An investigation of diet in early Anglo-Saxon England using carbon and nitrogen stable isotope analysis of human bone collagen

The principal aim of this work is to investigate whether protein sources in human diets in early Anglo-Saxon (5th-7th century AD) England varied with geographic location, or with respect to age or sex. The methodology used was analysis of δ¹⁵N and δ¹³C in human bone collagen from 76 adult skeletons from a total of 18 different cemeteries located in inland, riverine and coastal environments. In order to control for the possible effects of socio-economic status on diet, only high status individuals, as identified by grave goods, were studied. Data were analysed using statistical manipulation of the raw results and using IsoSource (Phillips and Gregg, 2003), a mathematical treatment which models the composition of the protein part of human diets using bone stable isotope ratios when the isotopic values for potential protein sources are known or can reasonably be inferred. The results suggest that protein sources varied little according to geographic location and that terrestrial foods dominated at all locations. However there were some subtle patterns. Slightly elevated δ¹³C in skeletons from coastal locations, and δ¹⁵N in those from riverine sites may indicate that communities in these locations made greater use of marine and freshwater resources respectively. If this is correct then it demonstrates that 5th-7th century communities made some use of wild resources even within a predominantly agrarian economy. In general the analysis of the raw data and the IsoSource results were mutually supportive, and this work demonstrates the value of this twin-pronged approach to analysis of human bone stable isotope data.     

Archaeological collagen: Why worry about collagen diagenesis?

DNA appears to decay by random chain scission resulting in a predictable range of fragment lengths. Collagen decay has also been modelled in this same way, although it has become increasingly evident that collagen decay does not follow this same pattern. Radiocarbon and stable isotope analysis now use ultra-filtration to isolate large fragments (>30% of original polymer length) even in Pleistocene bone. How then does collagen decay? This study contrasts experimentally degraded samples with collagen extracted from forensic, archaeological and fossil bone. In experimentally degraded bone, values for amino acid and elemental (C:N) composition, bulk δ ¹³C, δ ¹⁵N, and aspartic acid racemisation (AAR) changed very little until 99% of the collagen was lost, suggesting that the collagen triple helix and polypeptide chains remained remarkably intact. This suggestion was demonstrated directly by examining the integrity of individual polypeptide chains using cyanogen bromide (CNBr) cleavage followed by SDS-PAGE electrophoresis. In ancient samples, AAR values remain remarkably stable and the pattern of CNBr-cleavage was only replaced with a smear of smaller polypeptides in the oldest (Pleistocene) bones investigated. Smearing may reflect both modification of the methionine resides (the sites of CNBr-cleavage) and/or partial hydrolysis of the collagen molecule. The findings reveal why it is not usually necessary to worry about collagen diagenesis; it is mostly intact. However, evidence of partial deterioration of the oldest bone samples suggests that alternative purification strategies may increase yields in some samples.     

Human subsistence strategy at Liuzhuang site,Henan, China during the proto-Shang culture (∼2000–1600 BC) by stable isotopic analysis

Since the discovery of the proto-Shang culture, created mostly by ancestors of the Shang clan from the late Neolithic Age to the early Shang period (∼2000–1600 BC), the subsistence strategy and lifestyle of humans in China during their movement southwards have been a great focus. Chinese literature and archaeological findings suggest that the proto-Shang societies were composed of different cultural groups and had various subsistence strategies. For example, at the Liuzhuang site, three types of burials, i.e., stone coffin, wooden coffin and earthen shaft-pit, are found. The wooden coffin and earthen shaft-pit burials had been adopted locally in the Central Plains since the Neolithic Age while the stone coffin burials were usually used by people living in Northeast China and had never been found in the Central Plains before. In this study, stable carbon and nitrogen isotopic analyses were performed on human bones from the Liuzhuang site and animal bones from Zhangdeng site in Henan province, China to determine whether different social groups had various accesses to food resources and whether their dietary difference was related to inequality in social status. Humans have mean δ¹³C and δ¹⁵N values of −7.6 ± 0.6‰ and 9.6 ± 1.0‰ (n = 19) respectively, which strongly indicates that humans rely primarily on C₄-based food. The main contribution of C₄-based food in their diet is from millet agriculture or animals that consume millet by-products. The isotopic spacing of carbon and nitrogen isotopic values between pigs and humans, between dogs and humans, and between cattle and humans, all imply that these animals were the main meat resources for humans. Surprisingly, the δ¹³C values and δ¹⁵N values of humans with different types of burials are quite close, indicating that they had equal access to food resources. This result suggests that the proto-Shang humans had adapted to the local subsistence strategy, and the local cultural factors in the Central Plains were very well integrated into proto-Shang culture.     

Stable isotope analysis of dog,fox, and human diets at a Late Holocene Chumash village (CA-SRI-2) on Santa Rosa Island,California

Stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analyses of dog (Canis familiaris), island fox (Urocyon littoralis), and human bone collagen from CA-SRI-2 (AD 130–1830) on Santa Rosa Island, California provide a proxy of diet and the relationships between humans and these animals. Carbon isotopic signatures indicate that Native Americans and their dogs at CA-SRI-2 subsisted almost exclusively on marine resources, while the island fox ate primarily terrestrial foods. Nitrogen isotopes and archaeofaunal remains indicate that humans and dogs also ate higher trophic level foods, including finfishes, marine mammals, and seabirds with smaller amounts of shellfish. The CA-SRI-2 island foxes appear to have eaten higher amounts of terrestrial foods, similar to the diets observed in modern fox populations. These data generally confirm the commensal relationship assumed to exist between domesticated dogs and people, but the carbon isotopic composition of dogs is enriched ∼2‰ compared to humans. We hypothesize that the difference in carbon isotopes between dogs and humans may have resulted from a higher consumption of C3 plants with lower δ¹³C values by humans, or less likely from the ingestion by dogs of significant amounts of bone collagen, which is enriched by ∼4‰ over associated muscle.     

Stable isotope palaeodietary study of humans and fauna from the multi-period (Iron Age,Viking and Late Medieval) site of Newark Bay,Orkney

We report here on stable carbon and nitrogen isotope measurements of human and faunal bone collagen from the Iron Age, Viking Age, and Late Medieval site of Newark Bay, Orkney, Scotland. We found a wide range of results for humans in both δ¹³C (−15.4‰ to −20.3‰) and δ¹⁵N (8.6‰–15.6‰) values. The enriched carbon and nitrogen values indicate the consumption of significant amounts of marine protein, which is very unusual for post-Mesolithic (e.g. 4000 cal BC) UK and European populations. Also of interest is a statistically significant difference in δ¹³C (t = −2.48, p = 0.011) and δ¹⁵N (t = −2.44, p = 0.011) values, and therefore diets, between adult males and females at this site, with males (δ¹³C = −17.8 ± 1.2‰, δ¹⁵N = 13.2 ± 1.6‰) having, on average, a higher proportion of marine protein than females (δ¹³C = −18.9 ± 1.1‰, δ¹⁵N = 11.8 ± 1.8‰). The weaning age of the sub-adults was difficult to interpret due to the large isotopic variation in the adult females, but nearly all individuals between birth and 1.25 years have elevated δ¹⁵N values indicating that they were breastfed to some extent.     

Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen

Sulphur isotope measurements of bone collagen from archaeological sites are beginning to be applied more often, yet there are no clear criteria to assess the quality of the collagen and therefore the validity of the sulphur isotope values. We provide elemental data from different methods (DNA sequences, amino acid sequences and mass spectrometric measurements) which are used to establish a reliable system of quality criteria for sulphur isotope analyses of bone collagen. The difference in the amount of sulphur from fish and mammalian collagen type I led to the suggestion to use different criteria to assess the in vivo character of the collagen between these two categories. For establishing quality ranges, the bone collagen of 140 modern animals were analysed. The amount of sulphur in fish and mammalian bone collagen is 0.63 ± 0.08% and 0.28 ± 0.07%, respectively. Based on these results we define for mammalian bone collagen an atomic C:S ratio of 600 ± 300 and an atomic N:S ratio of 200 ± 100, and for fish bone an atomic C:S ratio of 175 ± 50 and an atomic N:S ratio of 60 ± 20. These quality criteria were then applied to 305 specimens from different archaeological contexts.     

Effects of Sodium Hydroxide Treatment and Ultrafiltration on the Removal of Humic Contaminants from Archaeological Bone

This study compared bone collagen extraction techniques that included treatment with sodium hydroxide and 30 kDa ultrafilters using a set of well‐preserved, humic‐contaminated archaeological marine mammal bones. Treatment with sodium hydroxide was effective at removing humic contaminants from archaeological bone, although yields were significantly decreased. Yields were also significantly decreased by ultrafiltration although this study produced no evidence that 30 kDa ultrafilters were effective at selectively removing humic contaminants from archaeological bone. The combination of sodium hydroxide treatment and ultrafiltration did not produce superior results to the treatment involving only sodium hydroxide. Archaeological samples exhibiting darker colouration indicative of humic contamination should be treated with sodium hydroxide to remove these contaminants. Copyright © 2017 John Wiley & Sons, Ltd.     

Stable isotope analysis of prehistoric populations from the cemeteries of the Middle and Lower Dnieper Basin,Ukraine

This paper presents the results of new stable isotopes (carbon and nitrogen) analysis of human, faunal and fish remains from thirteen cemeteries from the Middle and Lower Dnieper Basin, Ukraine. The results are integrated with earlier analyses, undertaken solely on human samples, in order to provide a comprehensive overview of subsistence across the Upper Palaeolithic through to Eneolithic periods in this region. This is the first time that a combined sample of human, faunal and fish remains has been studied in order to interpret subsistence strategies across these periods in Ukraine. The total dataset comprises 113 samples of human, faunal and fish remains, 59 of which include new analyses of faunal and fish remains that have not previously been made available for study (Table 1). The analysis of the faunal and fish remains allows for a consideration of trophic levels which indicates that the consumption of freshwater fish occurs from the Epipalaeolithic period onwards. Whilst the majority of the cemeteries and the individuals therein cluster in relatively tight groups, there is a significant offset between the human and faunal samples due to the consumption of freshwater resources. The fish samples analysed in the study appear to exhibit a relatively random distribution when compared to the other samples analysed. In general, freshwater resources remain significant through until the end of the Neolithic period and into the Eneolithic period at the sites investigated.     

A Bayesian AMS C chronology of the Classic Maya Center of Uxbenká, Belize

Archaeologists require precise chronologies of cultural change to understand the nature and timing, and causes and effects, of ecological and social transformations. The Uxbenká Archaeological Project (UAP) has made chronology building a main goal, employing epigraphic data, artifact seriation, and high-precision AMS ¹⁴C dating. With more than 60 ¹⁴C dates with measurement errors below ±25 ¹⁴C yr, primarily on single charcoal specimens, Uxbenká may be the most intensively dated Classic Period site in the tropical Maya Lowlands. Here we take the next step, combining this rich chronometric dataset with associated stratigraphic information in OxCal's Bayesian framework to generate a high-resolution chronology of Uxbenká's early development and expansion in southern Belize. We show how archaeologists may take advantage of complex architectural stratigraphy to constrain broad calibrated ranges during the Classic Period, and to estimate dates for observed construction events that can not be directly dated (e.g., clearing/leveling plazas, laying plaster floors, renovating/rebuilding structural elements). Our analysis confirms the antiquity of the ceremonial Stela Plaza (Group A), where first construction is estimated between 60 cal BC and cal AD 220 and is associated with a Pre-Classic structure obscured by later construction and reorientation of the group to a N-S alignment ca. AD 150–310. The analysis also indicates initial leveling and construction at plaza Group B from cal AD 60–310 and Group D from cal AD 20–240, both earlier than previously assumed. Though Late Classic contexts are not as well represented by direct dates, architectural modifications are documented at all three plaza groups after AD 550, including the extensive plastering, laying paving stones, and construction of facades on existing structures. The resulting chronology revises our previous thinking on the mode of settlement and development of Classic Maya society at Uxbenká and indicates specific areas of investigation to elucidate events in the Late and Terminal Classic at the site, including political and social fragmentation and the abandonment of the urban core late in the Classic Period.     

Stable isotope analysis of 14 individuals from the Mesolithic cemetery of Vasilyevka II,Dnieper Rapids region,Ukraine

Bone collagen extracted from 14 humans from the Mesolithic cemetery of Vasilyevka II was analysed for their δ¹³C and δ¹⁵N ratios. This cemetery is one of only two later Mesolithic cemeteries from the Dnieper Rapids region, being dated to 7300–6220 cal BC on the basis of three radiocarbon determinations. This analysis provides insights into the nature of the diet of the Dnieper populations at the very end of the Mesolithic period, prior to the adoption of pottery in the region, and the assumed shift towards the exploitation of domesticates in the economies of the populations of the Dnieper region, and considers these in relation to broader temporal indicators of diet in the region.     

Sorting the butchered from the boiled

Is it possible to identify cooked, rather than burnt, bone? Mild heating (≤100 °C,1 h) – typical of cooking – does not lead to detectable changes in any biochemical parameter of bone yet measured. If it is only possible to detect charred bone, how is it possible to detect cooking in the archaeological record? In a previous paper (Koon et al., 2003, J. Arch. Sci.), we used a Transmission Electron Microscopy (TEM) based approach to investigate changes in the organization of the bone protein, collagen, as it is heated, using bone from heating experiments and short term burials. The work revealed that mineralized collagen, despite requiring aggressive treatment to gelatinise the protein (e.g. 90 °C, 240+ h), readily accumulates minor damage. We believe that the presence of mineral matrix stabilises the collagen enabling the damage to accumulate, but preventing it from causing immediate gelatinisation. Once the mineral is removed, the damage can be observed using appropriate visualization methods.     

Investigation of the ‘canopy effect’ in the isotope ecology of temperate woodlands

Anomalously ¹³C-depleted carbon stable isotope values from closed forest ecosystems have been termed the ‘canopy effect’. Originally this was ascribed to recycling of depleted carbon from forest floor decomposition of organic material, although others have suggested that it is equally likely to be due to variations in leaf-level processes in response to increased shade. This depletion in the heavier carbon isotope is passed on to woodland herbivores feeding within the forest environments. A similar isotopic depletion has also been reported in the archaeological literature from more open temperate woodland settings, but no measurements have been made on the plants at the base of the food chain in order to quantify the effect. In this study we attempt this by examining the carbon and nitrogen stable isotopic values of different species of grasses from a range of open to closed habitat settings within Wytham Wood, Oxfordshire, UK. We find a strong relationship between carbon isotopic depletion of plant tissue and lowered light intensity with an up to 5‰ shift between grass grown in open and closed locations. In order to follow this up the food chain, we also report data on wool from sheep grazing in open pastures near the Wood, and on fallow deer living within the woodland, but which turn out not to show a strong canopy effect, probably related to their feeding strategies. We conclude that there is indeed a strong ‘canopy effect’ in temperate woodland, probably related to differential light levels, but that not all apparently woodland-dwelling mammals show such an effect. We also show considerable isotopic variation at the base of the food chain, which should counsel caution when attempting to interpret dietary isotopes using mixing models.     

Mummies, maize, and manure: multi-tissue stable isotope analysis of late prehistoric human remains from the Ayacucho Valley, Perú

This paper reports the δ¹³C and δ¹⁵N values of bone collagen, muscle and skin from several late prehistoric–early colonial (AD 1490–1640) mummies from Perú's Ayacucho Valley. The mean of the δ¹³C and δ¹⁵N values of bone collagen are −11.5 ± 1.4 and 11.1 ± 0.7‰, respectively. The mean of the δ¹³C values for Vinchos skin is −11.8 ± 1.2‰ and the mean of δ¹⁵N values is 13.2 ± 0.5‰. The samples of muscle tissue have a δ¹³C mean of −11.9 ± 0.9‰ and a δ¹⁵N mean of 12.7 ± 0.3‰. The data from bone collagen indicate maize was the basis of the region's subsistence economy. A significant correlation between δ¹³C and δ¹⁵N values of bone collagen (R² = 0.75) is consistent with the preferential fertilization of maize with composted manure. Both skin and muscle samples are consistently enriched in δ¹⁵N relative to paired samples of bone (2.1 ± 0.5 and 1.6 ± 0.7‰, respectively), possibly as a result of short term physiological stress or differential decomposition.     

A Bayesian C chronology of Early and Middle Bronze Age in Sicily. Towards an independent absolute dating

This paper proposes a Bayesian model for the ¹⁴C chronology of Sicilian Early and Middle Bronze Age, with a specific focus on the northeastern sector of the island. Building on the available ¹⁴C determinations, the model allows addressing a number of chronological questions left open in literature, making a first step towards an independent absolute chronology. The analysis put the start of the earlier part of Early Bronze Age (Capo Graziano 1-Casa Lopez phase) between 2400 and 2175 cal BC, and the end at about 1960 cal BC. The advanced stage of the same period (Capo Graziano 1-Filo Braccio phase) is likely to have started and ended around 1960 and 1730 cal BC respectively. The model indicates that the time slot with the highest posterior probability for the start of the Middle Bronze Age–Milazzese (Portella phase) is between 1490 and 1460 cal BC. This turns up to be earlier than held to date. Notably, the model enables for the first time to bracket the development of the later stage of Early Bronze (Capo Graziano 2-M. di Capo Graziano phase) between 1730 (end of Filo Braccio phase) and 1490/60 cal BC (start of Middle Bronze Age–Portella phase). The latter date is earlier than usually held for the end of Capo Graziano 2. The existence of a narrow gap between the end of the latter and the start of Middle Bronze Age-Portella phase is tentatively proposed only on stratigraphic grounds. Further, the analysis enables for the first time to pinpoint and quantify the lag that is likely to have existed between the start of those Sicilian prehistoric phases and the appearance of Late Helladic imports. The comparison with the Aegean ¹⁴C Bayesian chronology indicates that a time lag (about 45 yr) is likely to have occurred between the start of Capo Graziano 2 and of the Late Helladic period. A time lag between 20 and 70 yr is likely to have existed between the start of the Sicilian Middle Bronze Age and of the Late Helladic III. Arguments tentatively supporting the narrower interval are also discussed.     

Bone collagen preservation in the tropics: a case study from ancient Puerto Rico

Bone collagen is a well-characterized and generally robust protein that plays a key role in both radiometric dating and stable isotopic reconstruction of paleodiet. To be useful for such purposes, however, analyzed collagen must be relatively taphonomically unaltered. To date, little research has been conducted to document the taphonomic fate of bone collagen from archaeological sites in the tropics. In the present work, a large (n = 298) dataset of archaeological bone samples from sites on the island of Puerto Rico is examined by means of radiometric, chemical, and elemental analyses. The ultimate conclusion of this work is that while collagen loss may be accelerated in the tropics versus that seen in samples from higher latitudes, what collagen remains is typically sufficiently well-preserved and taphonomically unaltered as to make radiocarbon dating and/or stable isotopic analysis worthwhile.     

Correlation BetweenC Ages and Aspartic Acid Racemization at the Upper Palaeolithic Site of the Abri Pataud (Dordogne, France)

The Upper Palaeolithic site of the Abri Pataud (Dordogne, France) has been the subject of numerous¹⁴C age determinations which cover the period from 20,400±450 years (OXA-373) to 34,250±675 years (Grn-4507). The racemization ratios of aspartic acid have been determined in 26 fossil bones. The bones are present in two different sedimentary surroundings: levels (fine deposits with numerous artefacts and fossil bones) and “éboulis” (coarse deposits derived from the neighbouring limestone cliff). Racemization ratios for total aspartic acid have a poor correlation with¹⁴C ages (respectivelyr²=0·59 and 0·82 for the two environments) but apparent racemization rates are clearly dependent on the type of deposits (levels: K=4·4×10⁻⁶year⁻¹; éboulis: K=9×10⁻⁶year⁻¹).Racemization ratios of aspartic acid in a high molecular weight protein fraction (>10,000 Da) correlate much better with¹⁴C ages (r²=0·999) for the samples from the levels (k=2·1×10⁻⁵year⁻¹). From this regression line we estimate ages for two Aurignacian levels: level 6≈29,560 years and level 9≈30,440 years .It is noteworthy that none of the regression lines intercepts the origin. Racemization of aspartic acid appears to occur only after a delay of many thousands of years. This phenomenon could be the result of a coupling between the degradation of proteins and the racemization of amino acids.     

Possible isotopic fractionation of argon in source obsidians and archeological artifacts from Kulkuletti, Ethiopia

Despite uniform geochemistry ⁴⁰Ar/³⁹Ar ages of 0.6, 1.25, and 1.3 Ma on obsidians from a source flow (Worja) and from archeological artifacts at the sites of Kulkuletti (near Gademotta, Ethiopia) have been interpreted to suggest that they derive from different source flows of different ages. A further suggestion is that the magmatic source of the obsidian flows was chemically homogeneous between 1.3 Ma and 0.6 Ma. Chemical analysis of these obsidians indeed suggests that they derive from a single flow. Reexamination of the argon isotopic data reveals that argon isotopes may have been fractionated within the flow, so that the assumption of an atmospheric ⁴⁰Ar/³⁶Ar ratio of 295.5 is inappropriate to use for computing ages. Three groups of data are apparent, and internally all yield ages near 1.3 Ma if the intercept value for ⁴⁰Ar/³⁶Ar from an isotope correlation plot is used for computation instead of using the atmospheric ratio. The Soret thermal diffusion effect provides a possible mechanism of fractionation of argon within the obsidian flow that is of approximately the right magnitude to explain the observations by in-diffusion of already fractionated atmospheric argon.     

Hardwood ash nixtamalization may lead to false negatives for the presence of maize by depleting bulk δC in carbonized residues

Among the multiple proxies for detecting maize in precontact economies is the use of δ¹³C analysis of carbonized residues from ceramic cooking vessels. Although maize horticulture was widely established in Eastern North America (ENA) by A.D. 1000, there are carbonized residues from ceramic assemblages after this date that lack the elevated δ¹³C values indicative of the presence of maize. This may be due to the true absence of maize, or other factors including the masking of maize. Prior experimental research by Hart et al. demonstrated that the addition of C₃ plants or consumers to two part mixes with maize can mask maize presence even when maize is the dominant ingredient. Here we investigate the effect of alkali processing of maize (nixtamalization) on δ¹³C using the widespread ENA process of boiling maize kernels with wood ash, a C₃ product, to create hominy. Our experiments test whether or not the process of hardwood ash nixtamalization can mask the presence of maize in adhering carbonized residues by depleting δ¹³C values, and whether there is a reciprocal δ¹³C enrichment effect on the hardwood ash employed in nixtamalization. Overall, there is substantial δ¹³C depletion of residues when maize is cooked with hardwood ash, and hardwood ash cooked with maize shows the reciprocal enrichment. Therefore, the depleted values after the adoption of maize may be false negatives due to the nixtamalization process.     

A Late Holocene vertebrate food web from southern Haida Gwaii (Queen Charlotte Islands, British Columbia)

This study presents isotopic data (δ¹³C and δ¹⁵N from bone collagen) for 104 samples representing 29 vertebrate taxa from late pre-contact through to contact era (ca. 2000 – 100 BP) Haida Gwaii (British Columbia, Canada) from a wide variety of mostly marine organisms. The results demonstrate the considerable isotopic variability that characterizes potential prey items in coastal contexts and underscores the need for baseline faunal data to interpret human subsistence practices. Based on these data, a detailed marine food web was constructed, which provides insight into local ecological conditions. We present a simple method for quantifying the trophic level of ancient fauna using δ¹⁵N of bone collagen. Finally, we discuss the implications of this study for the reconstruction of human diet.     

Patterns of Resource Use and Isotopic Niche Overlap Among Guanaco (Lama guanicoe), Pampas Deer (Ozotoceros bezoarticus) and Marsh Deer (Blastocerus dichotomus) in the Pampas. Ecological,Paleoenvironmental and Archaeological Implications

ABSTRACT

In this paper, we analyze the isotopic niche and resource use of the guanaco (Lama guanicoe), pampas deer (Ozotoceros bezoarticus) and marsh deer (Blastocerus dichotomus) on the temperate plain of the northern Pampa region in the southeast of South America. The measured stable isotope compositions in bone tissue are δ¹³C, δ¹⁵N and δ¹⁸O. All the analyzed samples were recovered from different archaeological sites from the late Holocene. According to the results, the guanaco was strictly confined to the Pampa plain environment, developing a broad niche based on C₃ plants and a variable contribution of C₄ grasses within a grazing trend. The pampas deer preferentially used the Pampa plain, but also the prairies on the borders of the wetland, showing eurioic characteristics and a narrower niche based on C₃ plants. In turn, the marsh deer was strictly confined to wetland environments, developing a C₃ diet–base, within a narrow isotopic niche and stenoic characteristics. The three mammals showed a wide range of intraspecific variability, which was a key factor in their adaptability to spatial and temporal changes in the vegetation coverage. In fact, the temporal trends of their isotopic values were concurrent with the major climatic variations of the Holocene. Differences in the correlations between the values of both carbon sources in both deer species compared with the guanaco suggest a distinct chemical composition of their diet and/or differences in the allocation of nutrients. The isotopic values of nitrogen and spacing of the carbon sources in guanaco (pseudoruminant) and both deer species (ruminants) show no significant differences between them, thus establishing the values for local large herbivores. Significant correlations between δ¹³C and δ¹⁵N were found in the guanaco and marsh deer. The regional and extra–regional variability in the guanaco's δ¹³C_collagen and δ¹⁵N probably reflect the clinal variations in the vegetation coverage and the amount of rainfall. The collagen isotope values in the guanaco throughout the entire Holocene show that the humid Pampa would have shifted between being a more recurrent mesic and temperate plain with minor phases of dry–mesic conditions like during the Little Ice Age, and a humid and warm one at the peaks of the Holocene Thermal Maximum and the Medieval Climatic Anomaly. The guanaco and the pampas deer adapted to all the climatic changes that happened on the humid Pampa until the biological invasions of large European mammals changed the herbivores’ guild composition of this vast plain, pushing them into peripheral habitats due to competition. The within–species variability in isotopic signals through time and space make it necessary to carry out adequate sampling before reconstructing the diets of local past populations.