High-resolution palaeodietary reconstruction: Amino acid δ13C analysis of keratin from single hairs of mummified human individuals
Introduction
Stable isotope analysis of bone collagen preserved in archaeological remains has long been a major source of bioarchaeological information, providing direct palaeodietary and palaeoenvironmental information (Ambrose and DeNiro, 1986, Drucker et al., 2012). However, while the persistence of bone collagen in the archaeological record for tens of thousands of years in temperate environments is an advantage, a consequence of its turnover during the lifetime of an individual is an indistinct dietary signal. When a collagen sample is measured it represents the ultimate years or decades of dietary intake (Hedges et al., 2007). Information pertaining to more relevant human timescales, such as how much an individual's diet varied over the course of their life or even how it may have changed through the course of a year, is unresolved. So, whilst analysis of bone collagen isotope compositions is an invaluable part of bioarchaeology, alternative stable isotope approaches are constantly sought to add detail to dietary reconstructions of ancient individuals (Beaumont et al., 2013).
Mummified bodies are a powerful archaeological resource (Lynnerup, 2007) that enables detailed bioarchaeological investigations especially when stable isotope studies are included (Knudson and Stojanowski, 2008). Although a variety of tissues (skeletal and non-skeletal) can be examined in a mummified body, hair has been proposed as the ideal biomaterial for isotopic studies because hair is mainly constituted by protein (i.e. keratin) (Harkey, 1993) and it retains the unaltered isotope signature recorded incrementally as the tissue grows (Petzke et al., 2010); furthermore, sampling is minimally invasive. All these characteristics potentially allow the high-resolution reconstruction of the recent life history of ancient individuals, with respect to diet (and especially seasonal changes in diet), mobility and physio-pathological conditions.
Palaeodietary reconstructions conducted to date on South American mummy hair have mainly relied upon bulk stable isotope analysis, e.g. (Horn et al., 2009, Knudson et al., 2007, Webb et al., 2013, White et al., 2009, Williams and Katzenberg, 2012), requiring the use of 1 (Webb et al., 2013, Williams and Katzenberg, 2012) or 2 cm (Knudson et al., 2007, White et al., 2009) segments of multiple hairs. Detecting rapid dietary changes in the hair isotope signature is complex as the hair requires time to isotopically equilibrate to a new diet (O'Connell and Hedges, 1999), in part due to the buffering effect of the bodily endogenous amino acid pool (Jackson, 2007), meaning that the complete isotopic equilibrium between diet and scalp hair keratin after a dietary change may take up to ∼ 4–12 months for carbon and nitrogen isotope compositions (McCullagh et al., 2005, O'Connell and Hedges, 1999), although the latter appears to have a faster response (Huelsemann et al., 2009, O'Connell and Hedges, 1999). This is further complicated by the complexity of the hair growth cycle (Stenn and Paus, 2001) and by the inter-individual variability of growth rates (Loussouarn et al., 2005), which means that any two hairs may be temporally out of phase. The consequent misalignment of multiple hairs used in a bundle for isotopic analysis will generate an ‘averaged’ isotope signal that may be isotopically attenuated with respect to the original dietary signal (Remien et al., 2014).
The present study describes an improved method that refines human palaeodietary reconstructions by using compound-specific carbon stable isotope analysis of hair keratin amino acids from 0.5 cm segments of a single hair. This approach presents two major advantages over the traditional bulk method: (1) the analysis of a single hair avoids the potential problem of non-contemporaneous hairs being analyzed in bulk samples (i.e. growth cycle error) (Williams et al., 2011); (2) the use of minimal sample amount enhances the temporal resolution at an approximate fortnightly scale [considering a growth rate of approx. 1 cm/month (Krause and Foitzik, 2006)], which is favorable in detecting the onset of a new diet or short-term nutritional changes with respect to seasonality, mobility, environmental stress or disease (although diet-tissue equilibration time remains a potential source of error). Moreover, the application of a compound-specific approach can help to discriminate different sources of the isotopic signal via comparison of δ13C values of essential amino acids (EAAs) with non-essential AAs (NEAAs). Although these classifications are a matter of some debate (Wu, 2013), broadly speaking in healthy individuals, EAAs are amino acids whose carbon skeletons are not synthesizable by the human body and are routed from ingested proteins to body tissues, while NEAAs are produced through various metabolic pathways (i.e. de novo synthesis), in addition to direct routing. NEAAs in hair will therefore potentially contain carbon derived from the whole diet (proteins, lipids, carbohydrates), whereas EAAs will reflect only protein intake. Despite the potential of individual amino acid (AA) δ13C analysis to provide detailed information about distinct dietary components useful in reconstructing metabolic characteristics and nutritional stress among ancient populations, there are few published studies that contain reference data on compound-specific carbon isotope analysis of archaeological human tissues (Choy et al., 2010, Corr et al., 2005, Corr et al., 2009, Fogel and Tuross, 2003, Honch et al., 2012, McCullagh et al., 2006, Richards et al., 2007), especially for hair keratin, e.g. (Raghavan et al., 2010).
The technique was applied to hair samples from seven mummies from archaeological sites located along the coasts and the nearby valleys of the Atacama Desert, northern Chile (Table 1 and Fig. 1) and supplemented with bulk carbon, nitrogen and sulfur isotope analysis performed on multiple hairs. The individuals were selected in order to cover a wide chronological spectrum, from the Late Archaic Period (∼6000–4000 B.P.) to the Late Period (∼500-420 B.P.), and different geographic areas of the coasts (Arica, Camarones) and coastal valley (Azapa) of northern Chile. The study of intra-individual variation provides detailed insights into the subsistence strategies employed by pre-Columbian individuals from the perspective of the individual's life history, thus generating for the first time information on individual flexibility or persistence in dietary intake at a fortnightly scale from the analysis of a single hair.
Section snippets
Environmental context
Although the Atacama Desert has been characterized by unique dryness for millennia (Houston, 2006), the early human communities of the coastal areas had access to an exceptionally rich marine ecosystem. The maritime resources were abundant and reliable (except during El Niño-Southern Oscillation events), and complemented by estuarine resources retrieved at the mouths of rivers (Grosjean et al., 2007) (wetlands). Perennial and ephemeral rivers, recharged by melted snow from the Andes, ran
Archaeological background
The early inhabitants of the Pacific Ocean's rocky shore were specialized fishers, as well as skilled in hunting marine game and gathering mollusks (Olguín et al., 2015, Rebolledo et al., 2016, Santoro et al., 2012). This existence of a marine-focused diet is supported by studies on material culture (toolkits and grave goods) (Standen, 2003), chronic pathology (Arriaza, 1995a, Standen et al., 1984), bone injury (Arriaza, 1995a, Costa-Junqueira et al., 2000, Standen et al., 1984), and dental
Materials and methods
The scalp hairs analyzed in this study were taken from seven mummified individuals (Maderas Enco 1-C2, Morro 1-T28 C8, Camarones 15A-T14, Quiani 7-T13, Azapa 14-T31, Azapa 115-T9, Camarones 9-T39) that are part of the Archaeology Museum collection of the University of Tarapacá (UTA), Anthropology Department, Arica (Chile).
Methods are only outlined in brief here, further details can be found in Supporting Information 1.
Bulk stable isotopes
A summary of the hair keratin stable isotope compositions (δ13C, δ15N, δ34S) for each individual is provided in Table 2.
The C/N atomic ratios for the majority of hair segments fall inside the empirical range of values for modern hair (2.9–3.8) (O'Connell and Hedges, 1999). A few samples present slightly elevated C/N ratios (3.9), though such results are commonly accepted for archaeological hair (Knudson et al., 2015, Webb et al., 2013). Three hair segments (from 5 to 9 cm) of the AZ115-T9
Discussion
The following discussion details brief isotopic histories of the individuals according to their archaeological period and culture.
Conclusion
This study shows that stable carbon isotope analysis of keratin amino acids extracted from 0.5 cm segments of a single human hair highly enhances the resolution of palaeodietary reconstruction, permitting the discrimination of specific diet components at a resolution invisible to traditional methods. Moreover, the LC/IRMS technique requires a single strand of hair, reducing the damage to the archaeological remains.
This work highlights the importance of the relationship between δ13C Phe vs. δ13C
Acknowledgments
This research was supported by La Trobe University PhD scholarships, FHuSS-IRGS grant (#2014-HDR-2-7), FONDECYT grant 1130261, and ARC Future Fellowship FT0992258. Thanks are due to Andrew Gledhill for his technical support at the University of Bradford. The authors would also like to thank Prof. Fred Longstaffe, an anonymous reviewer, the editor, and the associate editor for their detailed comments and helpful suggestions.
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