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.

Urban form and scale shaped the agroecology of early 'cities' in northern Mesopotamia, the Aegean and Central Europe.

Agricultural extensification refers to an expansive, low-input production strategy that is land rather than labour limited. Here, we present a robust method, using the archaeological proxies of cereal grain nitrogen isotope values and settlement size, to investigate the relationship between agricultural intensity and population size at Neolithic to Bronze/Iron Age settlement sites in northern Mesopotamia, the Aegean and south-west Germany. We conclude that urban form-in particular, density of occupation-as well as scale shaped the agroecological trajectories of early cities. Whereas high-density urbanism in northern Mesopotamia and the Aegean entailed radical agricultural extensification, lower density urbanism in south-west Germany afforded more intensive management of arable land. We relate these differing agricultural trajectories to long-term urban growth/collapse cycles in northern Mesopotamia and the Aegean, on the one hand, and to the volatility of early Iron Age elite power structures and urban centralization in south-west Germany, on the other.

Crop manuring and intensive land management by Europe's first farmers.

The spread of farming from western Asia to Europe had profound long-term social and ecological impacts, but identification of the specific nature of Neolithic land management practices and the dietary contribution of early crops has been problematic. Here, we present previously undescribed stable isotope determinations of charred cereals and pulses from 13 Neolithic sites across Europe (dating ca. 5900-2400 cal B.C.), which show that early farmers used livestock manure and water management to enhance crop yields. Intensive manuring inextricably linked plant cultivation and animal herding and contributed to the remarkable resilience of these combined practices across diverse climatic zones. Critically, our findings suggest that commonly applied paleodietary interpretations of human and herbivore δ(15)N values have systematically underestimated the contribution of crop-derived protein to early farmer diets.

Practical considerations in the determination of compound-specific amino acid δ15N values in animal and plant tissues by gas chromatography-combustion-isotope ratio mass spectrometry, following derivatisation to their N-acetylisopropyl esters.

RATIONALE: Stable nitrogen isotope (δ(15)N) values of bone collagen are routinely used to inform interpretations of diet and trophic positions within contemporary and ancient ecosystems, yet the underlying physiological and biochemical factors which contribute to the bulk collagen δ(15)N value remain little understood. Determination of individual amino acid (AA) δ(15)N values in animal and plant proteins can help to elucidate the cycling of nitrogen and inform predictions of palaeodiet and ecology. METHODS: In this study we present a methodology for the measurement of amino acid δ(15)N values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Amino acid standards of known δ(15)N values were derivatised to their N-acetylisopropyl (NAIP) esters and purified through Dowex ion-exchange resin to determine any isotopic fractionation associated with derivatisation and ion-exchange chromatography. The effect of starch on AA δ(15)N values was also determined by hydrolysing bone collagen with and without the presence of starch. RESULTS: The amino acids derivatised to their NAIP esters give values within ±0.8‰ of their δ(15)N values measured separately by elemental analyser (EA)-IRMS, with a precision of better than 0.8‰. The δ(15)N values of AAs after Dowex ion-exchange chromatography were within ±0.9‰ of their values prior to ion-exchange chromatography. The AA δ(15)N values of bone collagen hydrolysed with and without starch were within ±0.8‰. CONCLUSIONS: Hydrolysis of lipid-extracted plant material followed by purification of AAs using Dowex ion-exchange resin and derivatisation to their NAIP esters is a suitable protocol for the accurate determination of individual plant and animal AA δ(15)N values by GC-C-IRMS.

Polyphenols: Bioavailability, Microbiome Interactions and Cellular Effects on Health in Humans and Animals.

Polyphenolic compounds have a variety of functions in plants including protecting them from a range of abiotic and biotic stresses such as pathogenic infections, ionising radiation and as signalling molecules. They are common constituents of human and animal diets, undergoing extensive metabolism by gut microbiota in many cases prior to entering circulation. They are linked to a range of positive health effects, including anti-oxidant, anti-inflammatory, antibiotic and disease-specific activities but the relationships between polyphenol bio-transformation products and their interactions in vivo are less well understood. Here we review the state of knowledge in this area, specifically what happens to dietary polyphenols after ingestion and how this is linked to health effects in humans and animals; paying particular attention to farm animals and pigs. We focus on the chemical transformation of polyphenols after ingestion, through microbial transformation, conjugation, absorption, entry into circulation and uptake by cells and tissues, focusing on recent findings in relation to bone. We review what is known about how these processes affect polyphenol bioactivity, highlighting gaps in knowledge. The implications of extending the use of polyphenols to treat specific pathogenic infections and other illnesses is explored.

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.

Isotope evidence for agricultural extensification reveals how the world's first cities were fed.

This study sheds light on the agricultural economy that underpinned the emergence of the first urban centres in northern Mesopotamia. Using δ13C and δ15N values of crop remains from the sites of Tell Sabi Abyad, Tell Zeidan, Hamoukar, Tell Brak and Tell Leilan (6500-2000 cal bc), we reveal that labour-intensive practices such as manuring/middening and water management formed an integral part of the agricultural strategy from the seventh millennium bc. Increased agricultural production to support growing urban populations was achieved by cultivation of larger areas of land, entailing lower manure/midden inputs per unit area-extensification. Our findings paint a nuanced picture of the role of agricultural production in new forms of political centralization. The shift towards lower-input farming most plausibly developed gradually at a household level, but the increased importance of land-based wealth constituted a key potential source of political power, providing the possibility for greater bureaucratic control and contributing to the wider societal changes that accompanied urbanization.

Cereal grain, rachis and pulse seed amino acid δ15N values as indicators of plant nitrogen metabolism.

Natural abundance δ(15)N values of plant tissue amino acids (AAs) reflect the cycling of N into and within plants, providing an opportunity to better understand environmental and anthropogenic effects on plant metabolism. In this study, the AA δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. It was found that the δ(15)N values of cereal grain and rachis AAs could be largely attributed to metabolic pathways involved in their biosynthesis and catabolism. The relative (15)N-enrichment of phenylalanine can be attributed to its involvement in the phenylpropanoid pathway and glutamate has a δ(15)N value which is an average of the other AAs due to its central role in AA-N cycling. The relative AA δ(15)N values of broad bean and pea seeds were very different from one another, providing evidence for differences in the metabolic routing of AAs to the developing seeds in these leguminous plants. This study has shown that AA δ(15)N values relate to known AA biosynthetic pathways in plants and thus have the potential to aid understanding of how various external factors, such as source of assimilated N, influence metabolic cycling of N within plants.

The effect of manuring on cereal and pulse amino acid δ(15)N values.

Amino acid δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown in manured and unmanured soil at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. Manuring was found to result in a consistent (15)N-enrichment of cereal grain amino acid δ(15)N values, indicating that manuring did not affect the metabolic routing of nitrogen (N) into cereal grain amino acids. The increase in cereal grain δ(15)N values with manuring is therefore due to a (15)N-enrichment in the δ(15)N value of assimilated inorganic-N. Greater variation was observed in the (15)N-enrichment of rachis amino acids with manuring, possibly due to enhanced sensitivity to changes in growing conditions and higher turnover of N in rachis cells compared to cereal grains. Total amino acid δ(15)N values of manured and unmanured broad beans and peas were very similar, indicating that the legumes assimilated N2 from the atmosphere rather than N from the soil, since there was no evidence for routing of (15)N-enriched manure N into any of the pulse amino acids. Crop amino acid δ(15)N values thus provide insights into the sources of N assimilated by non N2-fixing and N2-fixing crops grown on manured and unmanured soils, and reveal an effect of manure on N metabolism in different crop species and plant parts.