels will need to be amended to account for this difference, andestimations of past maize consumptionwill need to be reevaluated.

With respect to nitrogen, we find high variability in the d15N ofMesoamerican plants (range> 19&), and complete isotopic overlapbetween the legumes and non-legumes. The mean d15N values ofwild and market plants are statistically different, which may berelated to cultivation practices. This fact is further reflected in theisotopic differences observed between the common bean and otherlegumes measured in this study. The common bean is an inten-sively cultivated staple crop and the most commonly consumedlegume in Mesoamerica. The mean d15N of the common bean ismore than 3.5& higher than the mean for leguminous tree andshrub taxa, which requires little tending or cultivation effort. Our

findings are consistent with the growing body of evidence sug-gesting that cultivation practices are major drivers of plant d15N inagricultural systems (Bateman et al., 2005; Bogaard et al., 2007;Choi et al., 2002; Flores et al., 2007; Nakano et al., 2003), and ourresults indicate that this is true also for legumes. In prehistoricMesoamerican economies where populations consumed cultivatedplants and hunted wild animals, there may be little differentiationbetween d15N of plant and meat resources, leading to irresolvableproblems of isotopic equifinality in trophic level reconstruction.

The important excavation and analysis of the cave, Guila Naquitz(Flannery, 2009) in the 1960’s contains an analysis of wild food-stuffs available to humans in the area. Guila Naquitz containedsome of the earliest archaeological plant evidence in the Americasincluding directly AMS dated squash (Smith, 1997) at 10 kya, andcorn (Piperno and Flannery, 2001) at approximately 5 kya. In threeof the four ecotones surrounding Guila Naquitz, the dominant plantprotein source was the CAM utilizing prickly pear cactus (Opuntia).In all four ecotones, the estimates of plant based protein produc-tivity were far greater than that calculated for animal proteinavailability. Flannery (pg 262) is careful to point out that estimatesof food sources available in a given area do not necessarily translateto a human menu. However, the use of CAM plants such as pricklypear (Opuntia) and organ cactus (Stenocereus) in the human diet hasa long history in the Valley of Oaxaca documented at such sites as ElPalmillo (Feinman et al., 2007). The dominance of CAM plants in thecaves area coupled with the preponderance of C4 grasses, a foodsource for herbivores, suggests that human bone collagen carbonisotopic values may have been quite positive from the earliest timesof human habitation in this area of Oaxaca.

5. Conclusion

An empirical examination of wild and cultivated plants in theValley of Oaxaca reveals a high degree of carbon and nitrogenisotopic diversity, as well as a landscape rich in C4 and CAMresources. The floristic complexity and isotopic equifinality of someplant types in Oaxaca may compromise the application of carbonand nitrogen stable isotope analysis to three major paleodietaryquestions: the origin and spread of maize agriculture, the amountof maize consumed by farming populations, and the proportion ofplant and animal proteins in human diets (i.e., trophic level). Wesuggest that the issues of a more widely distributed C4 biome, therelatively enriched d13C values of CAM plants, and the potentialoverlap of nitrogen isotopic values in agricultural foodstuffs andanimals feeding off wild plant matter should be considered indietary reconstructions of Oaxaca.

Two other issues deserve a brief mention. A recent controlleddiet experiment using pigs (Warinner and Tuross, 2009) found thatnixtamalization (a traditional form of alkaline cooking used toprepare maize dough for foods such as tortillas) alters diet-tissueoffsets such that the amount of maize in the diet can be over-estimated as a function of food processing. Second, the use andincorporation of alcoholic beverages in isotopic studies is generallynot considered, although maize beer is considered a likelycontributor to consumer carbon isotopic composition in a study byUbelaker et al. (1995). In Oaxaca, the use of the fermented beveragepulque, derived from the CAMplant agave, has likely been in use formany centuries, if not millennia and would have further enrichedthe carbon isotopic values of consumers.

This study highlights some of the unknowns involved inreconstructing past plant isotopic values. Although the issuesraised in this paper complicate paleodietary reconstructions insome environments, we do not argue that stable isotope-basedpaleodietary studies in Oaxaca or Mesoamerica are out of reach.Careful diachronic studies that include human, faunal, and

C. Warinner et al. / Journal of Archaeological Science 40 (2013) 868e873872

archaeobotanical isotopic values can yield important informationabout changes in a region. Combining isotopic information withpaleoethnobotanical and zooarchaeological analyses can also yieldnew insights into past diets and environments. Finally, theemerging use of hydrogen and oxygen isotopes in bone collagenmay add to our ability to distinguish C4 and CAM subsistence andalso contribute to trophic level analyses.

Acknowledgments

This research was supported by grants from the David Rock-efeller Center for Latin American Studies, the Harvard UniversityOwens Fund, and the Harvard University Graduate Student Council.We’d like to thank Antonio Martínez Tuñón, Cheryl Makarewicz,Josh Wright, and David Diaz for their assistance with field collec-tion, Henry Kesner and Stephanie Zabel for assistance with theherbarium collections, Miriam Belmaker for statistical advice, andCynthia Kester for technical assistance with the mass spectrometer.We extend our sincere thanks to the Harvard University Herbaria,to Alejandro de Avila at the Jardín Etnobotanico de Oaxaca, and tothe many Mexican merchants who allowed us to sample theirproduce.

Appendix A. Supplementary data

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.jas.2012.07.003.

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