Abstract The genus Capsicum is New World in origin and represents a complex of a wide variety of both wild and domesticated taxa. Peppers or fruits of Capsicum species rarely have been identified in the paleoethnobotanical record in either Meso- or South America. We report here confirmation of Capsicum sp. residues from pottery samples excavated at Chiapa de Corzo in southern Mexico dated from Middle to Late Preclassic periods (400 BCE to 300 CE). Residues from 13 different pottery types were collected and extracted using standard techniques. Presence of Capsicum was confirmed by ultra-performance liquid chromatography (UPLC)/MS-MS Analysis. Five pottery types exhibited chemical peaks for Capsicum when compared to the standard (dihydrocapsaicin). No peaks were observed in the remaining eight samples. Results of the chemical extractions provide conclusive evidence for Capsicum use at Chiapas de Corzo during a 700 year period (400 BCE–300 CE). Presence of Capsicum in different types of culinary-associated pottery raises questions how chili pepper could have been used during this early time period. As Pre-Columbian cacao products sometimes were flavored using Capsicum, the same pottery sample set was tested for evidence of cacao using a theobromine marker: these results were negative. As each vessel that tested positive for Capsicum had a culinary use we suggest here the possibility that chili residues from the Chiapas de Corzo pottery samples reflect either paste or beverage preparations for religious, festival, or every day culinary use. Alternatively, some vessels that tested positive merely could have been used to store peppers. Most interesting from an archaeological context was the presence of Capsicum residue obtained from a spouted jar, a pottery type previously thought only to be used for pouring liquids.

Citation: Powis TG, Gallaga Murrieta E, Lesure R, Lopez Bravo R, Grivetti L, Kucera H, et al. (2013) Prehispanic Use of Chili Peppers in Chiapas, Mexico. PLoS ONE 8(11): e79013. https://doi.org/10.1371/journal.pone.0079013 Editor: John P. Hart, New York State Museum, United States of America Received: June 11, 2013; Accepted: September 18, 2013; Published: November 13, 2013 Copyright: © 2013 Powis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research was funded by a grant to Lesure from the Wenner Gren Foundation for Anthropological Research (Grant 8312). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Laboratory Analysis Methods Capsaicin, dehydrocapsaicin, 4-OH, 3-OMe-benzylamine, 3,4-Dihydroxybenzylamine and formic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). All solvents were mass spectrometry grade and all other chemicals used were of the highest grade available. Acquity UPLC HSS T3 1.7 µm (1×150 mm) column was purchased from Waters Corporation, Milford, MA. We confirm that we obtained permission from the Museo Regional de Chiapas to access the pottery vessels under analysis, and that they were loaned to us for one day while we conducted the residue study. Extraction of Chiapa de Corzo Pottery Samples A total of 13 pottery samples were extracted using the following procedure. Ninety –200 mg of burr from each sample was vortexed with 1 ml methanol: chloroform mixture (1∶1) for 3 minutes then centrifuged. The resulting precipitate from each sample was removed and the supernatant was concentrated with Speed vac. To the residue 100 ul methanol: water (1∶1) was added, vortexted and filtered with 5 kD membrane filters. The Filtrates were transferred to vials for UPLC/MS-MS analysis. Ultra Performance Liquid Chromatography (UPLC)/MS-MS Analysis A Xevo-TQ triple quadruple mass spectrometer (Waters, Milford, MA, USA) was used to record MS and MS-MS spectra using Electro Spray Ionization (ESI) in positive ion (PI) mode, capillary voltage of 3.0 kV, an extractor cone voltage of 3 V, and a detector voltage of 500 V. Cone gas flow was set at 50 L/h and desolvation gas flow was maintained at 600 L/h. Source temperature and desolvation temperature were set to 150 and 350°C, respectively. The collision energy was varied from 6 to 13 to optimize four different daughter ions. The acquisition range was 20–350 D. Pure standards (Figure 4) (Capsaicin, dehydrocapsaicin, 4-OH, 3-OMe-benzylamine and 3, 4-Dihydroxybenzylamine) were introduced to the source at a flow rate of 10 µl/min by using methanol:water (1∶1) and 0.1% formic acid mixture as the carrier solution to develop multiple reaction monitoring (MRM) method for UPLC/MS-MS operation (Table 2). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Major Capsaicinoids present in chili and proposed degradation pathway for biomarker analysis. https://doi.org/10.1371/journal.pone.0079013.g004 PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. MRM method parameters used in chilli analysis. https://doi.org/10.1371/journal.pone.0079013.t002 UPLC/MS-MS analyses of all the samples were carried out with a Waters Acquity UPLC system connected with Xevo-TQ triple quadruple mass spectrometer. Analytical separations on the UPLC system were conducted using a Acquity UPLC HSS T3 1.7 µm column (1×150 mm) at a flow rate of 0.15 ml/min. The gradient started with 100% A (0.1% formic acid in H 2 O) and 0% B (0.1% formic acid in CH 3 CN), changed to 50% A over 3 min, followed by a 4-min linear gradient to 10% A, resulting in a total separation time of 7 min. The elutions from the UPLC column were introduced to the mass spectrometer and resulting data was analyzed and processed using MassLynx 4.2 software. Pure standard mixture was used to optimize the UPLC conditions prior to analysis. After LCMS analysis the remaining extract of the samples was stored at −80°C in the Metabolomics Lab in the Department of Nutrition at UC-Davis for further evaluation.

Discussion and Conclusions The present study initially was conducted to search for chemical traces of cacao (Theobroma cacao) in a variety of vessel forms (e.g., bowls, jars, and vases), and most notably spouted jars, which were recovered from a variety of contexts (e.g., burials, caches) at Chiapa de Corzo. We also were interested in identifying flavorings and/or seasonings that could have been added to the cacao beverages. While we sought to recover chemical traces of cacao, none were found. We identified, however, traces of Capsicum in 38% of the sampled pottery. The results of the chemical extractions provide definitive proof that Middle and Late Preclassic vessels from the site contained Capsicum. The information obtained from the Chiapa de Corzo vessels extends the date of chili pepper use by the ancient Mesoamerican peoples by several hundred years into the Middle Preclassic period to about 400 BCE. While our scientific study has pushed back the antiquity of Mesoamerican chili pepper presence, we are most interested in how the pepper may have been used either from a culinary, pharmaceutical, or ritual perspective during this early time period. Finding positive Capsicum evidence in five samples of different pottery types and shapes – and where Capsicum was the only residue identified – a number of questions may be raised. It is important to mention that the analyses conducted on these samples were repeated to verify the presence of Capsicum in the positive samples. Was the chili ground up to produce a paste or a salsa and subsequently used as a seasoning in foods that were offered to the Zoquean gods or chiefs? Or, were the peppers left whole in the pots? We assume that the presence of chili is in the form of a sauce or paste, and not whole given that no seeds or other macrofossils were identified in the interiors of the vessels. If the peppers were served in paste form, then testing grinding implement (e.g., manos and metates) residues would be the next fruitful step in understanding the culinary practice involved. A starch grain analysis of such food preparation implements would complement our chemical extraction technique conducted on the pottery vessels. All of the vessels that tested positive for Capsicum were serving vessels and it becomes important to identify which pots may have been used in mixing the peppers into a paste form. At present, we do not have access to a full range of vessels associated with making such a seasoning. However, to date, there is not a single “molcajete’ found in the Chiapa de Corzo ceramic collection for the Preclassic period, like those found in Central Mexico for salsa making in later periods. This vessel form is found at Chiapa de Corzo but for the Postclassic period [34]. Further, were chili peppers consumed by elites and non-elites alike? All the chili-positive pottery vessels analyzed were discovered in different elite contexts from a long span of time from 400 BCE through CE 300, so was there restricted use of this pungent fruit 2,400 years ago? To address this question we would need to expand the dataset to include vessels derived from lower status or commoner contexts such as middens and burials. Why would there be evidence of chili peppers in a spouted jar? It is commonly assumed that spouted jars were used for pouring a liquid into another container. Perhaps the peppers were not made into a sauce but a spicy beverage or alternatively a chili sauce that was stored in the spouted jars and subsequently poured as a dining condiment. Given the pungency and “heat” associated with chili preparations, it would be logical to suggest that specific jars would be used to store and serve the products – jars whose only function was to store and serve chili-related products, at least for Samples 1 and 3. It is important to consider the possibility that all or at least some of the pots deposited at a specific event (tomb or offering) would be part of the community-wide feast and that afterwards the empty pots were deposited as part of the ritual deposition [24], [39]. If so, then the drained pots would leave minimal residues for archaeologists to examine. The sampled vessels were chosen by form rather than context, making the assumption that those particular types would be more likely to get a positive result for cacao (Theobroma cacao). The unexpected results provide a different scenario. From five different elites contexts (three burials: Tomb 7, 12, and 149, and two offerings) and each of them containing several vessels, at least one of the vessels chosen had chili residue on it. It would be interesting to sample the entire vessel assemblage from one or more of these contexts to see if other vessel types and forms had chili in them, or if other substance(s) were identified such as cacao. Information from hieroglyphic texts, both on vessels and murals, will help to shed light on the types of beverages prepared and consumed as well as which ingredients were used to flavor them. There is strong evidence for this in the Maya area where epigraphers have deciphered texts showing different kinds of cacao-based beverages and gruels [40]–[43], for example, but, to date, few identify chili peppers as an additive or flavoring. It is hoped that we can remedy this lack of information, from a biomolecular perspective, in the future when we conduct an indepth study of the remaining pottery vessels from Chiapa de Corzo. Is it possible that the chili substance inside these vessels was used for medicinal, ritual, or magical purposes rather than culinary? Recent studies show that Capsicum species were included in a number of herbal remedies for microbial origin ailments by the Maya [44]. Once again we have the problem that only Capsicum was identified, but still possible that the chili residue on the pot was placed to be used for medicinal purposes by its owner in the other world. Another explanation for this unexpected result is provided by Alfredo Lopez Austin. He suggested that sometimes the interior of the vessels were covered with a mixture of chili and ash as a measure to prevent/repeal insects to eat whatever is inside (Alfredo Lopez Austin, personal communication, 2013). So, in this case the chili could be just a part of the process to preserve something else that was not preserved or taken away at some point in time. The fortuitous finding of Capsicum species in these pots provides the earliest evidence of chili consumption in well-dated Mesoamerican archaeological contexts. We do not know exactly what the Chiapa de Corzo people were doing with it, but are clear that its use is important to be present on at least five different elite contexts and to be part of a ritual paraphernalia from at least 400 BCE to AD 300. Clearly, more research would be necessary to know what exactly Capsicum species are been used for, if it is a local species or is part of the well-known Mixe/Zoque trade network at Chiapa de Corzo. These questions, among others, would be promising but spicy areas of future research.

Acknowledgments We would like to thank Alfredo Lopez Austin, Aurora Montufar Lopez, David Lentz, Ruth Dickau, Dolores Piperno, Emily McClung de Tapia, Patricia Crown, John Clark, Marco Beteta, Paul Minnis, and Sonia Zarrillo for providing information about chili peppers in Mesoamerica and South America. Thanks to INAH and the Consejo de Arqueologia to give permission for the project and exportation of the samples for analysis, as well as Centro INAH Chiapas for providing the Chiapa de Corzo samples. Norbert Stanchly is thanked for providing comments on an earlier draft of this manuscript, and comments from the academic editor and reviewers made the final version better.

Author Contributions Conceived and designed the experiments: TP EGM RL LG HK NG. Performed the experiments: LG HK NG. Analyzed the data: TP EGM RL RLB LG HK NG. Contributed reagents/materials/analysis tools: LG HK NG. Wrote the paper: TP EGM RL RLB LG HK NG. Photographed all of the pottery vessels that were analyzed for ancient residues: EMG RLB.