Reconstructing herbivore diets: a multivariate statistical approach to interpreting amino acid nitrogen isotope values.

Recent studies have demonstrated that there exists significant variability in amino acid (AA) δ15N values of terrestrial plants, discriminating among plant types (i.e., legume seeds, grasses, tree leaves) as well as tissues of the same plant. For the first time, we investigate the potential of the spacing between the δ15N values of different AAs to differentiate between plant types and thus elucidate their relative importance in herbivore diet. Using principal component analysis, we show that it is possible to distinguish among five plant categories-cereal grains, rachis, legume seeds, herbaceous plants, and woody plants-whose consumption has different implications for understanding herbivore ecology and management practices. Furthermore, we were able to correctly classify the herbaceous plant diet of modern cattle using AA δ15N values of their tooth dentine adjusted for trophic enrichment. The AA δ15N patterns of wild and domestic herbivores from archaeological sites seem to be consistent with diets comprised predominantly of herbaceous plants, but there is variation in AA δ15N values among individuals that may reflect differing inputs of other plant types. The variation in AA δ15N values does not necessarily reflect the variation in herbivore bulk collagen δ13C and δ15N values, indicating that AA δ15N values have the potential to provide additional insights into plant dietary sources compared to bulk tissue isotope values alone. Future work should focus on defining trophic enrichment factors for a wider range of terrestrial herbivores and expanding libraries of primary producer AA δ15N values.

Dairying, diseases and the evolution of lactase persistence in Europe.

In European and many African, Middle Eastern and southern Asian populations, lactase persistence (LP) is the most strongly selected monogenic trait to have evolved over the past 10,000 years1. Although the selection of LP and the consumption of prehistoric milk must be linked, considerable uncertainty remains concerning their spatiotemporal configuration and specific interactions2,3. Here we provide detailed distributions of milk exploitation across Europe over the past 9,000 years using around 7,000 pottery fat residues from more than 550 archaeological sites. European milk use was widespread from the Neolithic period onwards but varied spatially and temporally in intensity. Notably, LP selection varying with levels of prehistoric milk exploitation is no better at explaining LP allele frequency trajectories than uniform selection since the Neolithic period. In the UK Biobank4,5 cohort of 500,000 contemporary Europeans, LP genotype was only weakly associated with milk consumption and did not show consistent associations with improved fitness or health indicators. This suggests that other reasons for the beneficial effects of LP should be considered for its rapid frequency increase. We propose that lactase non-persistent individuals consumed milk when it became available but, under conditions of famine and/or increased pathogen exposure, this was disadvantageous, driving LP selection in prehistoric Europe. Comparison of model likelihoods indicates that population fluctuations, settlement density and wild animal exploitation-proxies for these drivers-provide better explanations of LP selection than the extent of milk exploitation. These findings offer new perspectives on prehistoric milk exploitation and LP evolution.

Quinoa, potatoes, and llamas fueled emergent social complexity in the Lake Titicaca Basin of the Andes.

The Lake Titicaca basin was one of the major centers for cultural development in the ancient world. This lacustrine environment is unique in the high, dry Andean altiplano, and its aquatic and terrestrial resources are thought to have contributed to the florescence of complex societies in this region. Nevertheless, it remains unclear to what extent local aquatic resources, particularly fish, and the introduced crop, maize, which can be grown in regions along the lakeshores, contributed to facilitating sustained food production and population growth, which underpinned increasing social political complexity starting in the Formative Period (1400 BCE to 500 CE) and culminating with the Tiwanaku state (500 to 1100 CE). Here, we present direct dietary evidence from stable isotope analysis of human skeletal remains spanning over two millennia, together with faunal and floral reference materials, to reconstruct foodways and ecological interactions in southern Lake Titicaca over time. Bulk stable isotope analysis, coupled with compound-specific amino acid stable isotope analysis, allows better discrimination between resources consumed across aquatic and terrestrial environments. Together, this evidence demonstrates that human diets predominantly relied on C3 plants, particularly quinoa and tubers, along with terrestrial animals, notably domestic camelids. Surprisingly, fish were not a significant source of animal protein, but a slight increase in C4 plant consumption verifies the increasing importance of maize in the Middle Horizon. These results underscore the primary role of local terrestrial food resources in securing a nutritious diet that allowed for sustained population growth, even in the face of documented climate and political change across these periods.

Determination of Arginine δ15N Values in Plant and Animal Proteins by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry.

Nitrogen (N) stable isotope techniques are widely used in ecology, archaeology, and forensic science to explore trophic relationships and provenances of organisms and materials, most widely using bulk δ15N values of whole organisms, tissues, or other materials. However, compound-specific isotope values can provide more diagnostic isotope "fingerprints" and specific information about metabolic processes. Existing techniques for nitrogen isotope analysis allow the determination of δ15N values of 14 amino acids (AAs), accounting for ca. 75% of plant protein and collagen N. The majority of remaining N is from arginine, comprising 16 and 14% of collagen and plant protein N, respectively. We therefore aimed to develop a method to detect arginine and determine its δ15N value (δ15NArg) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), to further contribute to the understanding of the metabolic routing of this important AA. We demonstrate that arginine, as its N-acetyl isopropyl ester, is amenable to GC analysis using a 15 m midpolarity DB-35 column, eluting with baseline resolution from other AAs. The recorded δ15N value by GC-C-IRMS was within the error of that of the underivatized compound determined by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). The newly developed GC-C-IRMS method was applied to modern plant protein and cattle collagen, enabling their δ15NArg values to be related to AA biosynthesis. Determination of archaeological cattle collagen δ15NArg values confirmed the suitability of this method to provide further insights into past diets and ecosystems. Bulk collagen δ15N value reconstruction including δ15NArg values better reflect the measured bulk values, as the isotopic ratio of 91% of collagen N can now be determined at the compound-specific level.

Compound-specific δ15N values express differences in amino acid metabolism in plants of varying lignin content.

Amino acid δ15N values of foliage of various plant taxa, grown at the experimental farm stations of North Wyke, UK and Bad Lauchstädt, Germany were determined by GC-C-IRMS. The difference between δ15N values of glutamate (Glx) and phenylalanine (Phe) were found to differ significantly between woody and herbaceous plants, with mean Δ15NGlx-Phe (i.e. δ15NPhe - δ15NGlx) values of -9.3 ±â€¯1.6‰ and -5.8 ±â€¯2.1‰, respectively. These differences in values are hypothesised to be due to the involvement of Phe in the phenylpropanoid pathway, by which lignin and other phenolic secondary metabolites are produced, leading to isotopic fractionation and enrichment of the remaining Phe pool available for protein biosynthesis. This results in the more negative Δ15NGlx-Phe values observed in woody plants relative to herbaceous plants, as the former are assumed to produce more lignin. To test this assumption, plant leaf tissue lignin concentrations were estimated by solid state 13C cross-polarisation, magic-angle-spinning (CPMAS) NMR spectroscopy for a subset of plants, which showed that tree foliage has a higher concentration of lignin (12.6 wt%) than herbaceous foliage (6.3 wt%). The correlation of lignin concentration with Δ15NGlx-Phe values demonstrates that the difference in these values with plant type is indeed due to differential production of lignin. The ability to estimate the lignin content of plants from amino acid δ15N values will, to give one example, allow refinement of estimates of herbivore diet in present and past ecosystems, enabling more accurate environmental niche modelling.