Previous studies have demonstrated that enamel oxygen isotope values (δ18O) reflect rainfall variability experienced by wild primates and ancient humans, providing insights into local climatic conditions. However, it remains less clear whether seasonal patterns can be consistently detected in premodern human communities experiencing different local water dynamics, or how these patterns may vary regionally. Here we present 1958 ion microprobe δ18O measurements linked to enamel growth increments in 15 molars from 13 individuals, representing a combined total of 48 years of enamel formation sampled at approximately weekly resolution. We additionally integrate 87Sr/86Sr and trace-element datasets, contextualized by the δ18O enamel profiles, to assess seasonal adaptation and the likelihood that observed isotopic patterns reflect local rather than non-local signatures. Our dataset spans three Neolithic archaeological sites characterized by distinct water sources: Ali Kosh, Iran (ca. 7,500–6,500 cal BCE); Nemrik 9, Iraq (ca. 8,000–6,500 BCE); and Tepe Sialk North, Iran (ca. 5,800–4,900 BCE). Individuals from Ali Kosh exhibited the highest δ18O values in the dataset along with substantial intra-individual variability, consistent with reliance on evaporatively enriched transient and isotopically diverse water sources—possibly shaped by local mobility or water management strategies. In contrast, individuals from Nemrik 9 displayed clear, large-amplitude annual cycles in their enamel δ18O values, a pattern consistent with a settlement situated near a perennial river subjected to pronounced climatic seasonality. Tepe Sialk North individuals exhibited the lowest δ18O variability, consistent with consumption of isotopically buffered sources such as springs or artesian-fed systems. Predicted drinking water δ18O values for most of the Nemrik 9 and Ali Kosh individuals align well with modern regional rainfall isotope values, while those from Tepe Sialk North underestimate local precipitation δ18O values, as expected for groundwater-fed sources. Additionally, site-specific seasonal patterns were observed in Ba/Ca and Li/Ca elemental ratios, likely shaped by the interplay of regional geology and hydrology. Together, our findings provide valuable insights into Neolithic water access strategies in arid regions, while also underscoring the limitations of identifying bioarchaeological migrants based solely on δ18O values from tooth enamel.
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