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During the sixth millennium BCE, the first farmers of Central Europe rapidly expanded across a varied mosaic of forested environments. Such environments would have offered important sources of mineral-rich animal feed and shelter, prompting the question: to what extent did early farmers exploit forests to raise their herds? Here, to resolve this, we have assembled multi-regional datasets, comprising bulk and compound-specific stable isotope values from zooarchaeological remains and pottery, and conducted cross-correlation analyses within a palaeo-environmental framework. Our findings reveal a diversity of pasturing strategies for cattle employed by early farmers, with a notable emphasis on intensive utilization of forests for grazing and seasonal foddering in some regions. This experimentation with forest-based animal feeds by early farmers would have enhanced animal fertility and milk yields for human consumption, concurrently contributing to the expansion of prehistoric farming settlements and the transformation of forest ecosystems. Our study emphasizes the intricate relationship that existed between early farmers and forested landscapes, shedding light on the adaptive dynamics that shaped humans, animals and environments in the past., (© 2024. The Author(s).)

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 δ 15 N 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 δ 15 N 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 δ 15 N value (δ 15 N Arg ) 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 δ 15 N 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 δ 15 N Arg values to be related to AA biosynthesis. Determination of archaeological cattle collagen δ 15 N Arg values confirmed the suitability of this method to provide further insights into past diets and ecosystems. Bulk collagen δ 15 N value reconstruction including δ 15 N Arg 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.

Amino acid δ 15 N 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 δ 15 N values of glutamate (Glx) and phenylalanine (Phe) were found to differ significantly between woody and herbaceous plants, with mean Δ 15 N Glx-Phe (i.e. δ 15 N Phe - δ 15 N Glx ) values of -9.3 ± 1.6‰ and -5.8 ± 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 Δ 15 N Glx-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 13 C 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 Δ 15 N Glx-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 δ 15 N values will, to give one example, allow refinement of estimates of herbivore diet in present and past ecosystems, enabling more accurate environmental niche modelling., (Copyright © 2019. Published by Elsevier Ltd.)