Carbon and nitrogen stable isotope analysis using human bones and hair from Philippine burial sites

Stable isotope analysis has been utilized in archaeology since the 1970s, yet standardized protocols for terminology, sampling, pretreatment evaluation, calibration, quality assurance and control, data presentation, and graphical or statistical treatment still remain lacking in archaeological applications. Here, we present recommendations and requirements for each of these in the archaeological context of: bulk stable carbon and nitrogen isotope analysis of organics; bulk stable carbon and oxygen isotope analysis of carbonates; single compound stable carbon and nitrogen isotope analysis on amino acids in collagen and keratin; and single compound stable carbon and hydrogen isotope analysis on fatty acids. The protocols are based on recommendations from the Commission on Isotopic Abundances and Atomic Weights of the International Union of Pure and Applied Chemistry (IUPAC) as well as an expanding geochemical and archaeological science experimental literature. We hope that this will provide a useful future reference for authors and reviewers engaging with the growing number of stable isotope applications and datasets in archaeology.

Extreme climatic events can trigger sudden but often long-lasting and irreversible changes in ecosystems by causing mortality of foundation (habitat-forming) species. The magnitude and frequency of such events are likely to increase due to human-induced climate change, but the dynamics of such extreme biological events remain poorly understood, with only a limited number of case studies reported in the past. In many cases, assessing the impact of rare, extreme biological events can be challenging because these events can be unexpected and sudden, often making pre-event sampling not achievable. In late 2015 to early 2016, an extensive area of mangrove forest along ~ 1,000 km of coastline in the Gulf of Carpentaria, Australia, experienced severe dieback as a result of a climatic extreme event that included extreme temperatures, drought conditions and lower than average sea levels. My research aimed to address a knowledge gap in the effects of extreme climatic events on intertidal coastal ecosystems by assessing the ecological impacts of mass mortality of mangrove trees on the intertidal ecosystem. This aim was achieved through the use of a comparative experiment of an impacted forest and an adjacent unimpacted forest using traditional ecological survey techniques combined with conventional bulk stable isotope analyses and a more novel compound-specific isotope analysis of amino acids. My research also offers significant insights into the use of the more novel compound-specific isotope analysis of amino acids to complement the conventional bulk stable isotope analysis in mangrove ecosystem trophic analyses. Firstly, I used a combination of traditional ecological survey techniques and bulk stable isotope analysis of carbon (C) and nitrogen (N) to measure the effects of mangrove forest mortality on benthic faunal communities, with a focus on functional aspects of food web dynamics. I tested if changes in benthic faunal assemblages would be evident due to mangrove mortality, and if food web structure was impacted by the mangrove mortality and alternations in available food resources. I found that in the forest that experienced tree mortality, there were fewer crabs that relied on mangrove litter as a food source but more crabs that fed on the microphytobenthos. As the microphytobenthos was largely unaffected by the die-back event, they provided a buffer to the food-web responses. The infauna, e.g. burrowing crabs, was also largely unaffected by the mortality effect. However, overall, the habitat value for mangrove ecosystem services could be decreased due to lower physical habitat complexity following tree losses. Secondly, the initial dieback and recovery of the impacted mangrove ecosystem were evaluated using a combination of bulk stable C, N and S isotopes and a more novel amino acid compound-specific isotope method. I tested if tree mortality changed the overall circulation of C, N and S elements, and if this change would be reflected in δ13C, δ15N and δ34S values of mangrove ecosystem components such as mangrove plants, soil and associated animals. I also tested if/how these isotopic compositions change over time with the recovery of mangrove vegetation. Stable isotope analyses confirmed significant changes to the circulation of C, N and S elements following tree mortality. Recovery of the mangrove vegetation was evident from increased numbers of mangrove seedlings and saplings in the impacted forest over the two-year survey, but recovery of CNS cycling was not evident even after 30 months, suggesting a long-lasting effect of the mortality event. Finally, the use of bulk stable CNS isotopes and more novel compound-specific stable C and N isotopes of amino acids were compared to evaluate which isotopic compositions are more conservative tracers of mangrove organic matter and suited for analyses of mangrove food webs. I tested if stable isotopic compositions in essential amino acids that cannot be synthesised by animals would be more conservative in food web links between consumers and mangrove organic matter. Isotopic compositions in essential amino acids effectively separated mangrove organic matter from the microphytobenthos and helped to trace mangrove organic matter in a mangrove food web. These more sophisticated tracing techniques complemented traditional bulk stable isotope analyses by providing improved resolving power in mangrove trophic analyses. The outcome of this research will be an important contribution to the emerging global body of case studies that show significant ecological impacts driven by extreme climatic events, and how changes in habitat forming species result in significant impacts on ecosystem community dynamics as well as biogeochemical processes including C, N and S cycling. My research also provides a framework for combining the use of conventional and novel stable isotope measurements with traditional ecological survey techniques in reporting difficult to measure impacts of extreme biological events. The results of this research may also be used for planning and future-proofing coastal wetlands from future impacts of climatic extreme events, and support wetland conservation and restoration efforts.

We used the stable isotopes of carbon and nitrogen to examine the food webs of three small flood-plain lakes (billabongs) in south-eastern Australia. With few exceptions, stable carbon isotope analysis could not be used to discriminate among the conspicuous potential sources of fringing, emergent or floating vegetation or benthic detritus. These primary sources showed little spatial or temporal variation in δ13C values, with means ranging from-28.5 to-26.8‰ in spring and-29.1 to-25.4‰ in late summer. Submerged vegetation had similar δ13C values to the above sources in spring but showed greater spatial variation and were less 13C-depleted, considerably so in some species, in late summer. Epiphytes and algae were 13C-depleted in spring compared with the other primary sources but became more 13C-enriched in late summer. Mean δ13C values for primary and secondary consumers were not only far more variable (-37.4 to-22.7‰) but in general were more negative than the potential food sources, particularly in spring. Using the combined information from stable carbon and nitrogen isotope analysis, we could narrow down the list of potential primary sources driving food webs in these billabongs. The freshwater crayfish (Cherax) was one of the few taxa that appeared to obtain its biomass carbon from detrital material. Gastropods and leptocerid caddis larvae on emergent or submerged vegetation obtained a mixture of carbon from epiphytes and macrophytes; in both taxa, epiphytes contributed more to biomass carbon than did the macrophytes. However, other common grazers and collector/gatherers sampled from macrophytes, e.g. baetid mayflies, chironomid larvae and atyid shrimps, were often too 13C-depleted even to have derived their biomass carbon solely from epiphytes. Many other primary consumers, including zooplankton, and mussels (Velesunio), and most of the secondary consumers, including water mites (Hydracarina), phantom midge larvae (Chaoborus) and fish, were also 13C-depleted. The enormous biomass of littoral and fringing vegetation could contribute to metazoan food webs in these billabongs only if an additional highly 13C-depleted source was consumed simultaneously. Methane released from billabong sediments could provide such a 13C-depleted carbon source that is re-introduced into metazoan food webs via the consumption of methanotrophic bacteria. Alternatively, food webs in these water bodies are largely driven by an unknown and inconspicuous 13C-depleted primary producer, such as planktonic Chlorophyta.

"Late Holocene Climatic Changes Recorded In Macropod Bone Collagen Stable Carbon And Nitrogen Isotopes At Fromms Landing, South Australia." Australian Archaeology, 49(1), pp. 48–49

The impact of social status and biological sex on diet at 10th century CE Hungarian sites from stable carbon and nitrogen isotope analyses

Archaeodiet in the Greek World: Dietary Reconstruction from Stable Isotope Analysis

Stable carbon and nitrogen isotope analysis is one of the main techniques for assessing various aspects of life of the prehistoric populations including diet and economy. Here, we present the theoretical background of the method, and review the major stable isotope paleodietary studies relating to the Eurasian steppe and available by the end of 2013. Existing data show that the diet of various Chalcolithic to Early Iron Age populations in the region varied in time and space and included substantial amounts of fi sh. Variations in diet and economy between groups associated with the same archaeological culture were likely caused by adaptations to local environments and climates. Millet appeared in the area (in the Minusinsk Basin) only in the 14th century BC. The Minusinsk Basin thereby became one of the fi rst centres of millet cultivation in Siberia. The impact of climate, specifi cally precipitation, on the isotopic values of human bone tissue has also been recorded. Although studies of ancient Siberian and Eurasian steppe groups are numerous, paleodietary research using stable isotope analysis is still at the data acquisition stage. One of the main criteria of modern research in the region is a systematic and well-designed approach to the isotopic analysis of various archaeological populations. This analysis must include not only human bone samples, but also those relating to all potential dietary components such as terrestrial and aquatic animals as well as samples of associated plants.

The role of parasites in trophic ecology is poorly understood in marine ecosystems. Stable isotope analyses (SIA) have been widely used in studies of trophic ecology, but have rarely been applied to study the role of parasites. Considering that some parasites are associated with altered host foraging patterns, SIA can help elucidate whether parasitism influences host trophic interactions. French grunt (Haemulon flavolineatum), an abundant Caribbean coral reef fish, contributes greatly to trophic connectivity. They typically depart the reef at dusk, feed overnight in seagrass beds, and return to the reef at dawn. The large parasitic isopod Anilocra haemuli commonly infects French grunt, and infected fish are less likely to complete their diel migration, and are in poorer condition than uninfected conspecifics. Brown chromis (Chromis multilineata) are diurnally feeding planktivores and infection by Anilocra chromis does not influence host condition. To determine if Anilocra infection influences host diet and foraging locality, we conducted stable carbon and nitrogen isotope analyses on scale, muscle, heart and gill tissues of infected and uninfected French grunt and brown chromis. We determined that all French grunt had δ13C values representative of seagrass habitats, but infected French grunt were significantly enriched in 13C and 15N compared to uninfected conspecifics. This suggests that compared to uninfected conspecifics, infected French grunt forage in seagrass, but on isotopically enriched prey, and/or are in poorer condition, which can elevate δ13C and δ15N values. For brown chromis, infection did not significantly influence any δ13C and δ15N values; hence they all foraged in the same environment and on similar prey. This is the first study to use SIA to examine differences in resource use by Caribbean coral reef fishes associated with parasitism and to evaluate how closely related parasites might have host‐dependent effects on host trophic ecology.

Assessment of trophic dynamics of cephalopods and large pelagic fishes in the central North Atlantic Ocean using stable isotope analysis

Aims and ObjectivesMacroscopic skeletal analysis and stable carbon and nitrogen isotope analysis were employed to examine the relationship between skeletal “stress” lesions and changes in the isotopic life history profiles of six nonadults from the Gepid population buried at the Archiud “Hânsuri” Cemetery (4th–7th centuries AD).Materials and MethodsThe Gepids were a migratory barbarian population in the Carpathian Mountain basin of Transylvania, Romania. Macroscopic skeletal analysis was conducted on 32 individuals, and of those, six nonadults were selected for stable carbon and nitrogen isotope analysis of incrementally sampled dentine.ResultsMacroscopic skeletal analyses revealed 47% of the analysed population displayed evidence of childhood stress. Stable carbon and nitrogen isotope ranges were −17.7‰ to −11.8‰ for δ13C and 9.4‰ to 15.1‰ for δ15N.DiscussionThe overall dietary profile indicates a mixed terrestrial diet (C3/C4) with increased consumption of C4 plants during adolescence. The six nonadults appear to have been breastfed from 1 to 6 months and weaned by 3 years of age. High δ15N values seen in pre‐natal and post‐natal increments may suggest a level of nutritional/physiological stress during gestation and during the transitions from umbilical nutrients, breastmilk, and weaning foods. Although limited by the small sample size, this study supports the link between elevated δ15N values and nutritional stress and the relationship and timing of skeletal lesions with changes in the isotope profiles and was among the first to combine palaeopathological analyses and incremental stable isotope analyses on the Transylvania Gepids.

Stable carbon and nitrogen isotope analyses of bone collagen, and stable carbon and oxygen isotope analyses of the bone's structural carbonate, were performed on 120 individuals representing 33 vertebrate species, including a single human bone find, collected from the Late Neolithic settlement at Pestenacker, Bavaria, Germany. We were thus capable of reconstructing a rather complex food web and could also address particular issues, such as whether humans influenced the diet of their domestic animals as opposed to their wild relatives, or whether humans perhaps had to compete over food with their domesticates. A rather unexpected result was that freshwater fish, which could be captured in the nearby river Lech, a major tributary of the Danube, contributed to the human diet only occasionally. As for mammals, it was also possible to recognise different trophic levels for birds and aquatic vertebrates, applying stable isotope analyses to both bone collagen and structural carbonate. In the case of fish, δ18O values at least revealed a physiological regularity in terms of temperature preference, besides diet. Conceivably, variability of δ18O in surface water as reflected, for example, by species that avoided human settlements, may help to characterise past ecosystems and to define site catchment exploited by Neolithic man in the course of food acquisition. Copyright © 2006 John Wiley & Sons, Ltd.

Comparison of two methods of extracting bone collagen for stable carbon and nitrogen isotope analysis: comparing whole bone demineralization with gelatinization and ultrafiltration

Stable carbon and nitrogen isotope analysis was carried out on human and animal bones from four inland Early and Middle Bronze Age sites in Northern and Southern Italy. The main aims of the investigation were to explore the contribution of plant foods to the human diet and to examine any dietary differences between and within each of the sites. At two of the sites in Northern Italy, human and animal bones were significantly enriched in 13C. This finding was attributed to the consumption of domestic millets (Panicum miliaceum and/or Setaria italica), which are C4 pathway plants. Conversely, individuals from the two Bronze Age sites in Southern Italy were significantly depleted in 13C compared to those from the north. Here, millet was absent from the diet, and protein from C3 plants made a much greater dietary contribution than animal protein. This finding highlights the importance of cereal cultivation, most likely of wheat and barley, in the south of Italy during the Bronze Age. Overall, our results support the idea that the widespread cultivation of millet first occurred in Northern Italy, following its introduction from across the Alps in Central Europe. Finally, we found no significant differences in the stable isotope values between individuals at each site, when grouped by their sex or presence of grave goods. This leads to the conclusion that any status difference that may have existed is not reflected in the long-term dietary record, or at least not as measurable by stable isotope analysis.

Stable carbon and nitrogen isotope analysis as a tool for inferring beef cattle feeding systems in Japan

Bird communities reach their highest taxonomic and trophic diversity in tropical rain forest, but the use of different foraging strategies to meet food requirements in such competitive environments is poorly understood. Conventional dietary analyses are poorly suited to investigate dietary patterns in complex systems. We used stable carbon ((13)C/(12)C) and nitrogen ((15)N/(14)N) isotope analysis of whole blood to examine avian trophic patterns and sources of diet in the tropical rain forest of Los Tuxtlas, Veracruz, Mexico. We used stable nitrogen isotope analysis to delineate trophic levels, and stable carbon isotope analysis to distinguish the relative contribution of C-3 and CAM/C-4 ultimate sources of proteins to diets. There was large inter- and intraspecific variation in whole blood delta(13)C and delta(15)N values in 23 species of birds. Stable nitrogen isotope analysis separated birds into several trophic levels, including species that obtained their dietary protein mostly from plants, insects or a combination of both food sources. Stable carbon isotope analysis showed that most birds fed on C3-based foods but Stub-tailed Spadebills (Platyrinchus cancrominus) included C-3- and C-4/CAM-specialist individuals. Our analyses provided insights into the nutritional contribution of plant and animal sources of protein and distinguish their photosynthetic origin over relatively long average time periods.