During the last glacial period, rapidly changing environments posed substantial challenges to Neanderthal populations in Europe. Southern continental regions, such as Iberia, have been proposed as important climatic “buffer” zones during glacial phases. Contextualising the climatic and ecological conditions Neanderthals faced is relevant to interpreting their resilience. However, records of the environments and ecosystems they exploited across Iberia exhibit temporal and spatial gaps in coverage. Here we provide new evidence for palaeotemperatures, vegetation structure, and prey herbivore ecology during the late Pleistocene (MIS 5–3) in northern Spain, by applying multiple stable isotope tracers (δ18O, δ13C, δ15N, δ34S) to herbivore skeletal remains associated with Neanderthal occupations at Axlor Cave, Bizkaia. The results show little change over time and indicate stable climatic conditions and ecosystems across different occupations. Large within-layer isotopic variability in nitrogen and sulphur suggests the presence of a mosaic environment and a variety of isotopic ecotones that were exploited by Neanderthals and their prey. We implement a combination of carbonate and phosphate δ18O measurements to estimate palaeotemperatures using a cost-effective workflow. We show that the targeted use of phosphate δ18O measurements to anchor summer peak and winter trough areas enables high-precision seasonal palaeoclimatic reconstructions.

In the summer of 1986 a mass grave was discovered along the bank of the river Tryggevælde Å where it empties into Køge Bugt, the bay south of modern Copenhagen, Denmark. The human remains, dating to the late Mesolithic Ertebølle culture, consisted of eight individuals of multiple ages, ranging c. 35–45 years old to newborn children. Four were arranged on one side of the grave, with four on the other, placed head to foot. How they were related and what befell them is a mystery. Herein, we present a bioarchaeological assessment of these individuals for the first time and apply an acid etch-based analysis of dimorphic sex chromosome-linked tooth enamel peptides to confirm their biological sex. Our results allow a direct connection between engendered grave treatment and biological sex in non-adult individuals as young as c. 4 years of age. We conclude with a discussion of the possible circumstances of their deaths and their possible relationships to one another.

Megalithic cultures of central India provide important links between the southern Neolithic-Chalcolithic cultures and the early Historical period (∼500 BC to ∼AD 700) and reveal knowledge of ancient traditions of early inhabitants. Scientific dating of these Megalithic burial sites is a challenging task due to scarcity of dateable material and alterations. Here, we present multiple accelerator mass spectrometry radiocarbon (AMS 14C) dates from equine tooth-enamel and organic food remains recovered from pots from Megalithic burials of the Vidarbha region. Using δ13CTOC and δ15N values of organic food remains recovered from pots, we deduced past-diet (palaeo-vegetation) that indicates C4 type of vegetation and thus arid climate during life-spans of these burials. We also analyzed stable δ13C and δ18O isotopes of equine tooth-enamel to investigate hydro-climatic conditions of Maharashtra (Vidarbha region). A total of 10 AMS 14C dates of tooth enamel provide a time range of AD 250–874 for two Megalithic burials. Two AMS 14C dates of organic food remains recovered from pots corroborated aforementioned time-range. The average δ13C and δ18O of equine tooth-enamel samples were −5.3 ± 2.1‰ and −2.9 ± 0.8‰, respectively, both significantly enriched compared to their modern counterparts (−13.7‰ ± 0.7 and −4.3‰ ± 1.1), indicating intense arid conditions in the past.

In hot environments, collagen, which is normally targeted when radiocarbon (14C) dating bone, rapidly degrades. With little other skeletal material suitable for 14C dating, it can be impossible to obtain dates directly on skeletal materials. A small amount of carbonate occurs in hydroxyapatite, the mineral phase of bone and tooth enamel, and has been used as an alternative to collagen. Unfortunately, the mineral phase is often heavily contaminated with exogenous carbonate causing 14C dates to underestimate the true age of a sample. Although tooth enamel, with its larger, more stable crystals and lower porosity, is likely to be more robust to diagenesis than bone, little work has been undertaken to investigate how exogenous carbonate can be effectively removed prior to 14C dating. Typically, acid is used to dissolve calcite and etch the surface of the enamel, but it is unclear which acid is most effective. This study repeats and extends earlier work using a wider range of samples and acids and chelating agents (hydrochloric, lactic, acetic and propionic acids, and EDTA). We find that weaker acids remove carbonate contaminants more effectively than stronger acids, and acetic acid is the most effective. However, accurate dates cannot always be obtained.

Tooth enamel, the outermost layer of human teeth, is a complex, hierarchically structured biocomposite. The details of this structure are important in multiple human health contexts, from understanding the progression of dental caries (tooth decay) to understanding the process of amelogenesis and related developmental defects. Enamel is composed primarily of long, nanoscale crystallites of hydroxyapatite that are bundled by the thousands to form micron-scale rods. Studies with transmission electron microscopy show the relationships between small groups of crystallites and X-ray diffraction characterize averages over many rods, but the direct measurement of variations in local crystallographic structure across and between enamel rods has been missing. Here, we describe a synchrotron X-ray-based experimental approach and a novel analysis method developed to address this gap in knowledge. A ~500-nm-wide beam of monochromatic X-rays in conjunction with a sample section only 1 μm in thickness enables 2D diffraction patterns to be collected from small well-separated volumes within the enamel microstructure but still probes enough crystallites (~300 per pattern) to extract population-level statistics on crystallographic features like lattice parameter, crystallite size, and orientation distributions. Furthermore, the development of a quantitative metric to characterize relative order and disorder based on the azimuthal autocorrelation of diffracted intensity enables these crystallographic measurements to be correlated with their location within the enamel microstructure (e.g., between rod and interrod regions). These methods represent a step forward in the characterization of human enamel and will elucidate the variation of the crystallographic structure across and between enamel rods for the first time.

In temperate latitudes sheep have a seasonal reproductive behaviour, which imposes strong constraints on husbandry in terms of work organization and availability of animal products. During the last 50 years, researchers have focused on understanding the mechanisms driving small ruminants’ reproduction cycles and finding ways to control them. This characteristic is inherited from their wild ancestor. However, the history of its evolution over the 10 millennia that separates present day European sheep from their Near Eastern ancestors’ remains to be written. This perspective echoes archaeologists’ current attempts at reconstructing ancient pastoral societies’ socio-economical organization. Information related to birth seasonality may be retrieved directly from archaeological sheep teeth. The methodology consists of reconstructing the seasonal cycle record in sheep molars, through sequential analysis of the stable oxygen isotope composition (δ18O) of enamel. Because the timing of tooth development is fixed within a species, inter-individual variability in this parameter reflects birth seasonality. A review of the data obtained from 10 European archaeological sites dated from the 6th to the 3rd millennia BC is provided. The results demonstrate a restricted breeding season for sheep: births occurred over a period of 3 to 4 months, from late winter to early summer at latitudes 43°N to 48°N, while a later onset was observed at a higher latitude (59°N). All conclusions concurred with currently held expectations based on present day sheep physiology, which, aside from the historical significance, contributes to the reinforcing of the methodological basis of the approach. Further study in this area will permit regional variability attributable to technical choices, within global schemes, to be fully reported.

We investigated age estimation of persons using 45 Japanese human teeth that consisted of mainly the third molar. We measured the radiocarbon content in tooth enamel hydroxyapatite from the crown as well as those in collagen and bioapatite in the root of the same tooth. Age estimations from tooth enamel apatite yielded an absolute age shift of 1.7±1.4 yr from the true age. This value suggests that estimating the age of Japanese people from tooth enamel is as accurate as the previous studies conducted in Europe and the USA. The 14C analysis of roots suggested that bioapatite is formed slightly later than collagen in the same tooth roots. The appropriate formation age ranges suggested by the tooth enamel analysis can be optimized and confirmed by using the additional F14C values of the root samples. We also noticed a characteristic trend of enamel δ13C values in comparison with the formation ages of the crowns of the teeth. The δ13C value tends to increase gradually from –13.0‰ to –11.5‰ from the 1960s to the 1970s, probably being caused by changes in the Japanese diet. This trend may support the selection of proper tooth formation age as supplemental data in addition to 14C content analyses.

Fossils of megaherbivores from eight late Pleistocene 14C- and OSL-dated doline infillings of Ajoie (NW Switzerland) were discovered along the Transjurane highway in the Swiss Jura. Carbon and oxygen analyses of enamel were performed on forty-six teeth of large mammals (Equus germanicus, Mammuthus primigenius, Coelodonta antiquitatis, and Bison priscus), coming from one doline in Boncourt (~ 80 ka, marine oxygen isotope stage MIS5a) and seven in Courtedoux (51–27 ka, late MIS3), in order to reconstruct the paleoclimatic and paleoenvironmental conditions of the region. Similar enamel δ13C values for both periods, ranging from − 14.5 to − 9.2‰, indicate that the megaherbivores lived in a C3 plant-dominated environment. Enamel δ18OPO4 values range from 10.9 to 16.3‰ with a mean of 13.5 ± 1.0‰ (n = 46). Mean air temperatures (MATs) were inferred using species-specific δ18OPO4–δ18OH2O-calibrations for modern mammals and a present-day precipitation δ18OH2O-MAT relation for Switzerland. Similar average MATs of 6.6 ± 3.6°C for the deposits dated to ~ 80 ka and 6.5 ± 3.3°C for those dated to the interval 51–27 ka were estimated. This suggests that these mammals in the Ajoie area lived in mild periods of the late Pleistocene with MATs only about 2.5°C lower than modern-day temperatures.

The vertebrate fossil record of Cement Creek Cave, Colorado, spans from ≫ 45,000 yr ago to the present and represents the richest stratified series of high-elevation (≫ 2900 m) mammal remains known from the late Quaternary of North America. Stable carbon and oxygen isotope analyses of tooth enamel were used to assess potential ecological responses of two species found commonly throughout the cave, Yellow-bellied marmots (Marmota flaviventris) and Bushy-tailed woodrats (Neotoma cinerea), to late Quaternary climate and environmental changes of the Southern Rocky Mountains. Results indicate that despite such perturbations, the dietary ecologies of both species were maintained across this period. Neither taxon shifted to consuming C4 taxa or different C3 functional groups; similarly, no significant shifts in surface water use were detected. Variations in enamel δ13C were observed, however, that represent the physiological responses of high-elevation plants to changing levels of late Quaternary atmospheric CO2. While our findings extend both the geographic and elevational record of this plant CO2 response, they simultaneously highlight the ecological stability of high-elevation M. flaviventris and N. cinerea during climate changes of late Quaternary magnitude.

Pleistocene faunas from south China are difficult to subdivide based on the long temporal ranges of many taxa and a reduced number of genera in comparison to faunas from temperate north China. In south China, the Ailuropoda–Stegodon fauna is a very general one and includes a relatively stable suite of genera that apparently persisted for long periods of time. These attributes have made constraining its time range difficult. Application of electron spin resonance (ESR) dating of tooth enamel constrains the ages well where uranium uptake was minor. Where uranium uptake into teeth was significant, an approach combining ESR and230Th/234U isotopic analysis also yields excellent ages. Previous estimates of early, middle and late Pleistocene time ranges previously determined by biostratigraphic seriation for the Ailuropoda–Stegodon fauna are confirmed in all cases but are made more precise with our approach, including specific time ranges for certain archaic taxa. Absolute dating also yields an extended time range for Gigantopithecus blacki of 1200 to 310 ka.

Serial sampling of tooth enamel growth increments for carbon and oxygen isotopic analyses of Macropus (kangaroo) teeth was performed to assess the potential for reconstructing paleoseasonality. The carbon isotope composition of tooth enamel apatite carbonate reflects the proportional intake of C3 and C4 vegetation. The oxygen isotopic composition of enamel reflects that of ingested and metabolic water. Tooth enamel forms sequentially from the tip of the crown to the base, so dietary and environmental changes during the tooth's formation can be detected. δ13C and δ18O values were determined for a series of enamel samples drilled from the 3rd and 4th molars of kangaroos that were collected along a 900 km north–south transect in southern Australia. The serial sampling method did not yield pronounced seasonal isotopic variation patterns in Macropus enamel. The full extent of dietary isotopic variation may be obscured by attenuation of the isotopic signal during enamel mineralisation. Brachydont (low-crowned) Macropus teeth may be less sensitive to seasonal variation in isotopic composition due to time-averaging during mineralisation. However, geographic variations observed suggest that there may be potential for tracking latitudinal shifts in vegetation zones and seasonal environmental patterns in response to climate change.

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0·001). In conclusion, this study shows that the addition of 2·0–5·0 g CPP-ACP/l to milk substantially increases its ability to remineralize enamel subsurface lesions.

The primitive sheep of North Ronaldsay Island (Orkney), which feed almost exclusively on seaweed, have developed physiological features linked to that diet. The use of seaweed to feed domestic animals, attested for centuries in north-western Europe, may have appeared soon after the arrival of the first domestic herds during the 5th and 4th millennia BC. The use of isotope analysis of sheep tooth enamel as a means to investigate seaweed grazing in prehistoric times was tested. The teeth of five modern North Ronaldsay sheep were analysed for carbon (δ13C) and oxygen (δ18O) isotope ratios. Owing to differences in the δ13C of marine vs terrestrial plants, the seaweed-eating sheep have δ13C values clearly outside the range expected for terrestrial plant eaters. Results show that one group of individuals relied exclusively on seaweed throughout the year. In a second group, terrestrial plants provided about half the dietary carbon pool during the summer. In individuals relying exclusively on seaweed, the amplitude of seasonal change in the δ18O values of tooth enamel was low, possibly because of ingestion of marine water through fresh seaweed consumption. Analysis of a sheep tooth from the Neolithic site of Holm of Papa Westray (Orkney, Scotland), suggests that as early as at the beginning of the 4th millennium BC in the Orkney, seaweed contributed to the winter diet of domestic sheep.