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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.

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.)