<b>Uncovering ecological pathways of geographic differentiation through hierarchical analysis of multi-omics variation in a desert lizard</b>

Understanding how geographic populations differentiate is central to evolutionary biology and has critical conservation implications. However, most studies focus on single biological layers (e.g., genetics), limiting insights into how multiple traits co-vary across geographic gradients. Here, we investigated geographic variation in <i>Teratoscincus przewalskii</i>, a desert lizard, by integrating morphology and four internal biological profiles (i.e., neutral genetic diversity, dietary composition, gut microbiota, and liver metabolome) with ecological implications across nine populations spanning over 1,000 km. We found pronounced heterogeneity in both the extent and nature of geographic differentiation across biological layers. A clear hierarchy emerged: genetic markers showed minimal spatial structure, dietary composition and gut microbiota exhibited intermediate differentiation, while liver metabolome displayed the strongest differentiation. Climatic factors but not geographic distances drive these differentiations: with evapotranspiration best explaining diet, precipitation linking to microbial communities, and temperature best explaining liver metabolome and morphology. Evapotranspiration-dependent dietary changes likely reflect behavioral plasticity in response to spatial differences in arthropod communities. Temperature-dependent metabolic changes, particularly in cortisol, glutathione, and sphingosine levels, suggests adaptive responses to thermal gradient. Interestingly, strong correlations between diet and liver metabolites point to a cascading effect, whereby evapotranspiration indirectly influences metabolism through altering diets. In contrast, gut microbiota showed limited mediating effects. This reveals how climate shapes population differentiation both directly and via ecological intermediates. Overall, our findings reveal heterogeneous geographic differentiation across biological layers and partially uncover the underlying ecological pathways shaping this variation. These results underscore the importance of a systems-level approach to understanding population differentiation and informing effective conservation strategies.