Reconstructing past food webs is crucial for understanding evolutionary drivers and ecological dynamics. Stable isotope analysis of bone and trace elements has advanced this field, but nitrogen isotope analysis (δ¹⁵N), a powerful tool for modern trophic studies, has been limited in paleontological applications due to the poor preservation of nitrogen-containing organic matter in fossils. Historically, nitrogen isotope studies of fossil vertebrates have focused on relatively young, well-preserved bone or dentin. However, the organic matter in these tissues is susceptible to diagenetic alteration, especially in humid or acidic environments. In contrast, tooth enamel, with its dense, crystalline structure, offers better preservation of organic matter. Although enamel has low nitrogen content, recent advancements in analytical techniques, such as the oxidation-denitrification method coupled with nano-EA-IRMS, allow for precise δ¹⁵Nenamel measurements even with small samples. Previous studies have shown promise for using δ¹⁵Nenamel in modern mammals, but more data from diverse ecosystems and comparisons with other tissues were needed to fully validate its use in reconstructing ancient food webs. This study addresses these gaps by analyzing a large dataset of modern and fossil mammals using advanced analytical methods.

Extensive research has demonstrated the utility of nitrogen isotope ratios (δ¹⁵N) in studying modern animal trophic levels. A consistent trophic enrichment of 3-4‰ in ¹⁵N is observed between consumers and their diets. However, applying this method to the fossil record is challenging due to the degradation of organic matter, primarily collagen, in ancient bones. While recent studies have explored alternative proxies such as calcium and zinc isotopes, data on their isotopic fractionation across various tissues remain limited compared to the extensive work on nitrogen isotopes. Enamel, with its greater resistance to diagenetic alteration, presents a potential solution. However, its low nitrogen content and analytical challenges posed difficulties until recent developments in nano-EA-IRMS and other advanced analytical techniques made precise measurement of nitrogen isotopes in enamel feasible. Prior work in controlled experiments and a limited study of African mammals indicated that enamel nitrogen isotopes could indeed reflect trophic levels, but a broader study was needed to fully confirm this and assess the preservation of this signal in ancient fossils. Studies on sharks' teeth had shown success using this technique for older fossils, but it was unclear whether this would work similarly in mammalian fossils given differences in tooth structures and depositional environments.

This study employed a multifaceted approach combining analysis of modern and fossil mammalian teeth. The researchers collected tooth enamel samples from 54 modern African mammals representing various trophic levels (herbivores, carnivores, omnivores) and diverse ecosystems. For a subset of these modern mammals, bone collagen samples were also collected for comparison of δ¹⁵N values between enamel and collagen. Additionally, they analyzed 10 fossil mammalian teeth from a Late Pleistocene assemblage in Tam Ly Marklot Cave (THM), Laos. The oxidation-denitrification method was used to measure δ¹⁵Nenamel in both modern and fossil samples. This method involves careful cleaning procedures to remove exogenous nitrogen contamination and ensure accurate measurement of endogenous enamel-bound nitrogen. A gas chromatography-elemental analyzer-isotope ratio mass spectrometry (GC-EA-IRMS) was employed for precise isotope ratio measurement. For modern specimens where sufficient collagen was preserved, bone collagen δ¹⁵N and δ¹³C values were also measured. Statistical analyses, including ANOVA, Kruskal-Wallis tests, Dunn's post hoc tests, Pearson's correlation, and Spearman's correlation were used to analyze the data, examining the relationships between δ¹⁵Nenamel, δ¹⁵Ncollagen, δ¹³Cenamel, and δ¹³Ccollagen across dietary groups and ecosystems. The researchers also analyzed carbon isotope data (δ¹³C) from both enamel and collagen to provide further insights into dietary habits and habitat preferences.

The analysis of modern African mammals revealed a significant positive correlation between δ¹⁵Nenamel and δ¹⁵Ncollagen values (r = 0.865, p < 0.001), indicating that both tissues record similar isotopic information when collagen is preserved. The study also found a clear trophic enrichment in δ¹⁵Nenamel, with carnivores exhibiting significantly higher values (average 3.7‰) than herbivores. The δ¹⁵Nenamel values accurately reflected dietary differences among herbivores (browsers, grazers, mixed feeders), further validating its use as a trophic level indicator. The analysis of Late Pleistocene fossil teeth from THM Cave in Laos demonstrated that δ¹⁵Nenamel values preserved dietary and trophic level information despite the complete diagenetic loss of collagen. The fossil δ¹⁵Nenamel values showed a similar trophic enrichment pattern to modern mammals, with herbivores having lower values and the carnivore exhibiting a high value. Carbon isotope analysis (δ¹³C) provided complementary information on dietary preferences and habitat use, distinguishing between browsers and grazers in both modern and fossil samples. The N content of the fossil enamel samples was comparable to the modern samples, further reinforcing the idea that enamel effectively protects organic matter from diagenetic alteration. The positive correlations observed between δ¹³C values from enamel and collagen in both modern and fossil specimens confirm that carbon isotopes preserved in enamel reflect diet.

The strong correlation between δ¹⁵Nenamel and δ¹⁵Ncollagen in modern mammals, coupled with the clear trophic enrichment in δ¹⁵Nenamel values, firmly establishes tooth enamel as a reliable proxy for reconstructing dietary and trophic level information. The successful application of this method to Late Pleistocene fossils demonstrates the exceptional preservation potential of enamel-bound organic nitrogen, even in settings where collagen is entirely lost. The combined use of nitrogen and carbon isotope analysis provides a powerful tool for resolving finer-scale trophic dynamics and understanding dietary preferences within ecological communities. The findings have important implications for paleoecological studies, particularly where preservation of bone collagen is poor. This approach expands the temporal and taxonomic range of studies investigating ancient food webs, allowing for more accurate reconstructions of past ecosystems and dietary behaviors of extinct animals.

This study demonstrates the significant potential of using tooth enamel nitrogen isotope analysis (δ¹⁵Nenamel) for reconstructing past food webs. The strong correlation between δ¹⁵Nenamel and δ¹⁵Ncollagen in modern mammals, along with the successful application of this method to well-preserved fossil teeth, establishes δ¹⁵Nenamel as a powerful and robust proxy. Future research could focus on expanding the application of this method to a broader range of taxa, ecosystems, and geological time periods. Further investigations into the factors influencing isotopic fractionation in enamel, particularly concerning the influence of diagenetic processes, would further refine this powerful tool for paleoecological reconstructions.

The relatively small sample size of fossil teeth from THM Cave limits the strength of conclusions regarding the trophic structure of that specific assemblage. While the study demonstrates the potential of δ¹⁵Nenamel, additional studies with larger fossil samples are needed to confirm the broader applicability of this method. Additionally, the study primarily focused on mammals, and further research could explore its applicability to other vertebrate groups. Understanding the full extent of potential confounding factors such as diagenetic alteration and variations in baseline isotopic compositions across different environments will be an ongoing area of research.