Dietary and homeostatic controls of Zn isotopes in rats: A controlled-feeding experiment and modelling 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-modelling 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-modelling 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.

Spatial ecology of moose in Sweden: Combined Sr-O-C isotope analyses of bone and antler.

The study of spatial (paleo)ecology in mammals is critical to understand how animals adapt to and exploit their environment. In this work we analysed the 87Sr/86Sr, δ18O and δ13C isotope composition of 65 moose bone and antler samples from Sweden from wild-shot individuals dated between 1800 and 1994 to study moose mobility and feeding behaviour for (paleo)ecological applications. Sr data were compared with isoscapes of the Scandinavian region, built ad-hoc during this study, to understand how moose utilise the landscape in Northern Europe. The 87Sr/86Sr isoscape was developed using a machine-learning approach with external geo-environmental predictors and literature data. Similarly, a δ18O isoscape, obtained from average annual precipitation δ18O values, was employed to highlight differences in the isotope composition of the local environment vs. bone/antler. Overall, 82% of the moose samples were compatible with the likely local isotope composition (n = 53), suggesting that they were shot not far from their year-round dwelling area. 'Local' samples were used to calibrate the two isoscapes, to improve the prediction of provenance for the presumably 'non-local' individuals. For the latter (n = 12, of which two are antlers and ten are bones), the probability of geographic origin was estimated using a Bayesian approach by combining the two isoscapes. Interestingly, two of these samples (one antler and one bone) seem to come from areas more than 250 km away from the place where the animals were hunted, indicating a possible remarkable intra-annual mobility. Finally, the δ13C data were compared with the forest cover of Sweden and ultimately used to understand the dietary preference of moose. We interpreted a difference in δ13C values of antlers (13C-enriched) and bones (13C-depleted) as a joint effect of seasonal variations in moose diet and, possibly, physiological stresses during winter-time, i.e., increased consumption of endogenous 13C-depleted lipids.

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.

Formation and Replacement of Bone and Tooth Mineralized Tissues in Green Iguanas (Iguana iguana) Revealed by In-Vivo Fluorescence Marking.

Hard tissue formation patterns and rates reveal details of animal physiology, life history, and environment, but are understudied in reptiles. Here, we use fluorescence labels delivered in vivo and laser confocal scanning microscopy to study tooth and bone formation in a managed group of green iguanas (Iguana iguana, Linné 1758) kept for 1.5 years under experimentally controlled conditions and undergoing several dietary switches. We constrain rates of tooth elongation, which we observe to be slow when enamel is initially deposited (c. 9 µm/day), but then increases exponentially in the dentin root, reaching c. 55 µm/day or more after crown completion. We further constrain the total timing of tooth formation to ∼40-60 days, and observe highly variable timings of tooth resorption onset and replacement. Fluorescent labels clearly indicate cohorts of teeth recruited within Zahnreihen replacement waves, with faster sequential tooth recruitment and greater wave sizes posteriorly, where each wave initiates. Fluorescence further reveals enamel maturation after initial deposition. Rates of hard tissue formation in long bones range from 0.4 to 3.4 µm/day, correlating with animal weight gain and cortical bone recording the entire history of the experiment. We suggest additional labeling experiments to study hard tissue formation patterns in other reptiles, and propose strategies for chemical analyses of hard tissues in order to extract temporal information about past environments, behaviors, and diets from reptilian fossils throughout the Phanerozoic.

Macroscopic dental measures in guinea pigs (Cavia porcellus) fed natural and pelleted diets of different abrasiveness: implications for wear and compensatory growth in a hypselodont species.

The drivers of dental wear and compensatory hypselodont tooth growth are of current research interest. Expanding previous macroscopic dental wear measurements based on microtomographic scans of guinea pigs (Cavia porcellus) fed natural diets, we added diet groups with different predicted drivers of dental wear and analysed how measured variables relate to each other. The teeth of guinea pigs fed either pelleted diets containing external abrasives of various shapes, sizes and percentages (n = 66) or natural whole-leaf diets (n = 36, low-phytolith lucerne or grass or high-phytolith bamboo) were evaluated. The bamboo-fed animals showed the lowest tooth height with deep dentine basins, similar to the pellet-fed animals. Deeper dentine basins generally correlated with higher occlusal surfaces, allowing the hypothesis that changes in the pressure signal due to lower basins could initiate compensatory growth and broadening of the whole tooth surface in hypselodont teeth. Macroscopic dental wear did not categorically differ between whole-leaf or pelleted diets or between diets with internal phytoliths or with external silicate abrasives. Supporting interpretations that tooth wear should be viewed as a response to the biomechanical properties of ingested feed which may or may not be aptly summarized by broad descriptors such as 'whole/pelleted' or 'natural/artificial'.

Tooth enamel nitrogen isotope composition records trophic position: a tool for reconstructing food webs.

Nitrogen isotopes are widely used to study the trophic position of animals in modern food webs; however, their application in the fossil record is severely limited by degradation of organic material during fossilization. In this study, we show that the nitrogen isotope composition of organic matter preserved in mammalian tooth enamel (δ15Nenamel) records diet and trophic position. The δ15Nenamel of modern African mammals shows a 3.7‰ increase between herbivores and carnivores as expected from trophic enrichment, and there is a strong positive correlation between δ15Nenamel and δ15Nbone-collagen values from the same individuals. Additionally, δ15Nenamel values of Late Pleistocene fossil teeth preserve diet and trophic level information, despite complete diagenetic loss of collagen in the same specimens. We demonstrate that δ15Nenamel represents a powerful geochemical proxy for diet that is applicable to fossils and can help delineate major dietary transitions in ancient vertebrate lineages.

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.

Evidence for the cooking of fish 780,000 years ago at Gesher Benot Ya'aqov, Israel.

Although cooking is regarded as a key element in the evolutionary success of the genus Homo, impacting various biological and social aspects, when intentional cooking first began remains unknown. The early Middle Pleistocene site of Gesher Benot Ya'aqov, Israel (marine isotope stages 18-20; ~0.78 million years ago), has preserved evidence of hearth-related hominin activities and large numbers of freshwater fish remains (>40,000). A taphonomic study and isotopic analyses revealed significant differences between the characteristics of the fish bone assemblages recovered in eight sequential archaeological horizons of Area B (Layer II-6 levels 1-7) and natural fish bone assemblages (identified in Area A). Gesher Benot Ya'aqov archaeological horizons II-6 L1-7 exhibited low fish species richness, with a clear preference for two species of large Cyprinidae (Luciobarbus longiceps and Carasobarbus canis) and the almost total absence of fish bones in contrast to the richness of pharyngeal teeth (>95%). Most of the pharyngeal teeth recovered in archaeological horizons II-6 L1-7 were spatially associated with 'phantom' hearths (clusters of burnt flint microartifacts). Size-strain analysis using X-ray powder diffraction provided evidence that these teeth had been exposed to low temperature (<500 °C), suggesting, together with the archaeological and taphonomic data, that the fish from the archaeological horizons of Area B had been cooked and consumed on site. This is the earliest evidence of cooking by hominins.

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.

Post-mortem enamel surface texture alteration during taphonomic processes-do experimental approaches reflect natural phenomena?

Experimental approaches are often used to better understand the mechanisms behind and consequences of post-mortem alteration on proxies for diet reconstruction. Dental microwear texture analysis (DMTA) is such a dietary proxy, using dental wear features in extant and extinct taxa to reconstruct feeding behaviour and mechanical food properties. In fossil specimens especially, DMTA can be biased by post-mortem alteration caused by mechanical or chemical alteration of the enamel surface. Here we performed three different dental surface alteration experiments to assess the effect of common taphonomic processes by simplifying them: (1) tumbling in sediment suspension to simulate fluvial transport, (2) sandblasting to simulate mechanical erosion due to aeolian sediment transport, (3) acid etching to simulate chemical dissolution by stomach acid. For tumbling (1) we found alteration to be mainly dependent on sediment grain size fraction and that on specimens tumbled with sand fractions mainly post-mortem scratches formed on the dental surface, while specimens tumbled with a fine-gravel fraction showed post-mortem formed dales. Sandblasting (2) with loess caused only negligible alteration, however blasting with fine sand quartz particles resulted in significant destruction of enamel surfaces and formation of large post-mortem dales. Acid etching (3) using diluted hydrochloric acid solutions in concentrations similar to that of predator stomachs led to a complete etching of the whole dental surface, which did not resemble those of teeth recovered from owl pellets. The experiments resulted in post-mortem alteration comparable, but not identical to naturally occurring post-mortem alteration features. Nevertheless, this study serves as a first assessment and step towards further, more refined taphonomic experiments evaluating post-mortem alteration of dental microwear texture (DMT).

A simple CO2 equilibration method for measuring blood oxygen isotope compositions.

RATIONALE: Blood water oxygen isotope compositions can provide valuable insights into physiological processes and ecological patterns. While blood samples are commonly drawn for medical or scientific purposes, blood fractions are infrequently measured for oxygen isotopic compositions (δ18 O) because such measurements are time consuming and expensive. METHODS: We sampled blood from sheep, goats, and iguanas raised in field and animal laboratories into serum, EDTA, heparin, and uncoated plastic vials commonly used in medical and scientific research, then separated red blood cell (RBC) and plasma or serum blood fractions. These were injected into helium-flushed Exetainer tubes where they naturally outgassed endogenous CO2 (goat blood), or into He- and CO2 -flushed tubes (iguana blood). The CO2 gas was sampled on a GasBench II system, and δ18 O was measured by an isotope ratio mass spectrometer (IRMS). RESULTS: Repeated δ18 O measurements were stable over multiple days. The addition of desiccated blood solids to water standards had little impact on their δ18 O measurements, suggesting that organic molecular constituents within blood serum and plasma do not interfere with blood water δ18 O values. We observed slight but statistically significant δ18 O offsets between plasma, serum and RBC fractions. Mass-dependent body water turnover times for iguanas were derived from the data. CONCLUSIONS: We demonstrate that a simple blood-CO2 equilibration method using the GasBench can quickly, reliably and accurately characterize water δ18 O in the plasma, RBC, and whole blood fractions of mammalian and reptilian blood samples (precision ≤ 0.1‰). This method will expand the application of blood stable isotope analysis in physiological and medical research.

Digestion of bamboo compared to grass and lucerne in a small hindgut fermenting herbivore, the guinea pig (Cavia porcellus).

Bamboo is an enigmatic forage, representing a niche food for pandas and bamboo lemurs. Bamboo might not represent a suitable forage for herbivores relying on fermentative digestion, potentially due to its low fermentability. To test this hypothesis, guinea pigs (n = 36) were used as model species and fed ad libitum with one of three forages (bamboo, lucerne, or timothy grass) in a fresh or dried state, with six individuals per group, for 3 weeks. The nutrient composition and in vitro fermentation profile of bamboo displayed low fermentation potential, i.e. high lignin and silica levels together with a gas production (Hohenheim gas test) at 12 h of only 36% of that of lucerne and grass. Although silica levels were more abundant in the leftovers of (almost) all groups, guinea pigs did not select against lignin on bamboo. Dry matter (DM) intake was highest and DM digestibility lowest on the bamboo forage. Total short-chain fatty acid levels in caecal content were highest for lucerne and lowest for grass and bamboo. Bamboo-fed guinea pigs had a lower body weight gain than the grass and lucerne group. The forage hydration state did not substantially affect digestion, but dry forage led to a numerically higher total wet gut fill. Although guinea pigs increased DM intake on the bamboo diet, the negative effects on fermentation of lignin and silica in bamboo seemed overriding. For herbivores that did not evolutionary adapt, bamboo as an exclusive food resource can be considered as inadequate.

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.

Use of running plates by floor housed rats: A pilot study.

The outfit of husbandry facilities of, and the enrichment provided for, experimental rodents plays an important role in the animals' welfare, and hence also for the societal acceptance of animal experiments. Whether rats and mice benefit from being provided with running wheels or plates is discussed controversially. Here we present observations from a feeding experiment, where rats were provided a running plate. As a pilot study, six identical cages, with three animals per cage, were filmed for six days, and the resulting footage was screened for the number of bouts and the time the animals spent on the plates. The main activities observed on the plate in descending order were sitting (18.5 ± 13.8 bouts or 8.0 ± 13.7 min/animal per day), standing (10.2 ± 11.6 bouts, 3.8 ± 4.2 min), running (8.2 ± 13.3 bouts, 10.1 ± 21.4 min), grooming (2.0 ± 2.8 bouts, 6.7 ± 25.7 min), sleeping (1.0 ± 2.6 bouts, 24.0 ± 61.8 min) and playing (0.5 ± 0.9 bouts, 0.1 ± 0.5 min). Most of these activities (91% of all bouts, 90% of total time) occurred at night, similar to previous studies on running wheel usage. The running plate seems well-accepted as cage enrichment, even though in further studies, the motivating triggers and the effects of long-term use could be evaluated more in-depth.

Dental microwear texture gradients in guinea pigs reveal that material properties of the diet affect chewing behaviour.

Dental microwear texture analysis (DMTA) is widely used for diet inferences in extant and extinct vertebrates. Often, a reference tooth position is analysed in extant specimens, while isolated teeth are lumped together in fossil datasets. It is therefore important to test whether dental microwear texture (DMT) is tooth position specific and, if so, what causes the differences in wear. Here, we present results from controlled feeding experiments with 72 guinea pigs, which received either fresh or dried natural plant diets of different phytolith content (lucerne, grass, bamboo) or pelleted diets with and without mineral abrasives (frequently encountered by herbivorous mammals in natural habitats). We tested for gradients in dental microwear texture along the upper cheek tooth row. Regardless of abrasive content, guinea pigs on pelleted diets displayed an increase in surface roughness along the tooth row, indicating that posterior tooth positions experience more wear compared with anterior teeth. Guinea pigs feedings on plants of low phytolith content and low abrasiveness (fresh and dry lucerne, fresh grass) showed almost no DMT differences between tooth positions, while individuals feeding on more abrasive plants (dry grass, fresh and dry bamboo) showed a gradient of decreasing surface roughness along the tooth row. We suggest that plant feeding involves continuous intake and comminution by grinding, resulting in posterior tooth positions mainly processing food already partly comminuted and moistened. Pelleted diets require crushing, which exerts higher loads, especially on posterior tooth positions, where bite forces are highest. These differences in chewing behaviour result in opposing wear gradients for plant versus pelleted diets.

Shape, size, and quantity of ingested external abrasives influence dental microwear texture formation in guinea pigs.

Food processing wears down teeth, thus affecting tooth functionality and evolutionary success. Other than intrinsic silica phytoliths, extrinsic mineral dust/grit adhering to plants causes tooth wear in mammalian herbivores. Dental microwear texture analysis (DMTA) is widely applied to infer diet from microscopic dental wear traces. The relationship between external abrasives and dental microwear texture (DMT) formation remains elusive. Feeding experiments with sheep have shown negligible effects of dust-laden grass and browse, suggesting that intrinsic properties of plants are more important. Here, we explore the effect of clay- to sand-sized mineral abrasives (quartz, volcanic ash, loess, kaolin) on DMT in a controlled feeding experiment with guinea pigs. By adding 1, 4, 5, or 8% mineral abrasives to a pelleted base diet, we test for the effect of particle size, shape, and amount on DMT. Wear by fine-grained quartz (>5/<50 µm), loess, and kaolin is not significantly different from the abrasive-free control diet. Fine silt-sized quartz (∼5 µm) results in higher surface anisotropy and lower roughness (polishing effect). Coarse-grained volcanic ash leads to significantly higher complexity, while fine sands (130 to 166 µm) result in significantly higher roughness. Complexity and roughness values exceed those from feeding experiments with guinea pigs who received plants with different phytolith content. Our results highlight that large (>95-µm) external silicate abrasives lead to distinct microscopic wear with higher roughness and complexity than caused by mineral abrasive-free herbivorous diets. Hence, high loads of mineral dust and grit in natural diets might be identified by DMTA, also in the fossil record.

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.