The Biomolecular Archaeology of the Black Drink: Alkaloid Residue Analysis of Ilex vomitoria on Experimental Vessels and Applications for Prehistoric Specimens

Paper presented at the 2011 Southeastern Archaeological Conference, Jacksonville, Florida

Shannon Tushingham1*, Matthew Palumbo2, Charles H. McNutt3, and Ulla Anderson4

1Department of Anthropology, Washington State University’ 2Department of Biology, University of Florida; 3Department of Anthropology, University of Memphis; 4Department of Chemistry, UC Berkeley; *Corresponding Author: [email protected]

ABSTRACT

Early historic accounts document widespread use of a purgative known as “the black drink”, a caffeinated ceremonial tea famous for its use in purification rituals by elite males. The beverage was prepared from roasted leaves and twigs of yaupon holly (Ilex vomitoria), an evergreen shrub native to the southeastern coastal plain. Prehistoric use is poorly understood but often associated with shell cups found in mortuary contexts and the SECC. Our work, designed to track ancient use of psychoactive plants via Gas Chromatography‐Mass Spectrometry, includes identification of medicinal tea biomarkers, experimentation with brewing black drink, and residue extraction from pottery/shell specimens. Sampling included preparing and extracting a range of experimental vessels which served different functions in black drink cooking and consumption (cooking vessels, clay and shell cups), with extractions taken directly from the vessels and from tea residue on vessel interiors. Our findings suggest that chemical changes occur through the cooking process, but the presence of the biomarker caffeine remains stable and correlated the experimental vessels to the Ilex vomitoria plant in all but one case. We also describe the non-destructive method we developed where alkaloid residues can be extracted directly from the matrix of prehistoric shell cups and pottery vessels through a sonication process.

INTRODUCTION

Early historic accounts document widespread use of a purgative known as “the black drink”, a caffeinated ceremonial tea used as an everyday beverage by many southeastern Indians, but perhaps most famous for its use in dramatic purification rituals by elite males. The beverage was prepared from roasted leaves and twigs of Ilex vomitoria, a plant popularly known as the yaupon holly. The black drink seems to have been part of a religious or ceremonial system, reaching back perhaps thousands of years. As the black drink was consumed in Figure 1. 16th-century engraving by Jacques le Moyne of special marine shell cups that were often buried by high Timucan Black Drink Ceremony, Florida. Note seated status historic southeastern Indians, it is thought that individuals vomiting, women in right foreground prehistoric shell cups found in similar contexts, brewing black drink, and European observers in left foreground. Copy from Theodor de Bry (Frankfort: Typis particularly those engraved with Southeastern J. Wecheli, 1591). Smithsonian Institution National Ceremonial Complex iconographic symbols, may have Anthropological Archives negative no. 57,569. Tushingham et al. 2011-2 served a similar function. This is, however, speculation, as we simply have no direct evidence of the medicinal teas that may or may not have been used in these vessels.

In this paper we describe our research that is designed to track ancient use of the black drink and other ritual beverages via Gas Chromatography‐Mass Spectrometry. This work involves the identification of medicinal tea biomarkers, experimentation with brewing black drink, and residue extraction from pottery and shell specimens. We are specifically interested in understanding the chemistry of these plants, looking at how this chemistry might change through the cooking process, and how we might expect residues with identifiable alkaloid residues to preserve archaeologically. Our greater intent is to establish non-destructive extraction methods that can be used on prehistoric specimens.

HISTORIC NATIVE AMERICAN USE

There are ample historic accounts of Native American use of the black drink dating to as early as the 16th century (see Hudson 1979, Merrill 1979 and Fairbanks 1979 for summaries of this material). Perhaps the most famous image of a black drink ceremonial is shown in an engraving from the 1560s by a member of a Florida expedition where explorers recorded observations of local native people (Figure 1). In this illustration we see a chief and his advisors drinking the black drink in shell cups during a meeting. Note the men profusely regurgitating the beverage at their feet, the women preparing the drink in large pots in the lower right, and the European explorers observing the spectacle in the lower left.

From the ethnography, it is clear that the black drink was connected to purity, which found its most dramatic expression in those contexts where it was regurgitated. The beverage was consistently restricted to certain individuals, in particular men of a certain age or status. Though used to induce vomiting in some occasions, the black drink was used in other ritual contexts—ballgames, feasting, council meetings, burial ceremonies—and it was also often used simply as an everyday social beverage. In fact, Figure 2. Distribution of black drink use among ethno-historic Native there is no evidence that there is anything Americans (Herb Roe 2008; From Merrill 1979:44) about

Ilex vomitoria that actually causes vomiting. For example, it was consumed by many early colonials as a tea popularly known as cassina. It is possible that another emetic plant was added to the tea, or perhaps regurgitation occurred simply because Indians drank copious amounts of the hot fluid during these ceremonies—by some accounts over 6 gallons of the brew!

Tushingham et al. 2011-3

From the ethnographic record it seems that the black drink was used in most places where Ilex vomitoria was available (Figures 2, 3). There is evidence it was traded to interior groups, perhaps along with marine shell cups, and some groups tended patches of the plant in locations where it would not normally grow.

PREHISTORIC USE Figure 3. Natural Range of Ilex vomitoria Aiton (Merrill 1979:42) Prehistoric use of Ilex vomitoria and the emergence of the black drink ritual is poorly understood. It is often associated with marine shell cups, especially those found in mortuary contexts. As shell cups or dippers were used by historic Indians as black drink containers or drinking vessels, it is thought that they may have had a similar use in the past. Also, as shell cups were often buried with individuals of a certain stature (Figure 4), their presence in prehistoric mortuary contexts is similarly linked to high status ceremonialism.

Some of the best evidence for some sort of drinking ritual, possibly involving the black drink, dates to the Mississippian period. It is most famously associated with shell cups engraved with Southeastern Ceremonial Complex iconographic symbols (Figure 5). Occasionally these cups and associated pottery are found with a black residue on their interior walls.

In a 1979 survey of possible archaeological evidence for the black drink Jerald Milanich hypothesized that the origins of the black drink or some sort of drinking ritual date back earlier than the Southeastern Ceremonial Complex—perhaps as early as Hopewell. He based this assertion on the fact that the cups were widely distributed in mortuary and/or ceremonial contexts similar to those observed in the ethnographic record.

PLANT BIOLOGY Yaupon (Ilex vomitoria) is a dioecious shrub or small tree species of the holly family (Aquifoliaceae). This species is native to the coastal plain of the southeastern United States where it is distributed along the Atlantic coast from southern Virginia to central Florida and on the Gulf Coast from central Florida to east Texas. Determining the precise boundaries of this range is confounded by the probable Figure 4. Burial of Timucan warriors in a mound with weapons and shell cups as grave goods. From De Bry, Florida, pl. XIX. Smithsonian Institution, National Anthropological Archives, dispersal of yaupon by Native American trade to negative no. 57-559. Tushingham et al. 2011-4 areas beyond its natural distribution (Catesby 1974, Brickell 2000, MacDougal 2003). Such evidence for human dispersal of yaupon makes it difficult to determine whether yaupon’s distribution beyond the coastal plain is of natural or human origin. Within its native range, yaupon grows in coastal scrub, pineland, and hardwood forest habitats (Johnson and Barbour 1990). Yaupon foliage is consumed by numerous taxa of insect herbivores including a specialist holly leaf-miner (Phytomyza vomitoriae) and both leaves and twigs are browsed by white-tailed deer (Odocoileus virginianus). In spring (March- April), yaupon produces small white flowers that are insect pollinated (mainly hymenopteran species) and its fruit is a bird-dispersed drupe that turns red at maturity.

Ilex vomitoria is a psychostimulant plant that contains caffeine. Although another holly species, Ilex cassine, produces small quantities of caffeine, yaupon is the only known North American plant species that produces significant quantities of caffeine and is the source of cassina, the stimulating Amerindian beverage (Edwards and Bennett 2005). Two South American congeners produce caffeine, Ilex paraguariensis and I. guayusa. It is thought vomitoria has its origins from the south.

Mean foliar, dry-weight caffeine concentrations of wild-type yaupon detected in three studies are ~0.4 – 0.8% with a range of 0 – 1.9% (Edwards and Bennett 2005, Palumbo et al. 2007, Palumbo et al. 2009). (*note: yaupon varieties have been selected for cultivation as an ornamental hedge or small tree species) (*also note: water content of fresh yaupon leaves are typically ~50%) Such caffeine concentrations are similar to those reported for yerba maté prepared from leaves of I. paraguariensis (Mazzafera 1994, Reginatto et al. 1999, Coelho et al. 2001). Difficulty exists, however, in estimating caffeine concentrations consumed by Amerindians in cassina and other beverages prepared from leaves of yaupon. Such concentrations would depend on the amount of caffeine lost during processing and diluted during preparation of leaf material. For example, although leaves from Asian tea, C. sinensis, contain ~3.5% caffeine and Arabica coffee ‘beans’ contain only ~1.1% caffeine by weight, an 8 oz. (177 cm3) cup of tea contains two to seven times more caffeine due to the higher mass of product used in the preparation of tea (Weinberg and Bealer 2001). Historical and ethnographic accounts of Amerindians harvesting the young leaves of yaupon seem to be explained by chemical analyses demonstrating that the youngest, fully-expanded leaves contain the highest concentrations of caffeine (MJ Palumbo - unpublished data). Also, caffeine production in yaupon

Figure 5. Engraved shell cup from Spiro Mound, Oklahoma: Used to Brew the increases with availability of soil Black Drink? Tushingham et al. 2011-5 nitrogen and likely increase with light availability (Palumbo et al. 2007, Palumbo et al. 2009).

MATERIALS AND METHODS

GC-MS ANALYSIS is a technique that can identify certain alkaloids within a sample--if you know the chemical signature of a plant and find similar biomarkers in an artifact, you may reasonably suggest that the artifact was at one time associated with a particular plant. Recent archaeological applications of GC-MS include identification of tobacco and other smoke plants in Pacific Northwest Coast pipes

(Tushingham et al n.d.) Figure 6. Materials Used in the cooking experiments and identification of cacao imported from Mesoamerica in ceremonial drinking vessels from the American southwest (Crown and Hurst 2009, Washburn and Washburn 2011).

Our studies involve the identification of medicinal tea biomarkers, experimentation with brewing black drink, and residue extraction from pottery and shell specimens (Figure 6). We were interested in chemically characterizing the plants themselves, and looking at how this chemistry may or may not change through the cooking process. The greater goal was to establish non-destructive extraction methods that could be used on prehistoric specimens.

The study was modeled on a smoke plant study involving experimental smoking of a suite of smoke plants, including tobacco, GC MS characterization and identification of tobacco smoking in archaeological pipes and pipe fragments. In our approach we always use experimentation and look at more than one type of plant.

Leaves of Ilex vomitoria collected for this study were at the GTM Research Reserve in St. Johns County, Florida in late April 2011. The youngest, fully-expanded leaves of yaupon individuals growing in coastal scrub habitats at GTM were harvested with pruning clippers and placed in plastic bags. Leaves were then air-dried at ambient temperatures for three days before being shipped for chemical analysis. Tushingham et al. 2011-6

Plants and Experimental Vessels: Two psychoactive plants used by southeastern Native Americans as purgative ceremonial teas were used in the study, including Yaupon Holly (Ilex vomitoria), used to brew “the black drink” and button snake root (Eryngium yuccifolium), used by some groups to brew a black drink alternative. Tea leaves were then brewed as a tea in specially made experimental low fired clay cooking pots at the UC Davis archaeological laboratory. In brewing the tea we followed the process used by Native Americans in early historic accounts, which includes a period of roasting or parching the

leaves, before brewing. Tea was brewed in each pot 5x for 60-90 minutes. After about 30 minutes the tea begins forming a distinctive white froth (Figure 7). After brewing, the tea was poured into pottery and shell cups. Tea was let to dry in these cups. A distinctive black residue was left on the interior surface of all experimental vessels (Figure 8).

Extraction methods: Samples were prepared of the plants, the pottery cooking vessels, and the clay and shell cups. In this study a total of 10 samples were prepared at UC Davis prior to analysis at the GC MS laboratory at the Department of Chemistry, UC Berkeley (Table 1). Additional samples were prepared of residue scrapings from the cooking vessels and cups.

Two samples were taken directly from live plant specimens of Ilex vomitoria and Eryngium yuccifolium. Plant material was dried and crushed to a fine powder prior to sampling.

Figure 7. Experimentally Brewed “Black Drink” (Ilex vomitoria tea) Two residue samples from pottery sherds were prepared by removing residue with a Dremel drill. After removing residue from pottery sherds an additional 3 samples were prepared of crushed interior portions of sherds. One sample was prepared from residue dried inside a shell cup by simply flaking residue off with a small dentist’s tool. A final sample was prepared by placing extraction solvent directly in the shell cup and sonicating the solution for five minutes.

For all other samples 2 mg of material was added to 2 ml of extraction solvent and then sonicated for 5 minutes. Extracts were filtered using a .045 micron syringe filter and then dried with an evaporator.

Tushingham et al. 2011-7

Figure 8. Residue in experimental vessel (left), pottery cups (top right) and shell cup (bottom right)

Table 1. Study samples, With Caffeine Positive (+) and Negative (-) Results

Samples, ID# Starting Material Caffeine Ilex vomitoria samples PL-IV Crushed leaf, Ilex vomitoria + Residue scraping, interior of experimental clay cooking pot, in which I. vomitoria tea EV-CL-IV-B1-ResScrape brewed EV-CL-IV-B1-Sherd Crushed sherd, experimental clay cooking pot, in which I. vomitoria tea brewed + EV-CL-IV-C1-Body Crushed sherd, clay cup which held I. vomitoria tea + Crushed sherd, experimental (shell tempered) clay cooking pot, in which I. vomitoria tea EV-CL-IV-BPB-Boil brewed + EV-SH-1-ResScrape Residue scraping, shell (Busycon sp.) cup interior, in which I. vomitoria tea let to dry + EV-SH1-Shell Int Sonicated solution, shell (Busycon sp.) cup interior, in which I. vomitoria tea let to dry + Eryngium yuccifolium samples PL-EY Crushed root, Eryngium yuccifolium - EV-CL-EY-B3-ResScrape Residue scraping, experimental clay cooking pot, in which E. yuccifolium tea brewed - EV-CL-EY-B3-Sherd Crushed sherd, experimental clay cooking pot, in which E. yuccifolium tea brewed -

Notes: All residue scrapings taken from interior of experimental cooking pots and cups Tushingham et al. 2011-8

GC-MS RESULTS

Results confirm the expected pattern of caffeine presence in plant samples and experimental artifacts (Table 1). As expected, the analyzed Ilex vomitoria plant sample was found to contain the compound caffeine (Figure 9). Caffeine was also detected five of the experimentally prepared Ilex vomitoria samples (e.g., Figure 10). However, caffeine was not detected in the residue Figure 9. Section of gas chromatograph for Ilex vomitoria plant sample, with insets of scraping sample taken from the mass spectra at 9.94 minutes (in grey). ceramic sherd.

CONCLUSION

Through experimentation and chemical characterization of Ilex vomitoria plants, we have established a non-destructive method where chemical biomarkers, specifically caffeine, can potentially be extracted Figure 10. Section of gas chromatograph for caffeine positive Ilex vomitoria sonicated from prehistoric shell cups and shell cup sample, with inset of mass spectra at 9.94 minutes (in grey). pottery vessels. It is clear that the chemistry changes through the cooking process, but the presence of caffeine correlates the experimental vessels to the Ilex vomitoria plant.

Distinctive alkaloid biomarkers were found in GC-MS samples extracted directly from the matrix of experimental cooking pot sherds and clay cups. The results indicate, however, that extracting residues scrapings from pot and clay cup interiors might not work. We are not sure why, but residue scrapings from shell cup interiors did contain caffeine.

We also established a non-invasive process for extracting samples directly from shell cup interiors through a sonication process. This is important as we anticipate that many museums will be reluctant allow analyses on these cups, particularly engraved shell cups. There are many interesting potential applications of this process for prehistoric specimens. We are ready for the next phase of work, just need grant $ and some artifacts! Tushingham et al. 2011-9

REFERENCES CITED

Brickell, J. 2000 The natural history of North Carolina, with an account of the trade, manners, and customs of the Christian and Indian inhabitants. The Colonial Records Project Website, North Carolina Office of Archives and History, Raleigh, NC.

Catesby, M. 1974 The natural history of Carolina, Florida, and the Bahama islands. The Beehive Press, Savannah, GA.

Coelho, C.G. 2001 Methylxanthines of Ilex paraguariensis A. St.-Hill. Var. Loes. and var. paraguariensis. Revista brasileira de ciencias farmacéuticas. 37: 153-158.

Crown, Patricia L. and Jeffrey Hurst 2009 Evidence of cacao use in the Prehispanic American Southwest. Proceedings of the National Academy of Sciences 106(7):2110-2113.

Edwards, A.L. and B.C. Bennett. 2005 Diversity of methylxanthine content in Ilex cassine L. and Ilex vomitoria Ait: Assessing sources of the North American stimulant cassina. Economic Botany 59: 275-285.

Fairbanks, Charles H. 1979 The Function of Black Drink among the Creeks. In The Black Drink: A Native American Tea, edited by Charles Hudson, pp. 120-149. University of Georgia Press, Athens.

Hudson, Charles (editor) 1979 The Black Drink: A Native American Tea. University of Georgia Press, Athens.

Johnson AF and Barbour MG. 1990 Dune and Maritime Forests. In Ecosystems of Florida, edited by Meyers R.L.and J. J. Ewel, University of Central Florida Press, pp. 429-480.

MacDougall, A. 2003. Did Native Americans influence the northward migration of plants during the Holocene? Journal of Biogeography 30: 633-647.

Mazzafera, P. 1994 Caffeine, theobromine, and theophylline distribution in Ilex paraguariensis. Revista Braseleira de Fisiologia Vegetal 6: 149-151.

Tushingham et al. 2011-10

Merrill, William L. 1979 The Beloved Tree: Ilex vomitoria among the Indians of the Southeast and Adjacent Regions. In The Black Drink: A Native American Tea, edited by Charles Hudson, pp. 40-82. University of Georgia Press, Athens.

Milanich, Jerald 1979 Origins and Prehistoric Distributions of Black Drink and the Ceremonial Shell Drinking Cup. In The Black Drink: A Native American Tea, edited by Charles Hudson, pp. 83-119. University of Georgia Press, Athens.

Palumbo, M.J., F.E. Putz, and S.T. Talcott 2007 Nitrogen fertilizer and gender effects on the secondary metabolism of yaupon holly, a caffeine- containing North American holly. Oecologia 151: 1-9.

Palumbo MJ, Talcott ST, Putz FE. 2009 Ilex vomitoria Ait. (Yaupon): A native North American Source of a caffeinated and antioxidant- rich tea. Economic Botany 63: 130-137.

Reginatto, F.H., M.L. Athayde, G . Gosmann, and E.P. Schenkel 1999 Methylxanthine accumulation in Ilex species – Caffeine and theobromine in erva-mate (Ilex paraguariensis) and other Ilex species. Journal of the Brazilian Chemical Society 10: 443-446.

Weinberg, B.A., and B.K. Bealer 2002 The world of caffeine: The science and culture of the world’s most popular drug. Routledge Press, New York, NY.

Shannon Tushingham, Dominique Ardura, Mine Palazoglu, Jelmer Eerkens, Sevini Shahbaz, and Oliver Fiehn n.d. First Evidence of Tobacco Smoking in Ancient Pacific Northwest North America (manuscript in review with Science).

Washburn, Dorothy, William Washburn and Petia A. Shipkova 2011 The prehistoric drug trade: widespread consumption of cacao in Ancestral Pueblo and Hohokam communities in the American Southwest. Journal of Archaeological Science 38:1634- 1640.

2011 SOUTHEASTERN ARCHAEOLOGICAL CONFERENCE SLIDES ON FOLLOWING PAGES Biomolecular Archaeology of the Black Drink: Alkaloid residue analysis of ancient and experimental vessels

Shannon Tushingham1, Matthew Palumbo2, Charles H. McNutt3 and Ulla Anderson4 1Department of Anthropology, UC Davis 2Department of Biology, University of Florida 3Department of Anthropology, University of Memphis 4Department of Chemistry, UC Berkeley Plant biology Ethnography

Experimentation

Archaeology

Chemical Characterization 16th-century engraving by Jacques le Moyne of Timucan Black Drink Ceremony

Historic Native American Use of Black Drink

Cups in Southeastern Ceremonial Complex (SECC) iconography

Shamans with shell ups From Spiro shell cup engravings Hypothesized prehistoric use: shell cups and cooking vessels Marine shell cups traded inland to ceremonial centers

Often found in elite burials

Thought to have been buried with black drink

Residue on some cups Engraved shell cup from Spiro, OK used for the Black Drink?? Ilex Vomitoria, Yaupon Holly Ilex vomitoria= CAFFEINE!

• genetically related to two South American plants which caffeine, Ilex paraguariensis and I. guayusa.

• Mean foliar, dry-weight caffeine concentrations of wild-type yaupon detected in three studies are ~0.4 – 0.8% with a range of 0 – 1.9% (Edwards and Bennett 2005, Palumbo et al. 2007, Palumbo et al. 2009). South American Yerba maté • Ilex paraguariensis • National drink of Argentina Cooking experiments

Plant specimens: Guana-Tolomato-Matanzas (GTM) Research Reserve: 73,352 acres of coastal dune, marsh, estuarine, and upland habitats in northeastern Florida. Model: smoke plant study

GC-MS Extraction Identification of tobacco Experiment al “Smoking” & Characterization smoking in prehistoric pipes Brewing the tea

Historic brewing Then brew 1st roast…. 1 2

3 4 Yum! Residue

Cups

Cooking vessels Shell Ilex vomitoria plant

Crushed leaf of Ilex vomitoria

++Caffeine positive ++ Crushed sherd experimental clay cook pot I. vomitoria tea

++Caffeine positive ++ Residue scraping interior of experimental clay cook pot I. vomitoria tea

FAIL!

--Caffeine negative-- Crushed sherd Clay cup Ilex vomitoria tea let to dry

++Caffeine positive ++ Crushed sherd Bell Plain cook pot Ilex vomitoria

++Caffeine positive ++ Residue Shell cup ++Caffeine positive ++ I. vomitoria tea

++Caffeine positive ++ Sonicated solution ++Caffeine positive ++ Shell cup I. vomitoria

++Caffeine positive ++ Erygium Yuccifolium Button snake root Plant

Cooking pot sherd

Cooking pot residue scrape Conclusion

? ?