Stable isotope evidence of human diet in Mediterranean context during the Last Glacial Maximum.

The Final Gravettian in Europe overlapped with the cold and dry climatic event of Heinrich 2 (ca. 27-23.5 kyr cal BP), which caused the contraction of human distribution over refuge regions in the southern peninsulas of Europe. Here, we consider the human subsistence in the northeast Iberian Peninsula, where an extensive range of small to large prey was available. Four human remains from the Serinyà caves were investigated using the stable isotope ratios of carbon, nitrogen, and sulfur of bulk collagen (δ13Ccoll, δ15Ncoll, δ34Scoll) and of phenylalanine and glutamine amino acids (δ15NPhe, δ15NGlu). Direct AMS dating of the human and animal remains from the Final Gravettian levels of Mollet III, Reclau Viver, and Arbreda at Serinyà confirmed their chronological position from 27.5 to 22.6 kyr cal BP and the occurrence of four different human individuals. The δ13Ccoll and δ15Ncoll values showed a large contribution of terrestrial prey to the dietary protein of the individuals. The δ34Scoll values were consistent with a subsistence based on the local continental resources, without detectable contribution of marine resource. The δ15NPhe and δ15NGlu values confirm that freshwater resources were not a substantial component of the diet of the considered individuals. Contrast in the isotopic amounts in bulk collagen could be interpreted as the result of different proportions of terrestrial prey in human diet at Serinyà. Altogether, the isotopic investigation reveals the importance of terrestrial over aquatic resources in the subsistence of the studied Final Gravettian individuals from the Serinyà caves in northeastern Iberia during the Last Glacial Maximum. It would be consistent with a scenario of a productive enough terrestrial ecosystem to sustain hunter-gatherer subsistence in this refuge region.

A protocol for using attenuated total reflection Fourier-transform infrared spectroscopy for pre-screening ancient bone collagen prior to radiocarbon dating.

RATIONALE: Pre-screening of bone collagen quality is important to reduce the cost for analyses such as radiocarbon (14 C) dating with accelerator mass spectrometry in archaeological studies. We developed a pre-screening protocol based on attenuated total reflection (ATR) Fourier-transform infrared spectroscopy (FTIR) for assessing the chemical composition and mineralogy of ancient bone samples. METHODS: ATR-FTIR measurements were performed on bulk bones of diverse origin and age before collagen extraction. The percentage nitrogen of bulk bones, as well as the weight percentage, and the percentage carbon and nitrogen of extracted organic matter were noted. Several machine learning algorithms were applied to the spectral data and compared for their efficacy in screening for well preserved collagen. RESULTS: The results showed that (a) the first derivative of the spectral data was better suited to screening than the raw FTIR data, especially for a wider spectral range and (b) certain classification algorithms [e.g. gradient boosting machine (GBM)] were able to efficiently predict the degree of preservation in bone samples. CONCLUSIONS: This pre-screening protocol can serve as a fast, concise and inexpensive pre-screening tool for determining relative degrees of preservation before collagen extraction and subsequent 14 C dating. The screening power based on the machine learning techniques can be further improved by accumulating the FTIR spectral data of bones.

Changes in the Pharmacokinetics of Phenytoin in Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice.

We previously demonstrated that the expression levels of drug-metabolizing enzymes, cytochrome P450 (CYP) enzymes, in the liver are significantly decreased in a murine model of ulcerative colitis (UC). In this study, we investigated changes in the pharmacokinetics of phenytoin, a CYP2C substrate drug, in the presence of UC. Colitis was induced by feeding male mice 3.5% dextran sulfate sodium (DSS) dissolved in drinking water for 10 days. The messenger RNA (mRNA) expression of CYP2C29 and CYP2C37 and the protein expression of CYP2C in the liver were evaluated via real-time reverse transcription-polymerase chain reaction and Western blotting, respectively. In DSS-treated animals, both mRNA and protein expression levels of CYP2C in the liver were significantly reduced relative to those in control animals (by 20%-40%). Phenytoin (30 mg/kg) was administered orally in a single dose to mice, and plasma concentrations were measured. Plasma concentrations of phenytoin were higher in the DSS-treated group than in the control group at 12, 24, and 36 hours after administration. Animals given DSS also exhibited a higher area under the plasma concentration-time curve extrapolated to infinity (AUCinf, 315 µg·h/mL), a delayed elimination half-life ( T1/2, 8.1 hours), and a decreased body clearance (CL/F, 3.52 mL/h) compared with that of control animals (AUCinf, 215 µg·h/mL; T1/2, 3.6 h; CL/F, 5.58 mL/h). This study indicated that the presence of UC decreases CYP2C expression levels in the liver, thereby delaying the metabolism of CYP2C substrates, including phenytoin, and increasing blood concentrations of these substrates.

Ecological niche of Neanderthals from Spy Cave revealed by nitrogen isotopes of individual amino acids in collagen.

This study provides a refined view on the diet and ecological niche of Neanderthals. The traditional view is that Neanderthals obtained most of their dietary protein from terrestrial animals, especially from large herbivores that roamed the open landscapes. Evidence based on the conventional carbon and nitrogen isotopic composition of bulk collagen has supported this view, although recent findings based on plant remains in the tooth calculus, microwear analyses, and small game and marine animal remains from archaeological sites have raised some questions regarding this assumption. However, the lack of a protein source other than meat in the Neanderthal diet may be due to methodological difficulties in defining the isotopic composition of plants. Based on the nitrogen isotopic composition of glutamic acid and phenylalanine in collagen for Neanderthals from Spy Cave (Belgium), we show that i) there was an inter-individual dietary heterogeneity even within one archaeological site that has not been evident in bulk collagen isotopic compositions, ii) they occupied an ecological niche different from those of hyenas, and iii) they could rely on plants for up to ∼20% of their protein source. These results are consistent with the evidence found of plant consumption by the Spy Neanderthals, suggesting a broader subsistence strategy than previously considered.

Variation in vegetative morphology tracks the complex genetic diversification of the mycoheterotrophic species Pyrola japonica sensu lato.

PREMISE OF THE STUDY: Although the evolution of full mycoheterotrophy has attracted many plant researchers, molecular phylogenetic studies that focus on the transition from partial to full mycoheterotrophy are limited to a few taxa. Pyrola japonica sensu lato is an ideal model for examining the evolution of mycoheterotrophy, owing to its variable leaf size, which suggests that the species comprises several transitional stages. METHODS: To elucidate the molecular and morphological changes that occur during the evolutionary transition between partial and full mycoheterotrophy in P. japonica s.l. from 18 populations in Japan, we estimated a parsimony network of plastid haplotypes based on three noncoding regions, measured the leaf size and scape color of the shoots, and compared morphology among haplotypes. KEY RESULTS: The seven haplotypes exhibited star-like relationships, and at least three divergent haplotypes were associated with differences in morphology. The first was mainly observed in large-leaved and green-scaped populations, whereas the second was observed in extremely small-leaved and reddish-scaped populations, which indicated a high degree of mycoheterotrophy, and the last was detected among mixed populations with both green- and reddish-scaped shoots with intermediate leaf sizes. In addition, the inconsistent association between the haplotypes and morphology suggests a complex relationship. CONCLUSIONS: Pyrola japonica s.l. has at least three separate genetic lineages that have different leaf morphologies. The genetic lineages and their coexistence could have led to the variable leaf size and suggest the possibility that gene flow from partial to full mycoheterotrophs could reverse the evolutionary transition to full mycoheterotrophy.

Association between sex inequality in animal protein intake and economic development in the Papua New Guinea highlands: The carbon and nitrogen isotopic composition of scalp hair and fingernail.

OBJECTIVES: People in the Papua New Guinea Highlands consume sweet potatoes as their dietary staple; consumption of animal protein is limited. In such societies with marginal protein intake, the intra-household allocation of animal protein in terms of sex or age is of importance. The objective of this study was to investigate how the allocation pattern of protein-rich foods by sex and age is associated with economic development in the Papua New Guinea Highlands. METHODS: The carbon and nitrogen isotopic compositions of fingernails, collected in 1995 in two areas (Tari and Port Moresby [the national capital where Tari migrants resided]), and of scalp hair, collected in 2007, 2012, and 2013 in three areas of different degree of economic development (Levani, Tari, and Goroka) were analyzed. RESULTS: Analysis of fingernail samples showed that δ(15)N was lower in rural communities than in the urban migrant community, while a sex difference in δ(15)N (higher in males than in females) was found in the former but not in the latter community. Age was not associated with either δ(15)N or δ(13)C values. The analysis of scalp hair samples showed that δ(15)N values were lowest in Levani, the least developed area. Furthermore, there were statistically significant sex differences in δ(15)N values in Levani but not in Tari and Goroka. Age was not associated with either δ(15)N or δ(13)C values. DISCUSSION: The sex inequality in animal protein consumption seems to have decreased as the communities in the Papua New Guinea Highlands have experienced economic development.

Association of protein intakes and variation of diet-scalp hair nitrogen isotopic discrimination factor in Papua New Guinea highlanders.

OBJECTIVES: We present new nitrogen isotopic discrimination factor between diets and scalp hairs (Δ(15) NHair-Diet : δ(15) NHair - δ(15) NDiet ) for indigenous residents in three communities in the Papua New Guinea Highlands who consumed various amounts and qualities of protein. The Δ(15) N is important for precise evaluation of the dietary habits of human populations; in both contemporary and traditional lifestyles. Several hypotheses have been proposed regarding factors that affect Δ(15) N values, based largely on observations from animal feeding experiments. However, variations and factors controlling Δ(15) N in humans are not well understood, mainly due to the difficulty of controlling the diets of participants. MATERIALS AND METHODS: These residents were studied because they have maintained relatively traditional dietary habits, which allow quantitative recording of diets. Δ(15) N was estimated by comparing hair δ(15) N values to mean dietary δ(15) N values calculated from the recorded intake of each food item and their δ(15) N values. RESULTS: The results showed that: i) there was a significant difference in Δ(15) N among study locations (3.9 ± 0.9‰ for most urbanized, 5.2 ± 1.0‰ for medium and 5.0 ± 0.9‰ for least urbanized communities; range = 1.2-7.3‰ for all participants); and ii) estimated Δ(15) N values were negatively correlated with several indicators of animal protein intake (% nitrogen in diet: range = 0.9-7.6%). DISCUSSION: We hypothesize that a combination of several factors, which presumably included urea recycling and amino acid and protein recycling and/or de novo synthesis during metabolic processes, altered the Δ(15) N values of the participants.

Quantitative evaluation of marine protein contribution in ancient diets based on nitrogen isotope ratios of individual amino acids in bone collagen: an investigation at the Kitakogane Jomon site.

Nitrogen stable isotopes analysis of individual bone collagen amino acids was applied to archeological samples as a new tool for assessing the composition of ancient human diets and calibrating radiocarbon dates. We used this technique to investigate human and faunal samples from the Kitakogane shell midden in Hokkaido, Japan (5,300-6,000 cal BP). Using compound-specific nitrogen isotope analysis of individual amino acids, we aimed to estimate i) the quantitative contribution of marine and terrestrial protein to the human diet, and ii) the mean trophic level (TL) from which dietary protein was derived from marine ecosystems. Data were interpreted with reference to the amino acid trophic level (TL(AA)) model, which uses empirical amino acid delta(15)N from modern marine fauna to construct mathematical equations that predict the trophic position of organisms. The TL(AA) model produced realistic TL estimates for the Kitakogane marine animals. However, this model was not appropriate for the interpretation of human amino acid delta(15)N, as dietary protein is derived from both marine and terrestrial environments. Hence, we developed a series of relevant equations that considered the consumption of dietary resources from both ecosystems. Using these equations, the mean percentage of marine protein in the Kitakogane human diet was estimated to be 74%. Although this study is one of the first systematic investigations of amino acid delta(15)N in archeological bone collagen, we believe that this technique is extremely useful for TL reconstruction, palaeodietary interpretation, and the correction of marine reservoir effects for radiocarbon dating.

Author Correction: Heavy reliance on plants for Romanian cave bears evidenced by amino acid nitrogen isotope analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Heavy reliance on plants for Romanian cave bears evidenced by amino acid nitrogen isotope analysis.

Heavy reliance on plants is rare in Carnivora and mostly limited to relatively small species in subtropical settings. The feeding behaviors of extinct cave bears living during Pleistocene cold periods at middle latitudes have been intensely studied using various approaches including isotopic analyses of fossil collagen. In contrast to cave bears from all other regions in Europe, some individuals from Romania show exceptionally high δ15N values that might be indicative of meat consumption. Herbivory on plants with high δ15N values cannot be ruled out based on this method, however. Here we apply an approach using the δ15N values of individual amino acids from collagen that offsets the baseline δ15N variation among environments. The analysis yielded strong signals of reliance on plants for Romanian cave bears based on the δ15N values of glutamate and phenylalanine. These results could suggest that the high variability in bulk collagen δ15N values observed among cave bears in Romania reflects niche partitioning but in a general trophic context of herbivory.

Stable isotopes reveal patterns of diet and mobility in the last Neandertals and first modern humans in Europe.

Correlating cultural, technological and ecological aspects of both Upper Pleistocene modern humans (UPMHs) and Neandertals provides a useful approach for achieving robust predictions about what makes us human. Here we present ecological information for a period of special relevance in human evolution, the time of replacement of Neandertals by modern humans during the Late Pleistocene in Europe. Using the stable isotopic approach, we shed light on aspects of diet and mobility of the late Neandertals and UPMHs from the cave sites of the Troisième caverne of Goyet and Spy in Belgium. We demonstrate that their diet was essentially similar, relying on the same terrestrial herbivores, whereas mobility strategies indicate considerable differences between Neandertal groups, as well as in comparison to UPMHs. Our results indicate that UPMHs exploited their environment to a greater extent than Neandertals and support the hypothesis that UPMHs had a substantial impact not only on the population dynamics of large mammals but also on the whole structure of the ecosystem since their initial arrival in Europe.

Isotopic analyses suggest mammoth and plant in the diet of the oldest anatomically modern humans from far southeast Europe.

Relatively high 15N abundances in bone collagen of early anatomically modern humans in Europe have often been interpreted as a specific consumption of freshwater resources, even if mammoth is an alternative high 15N prey. At Buran-Kaya III, access to associated fauna in a secured archaeological context and application of recently developed isotopic analyses of individuals amino acids offer the opportunity to further examine this hypothesis. The site of Buran-Kaya III is located in south Crimea and has provided a rich archaeological sequence including two Upper Palaeolithic layers, from which human fossils were retrieved and directly dated as from 37.8 to 33.1 ka cal BP. Results from bulk collagen of three human remains suggests the consumption of a high 15N prey besides the contribution of saiga, red deer, horse and hare, whose butchered remains were present at the site. In contrast to bulk collagen, phenylalanine and glutamic acid 15N abundances reflect not only animal but also plant protein contributions to omnivorous diet, and allow disentangling aquatic from terrestrial resource consumption. The inferred human trophic position values point to terrestrial-based diet, meaning a significant contribution of mammoth meat, in addition to a clear intake of plant protein.

A High Burden of Asymptomatic Gastrointestinal Infections in Traditional Communities in Papua New Guinea.

Stool samples were collected from 148 healthy adults living a traditional subsistence lifestyle in Papua New Guinea and screened for enteric pathogens using real-time RT-PCR/PCR assays. Enteric pathogens were detected in a high proportion (41%) of individuals. Clear differences were observed in the detection of pathogens between highland and lowland communities. In particular, there was a marked difference in detection rates of norovirus GII (20% and 0%, respectively) and Shigella sp. (15% and 0%, respectively). Analysis of the relationship between enteric pathogen carriage and microbial community composition of participants, using box plots to compare specific normal flora population numbers, did not suggest that gut microbial composition was directly associated with pathogen carriage. This study suggests that enteric pathogens are common in healthy individuals in Papua New Guinean highland communities, presumably acting as a reservoir of infection and thus contributing to a high burden of gastrointestinal illnesses.

Characterization of the gut microbiota of Papua New Guineans using reverse transcription quantitative PCR.

There has been considerable interest in composition of gut microbiota in recent years, leading to a better understanding of the role the gut microbiota plays in health and disease. Most studies have been limited in their geographical and socioeconomic diversity to high-income settings, and have been conducted using small sample sizes. To date, few analyses have been conducted in low-income settings, where a better understanding of the gut microbiome could lead to the greatest return in terms of health benefits. Here, we have used quantitative real-time polymerase chain reaction targeting dominant and sub-dominant groups of microorganisms associated with human gut microbiome in 115 people living a subsistence lifestyle in rural areas of Papua New Guinea. Quantification of Clostridium coccoides group, C. leptum subgroup, C. perfringens, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus, and Lactobacillus spp. was conducted. Principle coordinates analysis (PCoA) revealed two dimensions with Prevotella, clostridia, Atopobium, Enterobacteriaceae, Enterococcus and Staphylococcus grouping in one dimension, while B. fragilis, Bifidobacterium and Lactobacillus grouping in the second dimension. Highland people had higher numbers of most groups of bacteria detected, and this is likely a key factor for the differences revealed by PCoA between highland and lowland study participants. Age and sex were not major determinants in microbial population composition. The study demonstrates a gut microbial composition with some similarities to those observed in other low-income settings where traditional diets are consumed, which have previously been suggested to favor energy extraction from a carbohydrate rich diet.