Dietary and homeostatic controls of Zn isotopes in rats: a controlled feeding experiment and modeling approach.

The stable isotope composition of zinc (δ66Zn), which is an essential trace metal for many biological processes in vertebrates, is increasingly used in ecological, archeological, and paleontological studies to assess diet and trophic level discrimination among vertebrates. However, the limited understanding of dietary controls and isotopic fractionation processes on Zn isotope variability in animal tissues and biofluids limits precise dietary reconstructions. The current study systematically investigates the dietary effects on Zn isotope composition in consumers using a combined controlled feeding experiment and box-modeling approach. For this purpose, 21 rats were fed one of seven distinct animal- and plant-based diets and a total of 148 samples including soft and hard tissue, biofluid, and excreta samples of these individuals were measured for δ66Zn. Relatively constant Zn isotope fractionation is observed across the different dietary groups for each tissue type, implying that diet is the main factor controlling consumer tissue δ66Zn values, independent of diet composition. Furthermore, a systematic δ66Zn diet-enamel fractionation is reported for the first time, enabling diet reconstruction based on δ66Zn values from tooth enamel. In addition, we investigated the dynamics of Zn isotope variability in the body using a box-modeling approach, providing a model of Zn isotope homeostasis and inferring residence times, while also further supporting the hypothesis that δ66Zn values of vertebrate tissues are primarily determined by that of the diet. Altogether this provides a solid foundation for refined (paleo)dietary reconstruction using Zn isotopes of vertebrate tissues.

Isotopic evidence of high reliance on plant food among Later Stone Age hunter-gatherers at Taforalt, Morocco.

The transition from hunting-gathering to agriculture stands as one of the most important dietary revolutions in human history. Yet, due to a scarcity of well-preserved human remains from Pleistocene sites, little is known about the dietary practices of pre-agricultural human groups. Here we present the isotopic evidence of pronounced plant reliance among Late Stone Age hunter-gatherers from North Africa (15,000-13,000 cal BP), predating the advent of agriculture by several millennia. Employing a comprehensive multi-isotopic approach, we conducted zinc (δ66Zn) and strontium (87Sr/86Sr) analysis on dental enamel, bulk carbon (δ13C) and nitrogen (δ15N) and sulfur (δ34S) isotope analysis on dentin and bone collagen, and single amino acid analysis on human and faunal remains from Taforalt (Morocco). Our results unequivocally demonstrate a substantial plant-based component in the diets of these hunter-gatherers. This distinct dietary pattern challenges the prevailing notion of high reliance on animal proteins among pre-agricultural human groups. It also raises intriguing questions surrounding the absence of agricultural development in North Africa during the early Holocene. This study underscores the importance of investigating dietary practices during the transition to agriculture and provides insights into the complexities of human subsistence strategies across different regions.

Late shellmound occupation in southern Brazil: A multi-proxy study of the Galheta IV archaeological site.

Brazilian coastal archaeology is renowned for its numerous and large shellmounds (sambaquis), which had been continuously occupied from at least 8000 to 1000 years cal BP. However, changes in their structure and material culture in the late Holocene have led to different hypotheses concerning their ecological and cultural changes. The archaeological site Galheta IV (ca. 1300 to 500 years cal BP) offers new insights into the complexity of the late coastal occupation in southern Brazil. Our attempt was to determine whether Galheta IV can be classified as a sambaqui site, or if it belongs to a Southern proto-Jê settlement. Here, we reassessed Galheta's collections and applied a multi-proxy approach using: new 14C dates, zooarchaeology, δ13C and δ15N isotopes in bulk collagen and 87Sr/86Srenamel isotopic ratios from eight human individuals, ceramics analysis, and FTIR. The results indicate an intense exploitation of marine resources, with an area designated for processing animals located at the opposite side of the funerary areas. Bone tools and specific species of animals were found as burial accompaniments. No evidence of human cremations was detected. 87Sr/86Sr results indicate that the eight human individuals always lived on the coast, and did not come from the inland. The pottery analysis confirms the association with Itararé-Taquara, but contrary to what was assumed by previous studies, the pottery seems related to other coastal sites, and not to the highlands. In light of these findings, we propose that Galheta IV can be considered a funerary mound resulting from long and continuous interactions between shellmound and Southern proto-Jê populations. This study not only enhances our understanding of the late coastal occupation dynamics in southern Brazil but also underscores its importance in reshaping current interpretations of shellmound cultural changes over time.

Stable isotopes show Homo sapiens dispersed into cold steppes ~45,000 years ago at Ilsenhöhle in Ranis, Germany.

The spread of Homo sapiens into new habitats across Eurasia ~45,000 years ago and the concurrent disappearance of Neanderthals represents a critical evolutionary turnover in our species' history. 'Transitional' technocomplexes, such as the Lincombian-Ranisian-Jerzmanowician (LRJ), characterize the European record during this period but their makers and evolutionary significance have long remained unclear. New evidence from Ilsenhöhle in Ranis, Germany, now provides a secure connection of the LRJ to H. sapiens remains dated to ~45,000 years ago, making it one of the earliest forays of our species to central Europe. Using many stable isotope records of climate produced from 16 serially sampled equid teeth spanning ~12,500 years of LRJ and Upper Palaeolithic human occupation at Ranis, we review the ability of early humans to adapt to different climate and habitat conditions. Results show that cold climates prevailed across LRJ occupations, with a temperature decrease culminating in a pronounced cold excursion at ~45,000-43,000 cal BP. Directly dated H. sapiens remains confirm that humans used the site even during this very cold phase. Together with recent evidence from the Initial Upper Palaeolithic, this demonstrates that humans operated in severe cold conditions during many distinct early dispersals into Europe and suggests pronounced adaptability.

The ecology, subsistence and diet of ~45,000-year-old Homo sapiens at Ilsenhöhle in Ranis, Germany.

Recent excavations at Ranis (Germany) identified an early dispersal of Homo sapiens into the higher latitudes of Europe by 45,000 years ago. Here we integrate results from zooarchaeology, palaeoproteomics, sediment DNA and stable isotopes to characterize the ecology, subsistence and diet of these early H. sapiens. We assessed all bone remains (n = 1,754) from the 2016-2022 excavations through morphology (n = 1,218) or palaeoproteomics (zooarchaeology by mass spectrometry (n = 536) and species by proteome investigation (n = 212)). Dominant taxa include reindeer, cave bear, woolly rhinoceros and horse, indicating cold climatic conditions. Numerous carnivore modifications, alongside sparse cut-marked and burnt bones, illustrate a predominant use of the site by hibernating cave bears and denning hyaenas, coupled with a fluctuating human presence. Faunal diversity and high carnivore input were further supported by ancient mammalian DNA recovered from 26 sediment samples. Bulk collagen carbon and nitrogen stable isotope data from 52 animal and 10 human remains confirm a cold steppe/tundra setting and indicate a homogenous human diet based on large terrestrial mammals. This lower-density archaeological signature matches other Lincombian-Ranisian-Jerzmanowician sites and is best explained by expedient visits of short duration by small, mobile groups of pioneer H. sapiens.

Multi-isotope zooarchaeological investigations at Abri du Maras: The paleoecological and paleoenvironmental context of Neanderthal subsistence strategies in the Rhône Valley during MIS 3.

The exploitation of mid- and large-sized herbivores (ungulates) was central to hominin subsistence across Late Pleistocene Europe. Reconstructing the paleoecology of prey-taxa is key to better understanding procurement strategies, decisions and behaviors, and the isotope analysis of faunal bones and teeth found at archaeological sites represent a powerful means of accessing information about past faunal behaviors. These isotope zooarchaeological approaches also have a near-unique ability to reveal environmental conditions contemporary to the human activities that produced these remains. Here, we present the results of a multi-isotope, multitissue study of ungulate remains from the Middle Paleolithic site of Abri du Maras, southern France, providing new insights into the living landscapes of the Rhône Valley during MIS 3 (level 4.2 = 55 ± 2 to 42 ± 3 ka; level 4.1 = 46 ± 3 to 40 ± 3 ka). Isotope data (carbon, nitrogen) reveal the dietary niches of different ungulate taxa, including the now-extinct giant deer (Megaloceros). Oxygen isotope data are consistent with a mild seasonal climate during level 4.2, where horse (Equus), bison (Bison), and red deer (Cervus elaphus) were exploited year-round. Strontium and sulfur isotope analyses provide new evidence for behavioral plasticity in Late Pleistocene European reindeer (Rangifer) between level 4.2 and level 4.1, indicating a change from the migratory to the sedentary ecotype. In level 4.1, the strong seasonal nature of reindeer exploitation, combined with their nonmigratory behavior, is consistent with a seasonally restricted use of the site by Neanderthals at that time or the preferential hunting of reindeer when in peak physical condition during the autumn.

A Neandertal dietary conundrum: Insights provided by tooth enamel Zn isotopes from Gabasa, Spain.

The characterization of Neandertals' diets has mostly relied on nitrogen isotope analyses of bone and tooth collagen. However, few nitrogen isotope data have been recovered from bones or teeth from Iberia due to poor collagen preservation at Paleolithic sites in the region. Zinc isotopes have been shown to be a reliable method for reconstructing trophic levels in the absence of organic matter preservation. Here, we present the results of zinc (Zn), strontium (Sr), carbon (C), and oxygen (O) isotope and trace element ratio analysis measured in dental enamel on a Pleistocene food web in Gabasa, Spain, to characterize the diet and ecology of a Middle Paleolithic Neandertal individual. Based on the extremely low δ66Zn value observed in the Neandertal's tooth enamel, our results support the interpretation of Neandertals as carnivores as already suggested by δ15N isotope values of specimens from other regions. Further work could help identify if such isotopic peculiarities (lowest δ66Zn and highest δ15N of the food web) are due to a metabolic and/or dietary specificity of the Neandertals.

Trophic position of Otodus megalodon and great white sharks through time revealed by zinc isotopes.

Diet is a crucial trait of an animal's lifestyle and ecology. The trophic level of an organism indicates its functional position within an ecosystem and holds significance for its ecology and evolution. Here, we demonstrate the use of zinc isotopes (δ66Zn) to geochemically assess the trophic level in diverse extant and extinct sharks, including the Neogene megatooth shark (Otodus megalodon) and the great white shark (Carcharodon carcharias). We reveal that dietary δ66Zn signatures are preserved in fossil shark tooth enameloid over deep geologic time and are robust recorders of each species' trophic level. We observe significant δ66Zn differences among the Otodus and Carcharodon populations implying dietary shifts throughout the Neogene in both genera. Notably, Early Pliocene sympatric C. carcharias and O. megalodon appear to have occupied a similar mean trophic level, a finding that may hold clues to the extinction of the gigantic Neogene megatooth shark.

Trophic ecology of a Late Pleistocene early modern human from tropical Southeast Asia inferred from zinc isotopes.

Tam Pà Ling, a cave site in northeastern Laos, has yielded the earliest skeletal evidence of Homo sapiens in mainland Southeast Asia. The reliance of Pleistocene humans in rainforest settings on plant or animal resources is still largely unstudied, mainly due to poor collagen preservation in fossils from tropical environments precluding stable nitrogen isotope analysis, the classical trophic level proxy. However, isotopic ratios of zinc (Zn) in bioapatite constitute a promising proxy to infer trophic and dietary information from fossil vertebrates, even under adverse tropical taphonomic conditions. Here, we analyzed the zinc isotope composition (66Zn/64Zn expressed as δ66Zn value) in the enamel of two teeth of the Late Pleistocene (63-46 ka) H. sapiens individual (TPL1) from Tam Pà Ling, as well as 76 mammal teeth from the same site and the nearby Nam Lot cave. The human individual exhibits relatively low enamel δ66Zn values (+0.24‰) consistent with an omnivorous diet, suggesting a dietary reliance on both plant and animal matter. These findings offer direct evidence of the broad utilization of resources from tropical rainforests by one of the earliest known anatomically modern humans in Southeast Asia.

"Here we go again": the inspection of collagen extraction protocols for 14C dating and palaeodietary analysis.

Archaeological bone collagen is highly useful for radiocarbon (14C) dating and palaeodietary reconstruction. However, collagen preservation and carbon contamination are essential considerations when extracting collagen, becoming especially crucial close to the limit of the method (50,000 years before present = BP). Strong progress has been achieved in the past two decades by 14C and stable isotopic laboratories in removing contamination from archaeological bones, but different pretreatment protocols have been proven to produce varying results. Here we compare three collagen extraction protocols used for palaeodietary studies and 14C dating, considering collagen yield, elemental and stable isotopic data, FTIR analysis, and 14C dates. We focus on the impact of ultrafiltration on the yield and quality of the extracted material. The results again underline the importance of rigorous decontamination methods to gain accurate 14C dates and demonstrate that different protocols have significant effects on the quality and yield of extracted collagen.

Strontium isotope evidence for Neanderthal and modern human mobility at the upper and middle palaeolithic site of Fumane Cave (Italy).

To investigate the mobility patterns of Neanderthals and modern humans in Europe during the Middle-to-Upper Palaeolithic transition period, we applied strontium isotope analysis to Neanderthal (n = 3) and modern human (n = 2) teeth recovered from the site of Fumane Cave in the Monti Lessini region of Northern Italy. We also measured a large number of environmental samples from the region, to establish a strontium 'baseline', and also micromammals (vole teeth) from the levels associated with the hominin teeth. We found that the modern humans and Neanderthals had similar strontium isotope values, and these values match the local baseline values we obtained for the site and the surrounding region. We conclude that both groups were utilizing the local mountainous region where Fumane Cave is situated, and likely the nearby Lessini highlands and Adige plains, and therefore the strontium evidence does not show differening mobility patterns between Neanderthals and modern humans at the Fumane site.

Zinc isotopes from archaeological bones provide reliable tropic level information for marine mammals.

In marine ecology, dietary interpretations of faunal assemblages often rely on nitrogen isotopes as the main or only applicable trophic level tracer. We investigate the geographic variability and trophic level isotopic discrimination factors of bone zinc 66Zn/64Zn ratios (δ66Zn value) and compared it to collagen nitrogen and carbon stable isotope (δ15N and δ13C) values. Focusing on ringed seals (Pusa hispida) and polar bears (Ursus maritimus) from multiple Arctic archaeological sites, we investigate trophic interactions between predator and prey over a broad geographic area. All proxies show variability among sites, influenced by the regional food web baselines. However, δ66Zn shows a significantly higher homogeneity among different sites. We observe a clear trophic spacing for δ15N and δ66Zn values in all locations, yet δ66Zn analysis allows a more direct dietary comparability between spatially and temporally distinct locations than what is possible by δ15N and δ13C analysis alone. When combining all three proxies, a more detailed and refined dietary analysis is possible.

The last battle of Anne of Brittany: Solving mass grave through an interdisciplinary approach (paleopathology, biological anthropology, history, multiple isotopes and radiocarbon dating).

Mass graves are usually key historical markers with strong incentive for archeological investigations. The identification of individuals buried in mass graves has long benefitted from traditional historical, archaeological, anthropological and paleopathological techniques. The addition of novel methods including genetic, genomic and isotopic geochemistry have renewed interest in solving unidentified mass graves. In this study, we demonstrate that the combined use of these techniques allows the identification of the individuals found in two Breton historical mass graves, where one method alone would not have revealed the importance of this discovery. The skeletons likely belong to soldiers from the two enemy armies who fought during a major event of Breton history: the siege of Rennes in 1491, which ended by the wedding of the Duchess of Brittany with the King of France and signaled the end of the independence of the region. Our study highlights the value of interdisciplinary approaches with a particular emphasis on increasingly accurate isotopic markers. The development of the sulfur isoscape and testing of the triple isotope geographic assignment are detailed in a companion paper [13].

Triple sulfur-oxygen-strontium isotopes probabilistic geographic assignment of archaeological remains using a novel sulfur isoscape of western Europe.

Sulfur isotope composition of organic tissues is a commonly used tool for gathering information about provenance and diet in archaeology and paleoecology. However, the lack of maps predicting sulfur isotope variations on the landscape limits the possibility to use this isotopic system in quantitative geographic assignments. We compiled a database of 2,680 sulfur isotope analyses in the collagen of archaeological human and animal teeth from 221 individual locations across Western Europe. We used this isotopic compilation and remote sensing data to apply a multivariate machine-learning regression, and to predict sulfur isotope variations across Western Europe. The resulting model shows that sulfur isotope patterns are highly predictable, with 65% of sulfur isotope variations explained using only 4 variables representing marine sulfate deposition and local geological conditions. We used this novel sulfur isoscape and existing strontium and oxygen isoscapes of Western Europe to apply triple isotopes continuous-surface probabilistic geographic assignments to assess the origin of a series of teeth from local animals and humans from Brittany. We accurately and precisely constrained the origin of these individuals to limited regions of Brittany. This approach is broadly transferable to studies in archaeology and paleoecology as illustrated in a companion paper (Colleter et al. 2021).

Strontium (87 Sr/86 Sr) isotope analysis of the Namu skeletal assemblage: A study of past human migration on Taumako, a Polynesian Outlier in the eastern Solomon Islands.

OBJECTIVES: This study aims to assess if inter-island mobility can be identified during the Namu period (ca. 1,510-1800 AD) using 87 Sr/86 Sr analysis of dental enamel for individuals from the Namu burial ground on Taumako Island in the eastern Solomon Island Chain. Historic evidence from this region suggests that females migrated between the Duff, Reef, and Santa Cruz islands for marriage purposes. We hypothesize that observable trends in migrational (87 Sr/86 Sr) and dietary (δ13 C and δ15 N) isotopes can reveal the relationship between demographic factors, social status, diet, and female mobility on Taumako. METHODS: This research analyzes enamel 87 Sr/86 Sr for 58 individuals in the Namu skeletal sample. The 87 Sr/86 Sr results were compared with published dietary isotope data (bone collagen and dentin δ13 C and δ15 N values) and type/number of grave goods to assess whether trends within the data may be related to sex, age, or burial wealth. RESULTS: The results show that females display significantly higher 87 Sr/86 Sr values compared to males. One young adult female displayed a 87 Sr/86 Sr value that was +2SD outside the mean for the sampled individuals. A linear mixed-effects model and principle components analysis of 87 Sr/86 Sr, δ13 C, and δ15 N values suggest that wealth, sex, and age-cohort membership have an observable influence on the isotopic variation for the Taumako population. CONCLUSION: We suggest that during the Namu period, Taumako was patrilocal and that some females migrated there from the nearby Santa Cruz and Reef islands. One female immigrated to Taumako from a geologically distinct region outside of the Duff, Reef, and Santa Cruz Island groups.

Zinc isotope variations in archeological human teeth (Lapa do Santo, Brazil) reveal dietary transitions in childhood and no contamination from gloves.

Zinc (Zn) isotope ratios of dental enamel are a promising tracer for dietary reconstruction in archeology, but its use is still in its infancy. A recent study demonstrated a high risk of Zn contamination from nitrile, and latex gloves used during chemical sample preparation. Here we assess the potential impact of the use of such gloves during enamel sampling on the Zn isotope composition of teeth from a population of early Holocene hunter gatherers from Lapa do Santo, Lagoa Santa, Minas Gerais, Brazil. We first examined the amount of Zn and its isotopic composition released from the gloves used in this study by soaking them in weak nitric acid and water. We compared Zn isotope ratios obtained from teeth that were sampled wearing nitrile, latex or no gloves. Finally, we performed a linear mixed model (LMM) to investigate post hoc the relationship between the gloves used for sampling and the Zn isotope variability in dental enamel. We found that the gloves used in this study released a similar amount of Zn compared to previous work, but only in acidic solution. Zn isotope ratios of teeth and the LMM identified no sign of significant Zn coming from the gloves when teeth were handled for enamel sampling. We hypothesize that Zn in gloves is mostly released by contact with acids. We found that the main source of Zn isotope variability in the Lapa do Santo population was related to the developmental stage of the tooth tissues sampled. We report identical results for two individuals coming from a different archeological context. Tooth enamel formed in utero and/or during the two first years of life showed higher Zn isotope ratios than enamel formed after weaning. More work is required to systematically investigate if Zn isotopes can be used as a breastfeeding tracer.

Zinc isotopes in Late Pleistocene fossil teeth from a Southeast Asian cave setting preserve paleodietary information.

Stable carbon and nitrogen isotope ratios of collagen from bone and dentin have frequently been used for dietary reconstruction, but this method is limited by protein preservation. Isotopes of the trace element zinc (Zn) in bioapatite constitute a promising proxy to infer dietary information from extant and extinct vertebrates. The 66Zn/64Zn ratio (expressed as δ66Zn value) shows an enrichment of the heavy isotope in mammals along each trophic step. However, preservation of diet-related δ66Zn values in fossil teeth has not been assessed yet. Here, we analyzed enamel of fossil teeth from the Late Pleistocene (38.4-13.5 ka) mammalian assemblage of the Tam Hay Marklot (THM) cave in northeastern Laos, to reconstruct the food web and assess the preservation of original δ66Zn values. Distinct enamel δ66Zn values of the fossil taxa (δ66Zncarnivore < δ66Znomnivore < δ66Znherbivore) according to their expected feeding habits were observed, with a trophic carnivore-herbivore spacing of +0.60‰ and omnivores having intermediate values. Zn and trace element concentration profiles similar to those of modern teeth also indicate minimal impact of diagenesis on the enamel. While further work is needed to explore preservation for settings with different taphonomic conditions, the diet-related δ66Zn values in fossil enamel from THM cave suggest an excellent long-term preservation potential, even under tropical conditions that are well known to be adverse for collagen preservation. Zinc isotopes could thus provide a new tool to assess the diet of fossil hominins and associated fauna, as well as trophic relationships in past food webs.

Dynamic homeostasis modeling of Zn isotope ratios in the human body.

Recent research performed on volunteers and patients suggested that diet, health, and basal metabolic rates (BMR) are factors controlling the bodily Zn isotope compositions (isotopic homeostasis). However, our poor understanding of the variability of Zn distribution among the different organs and fluids of the human body, and the ensuing isotope fractionation, limits the use of this isotopic system as a typical diagnostic tool for cancers and for past hominin diet reconstructions. Using box model calculations, we investigated the dynamics of Zn isotope variability in blood and other body tissues as well as the consistency of the hypothesis of heavy Zn isotope accumulation through time in the human body. We compare the results of the model with data obtained from control feeding experiments and from archeological samples. Model simulations indicate that the absence of an aging drift in non-circumpolar populations cannot be explained by their lower BMR. We argue that the drift observed in the blood of a circumpolar population results from a differential diet between young and older individuals in this population. When applied to the δ66Zn measured in blood, bones, or teeth, the present box model also offers insight into the isotope composition of the human diet, and therefore into its nature. Applying the model to isotopic observations on the remains of past hominins is a promising tool for diet reconstruction.