An Ancient Residue Metabolomics-Based Method to Distinguish Use of Closely Related Plant Species in Ancient Pipes
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ORIGINAL RESEARCH published: 26 June 2020 doi: 10.3389/fmolb.2020.00133 An Ancient Residue Metabolomics-Based Method to Distinguish Use of Closely Related Plant Species in Ancient Pipes Korey J. Brownstein 1†, Shannon Tushingham 2*, William J. Damitio 2, Tung Nguyen 3 and David R. Gang 1* 1 Institute of Biological Chemistry, Washington State University, Pullman, WA, United States, 2 Department of Anthropology, Washington State University, Pullman, WA, United States, 3 Department of Civil and Environmental Engineering, Washington State University, Pullman, WA, United States Edited by: Robert David Hall, Wageningen University and Residues from ancient artifacts can help identify which plant species were used Research, Netherlands for their psychoactive properties, providing important information regarding the Reviewed by: deep-time co-evolutionary relationship between plants and humans. However, relying Takayuki Tohge, Nara Institute of Science and on the presence or absence of one or several biomarkers has limited the ability Technology (NAIST), Japan to confidently connect residues to particular plants. We describe a comprehensive Lena Fragner, University of Vienna, Austria metabolomics-based approach that can distinguish closely related species and provide *Correspondence: greater confidence in species use determinations. An ∼1430-year-old pipe from central Shannon Tushingham Washington State not only contained nicotine, but also had strong evidence for the [email protected] smoking of Nicotiana quadrivalvis and Rhus glabra, as opposed to several other species David R. Gang [email protected] in this pre-contact pipe. Analysis of a post-contact pipe suggested use of different plants, including the introduced trade tobacco, Nicotiana rustica. Ancient residue metabolomics †Present address: Korey J. Brownstein, provides a new frontier in archaeo-chemistry, with greater precision to investigate the Department of Molecular Genetics evolution of drug use and similar plant-human co-evolutionary dynamics. and Cell Biology, The University of Chicago, Chicago, IL, United States Keywords: ancient residue metabolomics, archaeology, caffeine, nicotine, pre-contact pipe, psychoactive compound, smooth sumac, tobacco Specialty section: This article was submitted to Metabolomics, INTRODUCTION a section of the journal Frontiers in Molecular Biosciences There is an increasing recognition of the deep-time co-evolutionary relationship between humans Received: 20 February 2020 and certain psychoactive and medicinal plants, yet most of what we know about their past uses Accepted: 04 June 2020 is rooted in analogy with practices observed in the present or recent past. Despite the fact that Published: 26 June 2020 tobacco use is a concern worldwide, with significant consequences to health and society, very little is Citation: known about its early adoption and use. Using what is called the biomarker approach, we addressed Brownstein KJ, Tushingham S, this question in the Pacific Northwest of North America, where we found nicotine-positive smoke Damitio WJ, Nguyen T and Gang DR implements predating the contact period (Tushingham et al., 2018). Although we could conclude (2020) An Ancient Residue that deep-time continuity of indigenous smoking existed in a place where tobacco has been Metabolomics-Based Method to Distinguish Use of Closely Related depicted as being introduced by early Euro-American traders and explorers and that “the spread Plant Species in Ancient Pipes. of domesticated trade tobacco seems to have overtaken and obscured ancient indigenous tobacco Front. Mol. Biosci. 7:133. practices” (Tushingham et al., 2018), the approach we previously used, based on biomarkers, could doi: 10.3389/fmolb.2020.00133 not distinguish between trade tobacco and local native Nicotiana species. Frontiers in Molecular Biosciences | www.frontiersin.org 1 June 2020 | Volume 7 | Article 133 pipe, which datesby to the end 1334–1524 of calibratedand the their radiocarbon eighteenth century trade years C.E.)(dating goods, and to which a occurs the “pre-contact” was in time applied the following to Northwest the twoof region archaeological arrival specific pipes, of plants amethod Euro-Americans, from “post-contact” for closely pipe ancient related residue species. analysis This thatcertain approach can plant species differentiate with specific use artifactabout types. sites, artifactbroad function, time scales, and giving researchers ritual the tools activity to test assumptions that associate e ( biomarkers, such as nicotine,specific anabasine, application. arbutin, Beyond andindividual cotinine smoke metabolites plant is use connected and to associated specific plant use or Frontiers in Molecular Biosciences | www.frontiersin.org four by Euro-American tradersplants and people explorers smoked prior after tocontexts the the throughout introduction 1700s. the of region, trade The as we tobacco know 4,000 very to littles 5,000 about what years havefrom been excavated inT archaeological smoke plants used(RGL), by indigenousNicotiana peoples rustica (NRU), ( Nicotiana tabacum (NTA),(CSE), Nicotiana Rhus attenuata (NAT), glabra Nicotiana quadrivalvis (NQU), of North America, c ( North America smoked as many as 100are different still largely plant inaccessible species with thatcultivation, approach and ritual as and well. ceremonial use. Medicinalabout applications human management,t domestication, trade, conveyance, e southeastern Unitedto States cacao ( andof yaupon theobromine, holly theophylline, and trade caffeine in being the connected southwestern and a Brownstein et al. and conveyance dynamics of anthropologically significant questions about the past use,ancient spread, residue metabolomics provideslevel expanded has ability a to wide ask range ofpotential potential to distinguish applications. species. ForA example, metabolomics-based approach,multiple as described plant herein, speciesof has have the one been orapproach, used by a focusing in few oncommon a positive specific to particular detection metabolites, all andlargely pipe. cannot relies identification of on reliably these the detectionusing tell species. the of traditional nicotine, In if biomarker which approach is addition, because a this the metabolite approach biomarker b o distinguish between related species, leaving questions Eerkens et al., 2012; Rafferty et al., 2012; Tushingham Moerman, 1998 pecies in the United States. Furthermore, while pipes as old ontact use of these different species. In the Pacific Northwest t al., 2015 t al., 2013, 2018; Carmody et al., 2018 lso followed the biomarker approach, where the presence of een addressed with biomarker analysis, such as identification hese species are listed in We outline here a comprehensive, metabolomics-based At the time of Euro-American contact, indigenous peoples in Other early forays into ancient psychoactive plant use The ability to identify ancient plant use to the species- Nicotiana Kartesz, 2015 ). However, the biomarker approach has failed and Taxus brevifolia (TBR) species cannot be distinguished from each other ), but much remains to be known about the pre- ) show the current distribution of these plant Arctostaphylos uva-ursi (AUV), Cornus sericea Washburn et al., 2014; Crown ), decoction use has Table 1 specific plant species Moerman, 1998 . Figures S1–S8 were the most common ). by humans over (maps adapted 2 TABLE 1 | Compounds shared between the ancient pipes (i.e., PIPE_116 and PIPE_108) and plants smoked in this study. Common name Species Abbreviation Family Commonly Tissue Compounds exclusive Compounds common to the Compounds common to the associated smoked to each experimental pre-contact pipe (PIPE_116) post-contact pipe (PIPE_108) biomarkers pipe and each experimental pipe and each experimental pipe Bearberry/kinnikinnick Arctostaphylos AUV Ericaceae Arbutin leaves 108 5 1 uva-ursi (L.) Spreng. Red osier dogwood Cornus sericea L. CSE Cornaceae * Bark 65 6 1 Coyote tobacco Nicotiana attenuata NAT Solanaceae Nicotine leaves 71 0 0 Torr. ex S. Watson Indian tobacco Nicotiana NQU Solanaceae Nicotine leaves 152 8 0 quadrivalvis J une 2020 | Volume 7 | Article 133 Pursh. A Aztec tobacco Nicotiana rustica L. NRU Solanaceae Nicotine leaves 46 3 5 ncient Residue Metabolomics Cultivated tobacco Nicotiana tabacum NTA Solanaceae Nicotine leaves 39 2 1 L. Smooth sumac Rhus glabra L. RGL Anacardiaceae * Autumn 452 33 2 leaves Pacific yew Taxus brevifolia Nutt. TBR Taxaceae Paclitaxel Needles 545 5 0 Commonly associated biomarkers for archaeometric studies were found in KNApSAcK (Afendi et al., 2011). The asterisks (*) indicate that the biomarkers for Cornus sericea (CSE) and Rhus glabra (RGL) could not be determined in KNApSAcK. Brownstein et al. Ancient Residue Metabolomics before present (cal BP) (Pouley, 2001; Damitio, 2018). For the temperatures, and clay composition. The dimensions of the pre-contact pipe, we identified with confidence which among experimental pipes were as follows—bowl diameter: 1.5 cm, several closely related Nicotiana species, as well as additional bowl depth: 2.0 cm, stem length: 9.5 cm, and total length: non-solanaceous species, were smoked in the pipe. Analysis of 11.0 cm. The pipes were inserted into a vice and connected the post-contact pipe identified the use of different plant species. to catheter tip disposable syringes by adjustable rubber tubing. The developed methods (i.e., experimental smoking of pipes, Plant tissues—either leaves, needles, or bark shavings—were lit sequential extraction method, and chemical