Abstract

Cacao (Theobroma cacao L.) is an important economic crop, yet studies of its domestication history and early uses are limited. Traditionally, cacao is thought to have been first domesticated in Mesoamerica. However, genomic research shows that T. cacao’s greatest diversity is in the upper Amazon region of northwest South America, pointing to this region as its centre of origin. Here, we report cacao use identified by three independent lines of archaeological evidence—cacao starch grains, absorbed theobromine residues and ancient DNA—dating from approximately 5,300 years ago recovered from the Santa Ana-La Florida (SALF) site in southeast Ecuador. To our knowledge, these findings constitute the earliest evidence of T. cacao use in the Americas and the first unequivocal archaeological example of its pre-Columbian use in South America. They also reveal the upper Amazon region as the oldest centre of cacao domestication yet identified.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

NCBI GenBank accession numbers of the Theobroma mitochondrial reference sequences are MF462389, MF462390 and MF462396 to MF462398. Examples of PCR amplified mitochondrial ancient DNA sequences identified as T. cacao sequences are reported in Supplementary Figs. 24. All results on ancient DNA sequences obtained after DNA capture and containing SNPs are reported in Supplementary Information; corresponding SNPs from the collection of modern accessions, used as controls, are reported in the Tropgene database (http://tropgenedb.cirad.fr/tropgene/)52. Additional data that support the findings of this study are available from the corresponding author on reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Powis, T. G., Cyphers, A., Gaikwad, N. W., Grivetti, L. & Cheong, K. Cacao use and the San Lorenzo Olmec. Proc. Natl Acad. Sci. USA 108, 8595–8600 (2011).

  2. 2.

    Powis, T. G. et al. Oldest chocolate in the New World. Antiquity 81 (2007); https://www.antiquity.ac.uk/projgall/powis314/

  3. 3.

    Henderson, J. S., Joyce, R. A., Hall, G. R., Hurst, W. J. & McGovern, P. E. Chemical and archaeological evidence for the earliest cacao beverages. Proc. Natl Acad. Sci. USA 104, 18937–18940 (2007).

  4. 4.

    Sheets, P. D. The Ceren Site: An Ancient Village Buried by Volcanic Ash in Central America 2nd edn (Wadsworth Publishing, Belmont, 2005).

  5. 5.

    Coe, S. D. & Coe, M. D. The True History of Chocolate 3rd edn (Thames and Hudson, London, 2013).

  6. 6.

    Stuart, D. in Chocolate in Mesoamerica: A Cultural History of Cacao (ed. McNeil, C. L.) 184–201 (Univ. Press of Florida, Gainesville, 2009).

  7. 7.

    Crown, P. L. & Hurst, W. J. Evidence of cacao use in the Prehispanic American Southwest. Proc. Natl Acad. Sci. USA 106, 2110–2113 (2009).

  8. 8.

    Washburn, D. K., Washburn, W. N. & Shipkova, P. A. Cacao consumption during the 8th century at Alkali Ridge, southeastern Utah. J. Arch. Sci. 40, 2007–2013 (2013).

  9. 9.

    Washburn, D. K., Washburn, W. N., Shipkova, P. A. & Pelleymounter, M. A. Chemical analysis of cacao residues in archaeological ceramics from North America: considerations of contamination, sample size and systematic controls. J. Arch. Sci. 50, 191–207 (2014).

  10. 10.

    Grivetti, L. H. & Shapiro, H.-Y. (eds) Chocolate, History, Culture, and Heritage (John Wiley & Sons, Hoboken, 2009).

  11. 11.

    Hurst, W. J., Tarka, S. M. Jr, Powis, T. G., Valdez, F.Jr & Hester, T. R. Cacao usage by the earliest Maya civilization. Nature 418, 289–290 (2002).

  12. 12.

    Powis, T. G., Valdez, F., Hester, T. R., Hurst, W. J. & Tarka, S. M. Spouted vessels and cacao use among the Preclassic Maya. Lat. Am. Antiq. 13, 85–106 (2002).

  13. 13.

    McNeil, C. L. Chocolate in Mesoamerica: A Cultural History of Cacao (Univ. Press of Florida, Gainesville, 2009).

  14. 14.

    Motamayor, J. C. et al. Cacao domestication I: the origin of the cacao cultivated by the Mayas. Heredity 89, 380–386 (2002).

  15. 15.

    Motamayor, J. C. et al. Geographic and genetic population differentiation of the Amazonian chocolate tree (Theobroma cacao L.). PLoS ONE 3, e3311 (2008).

  16. 16.

    Loor Solorzano, R. G. et al. Insight into the wild origin, migration and domestication history of the fine flavour Nacional Theobroma cacao L. variety from Ecuador. PLoS ONE 7, e48438 (2012).

  17. 17.

    Thomas, E. et al. Present spatial diversity patterns of Theobroma cacao L. in the neotropics reflect genetic differentiation in Pleistocene refugia followed by human-influenced dispersal. PLoS ONE 7, e47676 (2012).

  18. 18.

    Valdez, F. in The Handbook of South American Archaeology (eds Silverman, H. & Isbell, W. H.) 865–888 (Springer, New York, 2008).

  19. 19.

    Cuatrecasas, J. Cacao and its allies, a taxonomic revision of the genus.Theobroma. Contr. US Natl Herb. 35, 379–614 (1964).

  20. 20.

    Walker, T. in Chocolate: History, Culture, and Heritage (eds Grivetti, L. E. & Shapiro, H.-Y.) 543–558 (John Wiley & Sons, Hoboken, 2009).

  21. 21.

    Bletter, N. & Daly, D. C. in Chocolate in Mesoamerica: A Cultural History of Cacao (ed. McNeil, C. L.) 31–68 (Univ. Press of Florida, Gainesville, 2009).

  22. 22.

    Valdez, F., Guffroy, J., de Saulieu, G., Hurtado, J. & Yepes, A. Découverte d’un site cérémoniel formatif sur le versant oriental des Andes. C. R. Palevol. 4, 369–374 (2005).

  23. 23.

    Zarrillo, S. Human Adaptation, Food Production, and Cultural Interaction During the Formative Period in Highland Ecuador. PhD dissertation, Univ. Calgary (2012).

  24. 24.

    Lanaud, C., Loor, R. G., Zarrilo, S. & Valdez, F. Origen de la domesticacion del cacao y su uso temprano en el Ecuador. Nuestro Patrimonio 12, 12–14 (2012).

  25. 25.

    Zarrillo, S. & Valdez, F. in Arqueologia Amazonica: las Civilizaciones Ocultas del Bosque Tropical (ed. Valdez, F.) 147–171 (Abya-Yala, Ecuador, 2013).

  26. 26.

    Reichert, E. T. The Differentiation and Specificity of Starches in Relation to Genera, Species, Etc. (Carnegie Institution of Washington, Washington DC, 1913).

  27. 27.

    Schmieder, R. L. & Keeney, G. Characterization and quantification of starch in cocoa beans and chocolate products. J. Food. Sci. 45, 555–557 (1980).

  28. 28.

    Pagán Jiménez, J. R. Almidones: Guía de Material Comparativo Moderno del Ecuador Para los Estudios Paleoetnobotánicos en el Neotrópico (Aspha, Buenos Aires, 2015).

  29. 29.

    Hammerstone, J. F., Romanczyk, L. J. & Martin Aitken, W. Purine alkaloid distribution with Herrania and Theobroma. Phytochemistry 35, 1237–1240 (1994).

  30. 30.

    Pääbo, S. et al. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645–679 (2004).

  31. 31.

    Parducci, L. et al. Molecular- and pollen-based vegetation analysis in lake sediments from central Scandinavia. Mol. Ecol. 22, 3511–3524 (2013).

  32. 32.

    Willerslev, E. et al. Fifty thousand years of Arctic vegetation and megafaunal diet. Nature 506, 47–51 (2014).

  33. 33.

    Carpenter, M. L. et al. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries. Am. J. Hum. Genet. 93, 852–864 (2013).

  34. 34.

    Argout, X. et al. The genome of Theobroma cacao. Nat. Genet. 43, 101–108 (2011).

  35. 35.

    Pritchard, J. K., Stephens, M. & Donnelly, P. Inference of population structure using multilocus genotype data. Genetics 155, 945–959 (2000).

  36. 36.

    Hansen, A., Willerslev, E., Wiuf, C., Mourier, T. & Arctander, P. Statistical evidence for miscoding lesions in ancient DNA templates. Mol. Biol. Evol. 18, 262–265 (2001).

  37. 37.

    Silva, P. & Oliveira-Cardoso, A. Historico das introduçoes de cacaueiro (Theobroma cacao L.) no Reconcavo da Bahia, Brasil. Revista Theobroma 10, 135–140 (1980).

  38. 38.

    Mackensen, A. K., Brey, T., Bock, C. & Luna, S. Spondylus crassisquama Lamarck, 1819 as a microecosystem and the effects of associated macrofauna on its shell integrity: isles of biodiversity or sleeping with the enemy? Mar. Biodivers. 42, 443–451 (2012).

  39. 39.

    Piperno, D. R. & Pearsall, D. M. The Origins of Agriculture in the Lowland Neotropics (Academic Press, New York, 1998).

  40. 40.

    Hilbert, L. et al. Evidence for mid-Holocene rice domestication in the Americas. Nat. Ecol. Evol. 1, 1693–1698 (2017).

  41. 41.

    Biehl, B. Veränderungen der subcellulären struktur in keimblättern von kakaosamen (Theobroma cacao L.) während der fermentation und troeknung. Z. Lebensm. Unters. Forch. 153, 137–150 (1973).

  42. 42.

    Redgwell, R. J. & Hansen, C. E. Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans. Planta 210, 823–830 (2000).

  43. 43.

    King, A., Powis, T. G., Cheong, K. & Gaikwad, N. Cautionary tales on the identification of caffeinated beverages in North America. J. Archaeol. Sci. 85, 30–40 (2017).

  44. 44.

    Kufer, J. & McNeil, C. L. in Chocolate in Mesoamerica: A Cultural History of Cacao (ed. McNeil, C. L.) 384–407 (Univ. Press of Florida, Gainesville, 2009).

  45. 45.

    Cieslak, M. et al. Origin and history of mitochondrial DNA lineages in domestic horses. PLoS ONE 5, e15311 (2010).

  46. 46.

    Pääbo, S. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl Acad. Sci. USA 86, 1939–1943 (1989).

  47. 47.

    Dabney, J., Meyer, M. & Pääbo, S. in DNA Repair, Mutagenesis, and Other Responses to DNA Damage (eds Friedberg, E. C., Elledge, S. J., Lehmann, A. R., Lindahl, T. & Muzi-Falconi, M.) 19–26 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2013).

  48. 48.

    Argout, X. et al. Towards the understanding of the cocoa transcriptome: production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions. BMC Genomics 9, 512 (2008).

  49. 49.

    Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 13, 134 (2012).

  50. 50.

    Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G .J. Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

  51. 51.

    Ewing, B., Hillier, L., Wendl, M. C. & Green, P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185 (1998).

  52. 52.

    Hamelin, C., Sempere, G., Jouffe, V. & Ruiz, M. TropGeneDB, the multi-tropical crop information system updated and extended. Nucleic Acids Res. 41, D1172–D1175 (2013).

  53. 53.

    Argout, X. et al. The cacao Criollo genomev2.0: an improved version of the genome for genetic and functional genomic studies. BMC Genomics 18, 730 (2017).

  54. 54.

    Haak, W. et al. Massive migration from the steppe was a source for Indo-European languages in Europe. Nature 522, 207–211 (2015).

  55. 55.

    Cruz-Dávalos, D. I. et al. Experimental conditions improving in-solution target enrichment for ancient DNA. Mol. Ecol. Resour. 17, 508–522 (2017).

  56. 56.

    Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).

  57. 57.

    Jónsson, H., Ginolhac, A., Schubert, M., Johnson, P. L. F. & Orlando, L. mapDamage 2.0: fast approximate Bayesian estimates of ancient DNA damage parameters. Bioinformatics 29, 1682–1684 (2013).

  58. 58.

    GBIF Backbone Taxonomy (GBIF Secretariat, 2017); https://doi.org/10.15468/39omei

Download references

Acknowledgements

S.Z.’s research was funded by a Social Sciences and Humanities Research Council of Canada Doctoral Fellowship and an Honorary Isaak Walton Killam Memorial Scholarship, as well as the Martha Biggar Anders Memorial Award (2008, 2009, 2011), a Graduate Research Scholarship, a Faculty of Graduate Studies Travel Award, a Graduate Studies Scholarship and a Graduate Student Association Professional Development Grant from the University of Calgary. We thank the Universidad San Francisco de Quito, Riobamba and the University of British Columbia, Okanagan for use of laboratory facilities for some of S.Z.’s research. Funding for this project was provided by a Hampton Research Grant (no. F11-00878) from the University of British Columbia. Funding for archaeological excavations at SALF was provided by IRD. This project is supported by Agropolis Fondation under the reference ID 1202-029 through the Investissements d’avenir programme (Labex Agro: no. ANR-10-LABX-0001-01). The Hiseq sequencing activities were made in collaboration with the GeT platform, a partner of the National Infrastructure France Génomique, with thanks for support by the Commissariat aux Grands Investissements (no. ANR-10-INBS-0009). We are grateful to P. Lachenaud for providing genotyping by sequencing data on Guina group, to H. Kucera for assisting with Biochemical analyses, to N. Waber for preparing Fig. 1 and M. Berard for assistance in preparing Figs. 2 and 3. We also thank The United States Department of Agriculture, Agricultural Research Services, Plant Genetic Resources Conservation Unit for generously supplying Theobroma and Herrania pods and the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE—Costa Rica) and the Cocoa Research Center, University of West Indies (CRC, Trinidad and Tobago) for providing Theobroma and Herrania leaves for modern DNA analyses.

Author information

Author notes

  1. These authors contributed equally: Sonia Zarrillo, Nilesh Gaikwad, Claire Lanaud.

Affiliations

  1. Department of Anthropology and Archaeology, University of Calgary, Calgary, Alberta, Canada

    • Sonia Zarrillo
  2. Department of Anthropology, University of British Columbia, Vancouver, British Columbia, Canada

    • Sonia Zarrillo
    •  & Michael Blake
  3. Department of Nutrition and Department of Environmental Toxicology, West Coast Metabolomics Center, University of California, Davis, CA, USA

    • Nilesh Gaikwad
  4. Gaikwad Steroidomics Laboratory, Davis, CA, USA

    • Nilesh Gaikwad
  5. CIRAD, UMR AGAP, Montpellier, France

    • Claire Lanaud
    • , Christopher Viot
    • , Olivier Fouet
    • , Xavier Argout
    •  & Hélène Vignes
  6. AGAP, University Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France

    • Claire Lanaud
    • , Christopher Viot
    • , Olivier Fouet
    • , Xavier Argout
    •  & Hélène Vignes
  7. Department of Geography and Anthropology, Kennesaw State University, Kennesaw, GA, USA

    • Terry Powis
  8. INRA-UMR BIOGECO, Cestas, France

    • Isabelle Lesur
    • , Erwan Guichoux
    •  & Franck Salin
  9. HelixVenture, Mérignac, France

    • Isabelle Lesur
  10. Instituto Nacional de Investigación Agropecuaria Estación Experimental Tropical Pichilingue, Quevedo, Provincia de Los Ríos, Ecuador

    • Rey Loor Solorzano
  11. INRA, GeT-PlaGe, Genotoul, Castanet-Tolosan, France

    • Olivier Bouchez
  12. New South Associates Inc., Stone Mountain, GA, USA

    • Patrick Severts
  13. Ministerio de Cultura y Patrimonio, Ecuador/IRD, Quito, Ecuador

    • Julio Hurtado
    •  & Alexandra Yepez
  14. Department of Nutrition, University of California, Davis, CA, USA

    • Louis Grivetti
  15. Institut de Recherche pour le Développement, UMR 208 PALOC, MNHN-IRD, Marseille, France

    • Francisco Valdez

Authors

  1. Search for Sonia Zarrillo in:

  2. Search for Nilesh Gaikwad in:

  3. Search for Claire Lanaud in:

  4. Search for Terry Powis in:

  5. Search for Christopher Viot in:

  6. Search for Isabelle Lesur in:

  7. Search for Olivier Fouet in:

  8. Search for Xavier Argout in:

  9. Search for Erwan Guichoux in:

  10. Search for Franck Salin in:

  11. Search for Rey Loor Solorzano in:

  12. Search for Olivier Bouchez in:

  13. Search for Hélène Vignes in:

  14. Search for Patrick Severts in:

  15. Search for Julio Hurtado in:

  16. Search for Alexandra Yepez in:

  17. Search for Louis Grivetti in:

  18. Search for Michael Blake in:

  19. Search for Francisco Valdez in:

Contributions

F.V., M.B., S.Z., T.P., N.G. and C.L. designed the research. F.V., J.H., A.Y. and S.Z. performed excavations at Santa Ana-La Florida. S.Z. designed starch investigation methods and performed starch granule analysis. T.P. and P.S. sampled artefacts for theobromine analysis. N.G. designed mass spectrometry analysis, performed UPLC-MS/MS analyses and processed and analysed mass spectrometry data. L.G. provided general input to the overall project. C.L., C.V., I.L., O.F., X.A., E.G., F.S. and R.L.S., performed aDNA experiments and analyses. H.V. and O.B. performed NGS aDNA sequencing. S.Z., C.L., N.G., T.P., M.B. and F.V. led the writing of the paper with inputs from all other authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Michael Blake.

Supplementary information

  1. Supplementary Information

    Supplementary Notes; Supplementary Figures 1–8; Supplementary Tables 3–9 and 11–16

  2. Reporting Summary.

  3. Supplementary Table 1

    Radiocarbon dates from the Santa Ana-La Florida site, Palanda (Zamora-Chinchipe, Ecuador)

  4. Supplementary Table 2

    Total SALF samples analysed for starch grains, theobromine and aDNA

  5. Supplementary Table 10

    List of aDNA sequences specific to Theobroma and containing SNPs identified by GBS

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41559-018-0697-x