Abstract
<jats:p>Background: Seasonal climate variation drives physiological change across levels of biological organization in ectotherms, yet whether these responses follow a predictable temporal hierarchy remains untested in wild populations. In a year-round field study of the Lesser Treefrog (Dendropsophus minutus, Anura: Hylidae) in Sao Jose do Rio Preto, southeastern Brazil, we sampled 40 to 68 adult males across a full annual cycle to quantify seasonal variation in four hepatic phenotypic modules spanning multiple organizational levels: liver histochemistry (pigments and glycogen; intracellular), cell and nucleus morphometry (cellular), tissue volumetric composition of hepatocytes, sinusoids, melanomacrophage centres and portal structures (tissue), and whole-body somatic indices of liver mass and body condition (organismal). We asked whether lower organizational levels respond faster to seasonal climate, as predicted by the bottom-up cascade framework originally proposed for toxicant exposure, or whether alternative hierarchies emerge when climate acts first through whole-organism physiology. To test whether climatic change precedes each biological response, we extended Procrustean superimposition to incorporate time lags, tested both forward and reverse directions, and complemented it with phenotypic trajectory analysis, phase analysis of monthly change, and lagged regression against temperature, precipitation, humidity and drought. Results: Somatic indices and tissue composition tracked climate with no detectable delay, whereas histochemistry and cell morphometry accumulated a three-month delay, and tissue composition in turn led somatic indices by one month. Trajectory analysis separated the modules into a fast-responding group (somatic indices, histochemistry and morphometry) and a slower tissue-composition tier, a two-tier structure confirmed by paired bootstrap resampling in which the slow tier differed from every fast module in both displacement rate and total path length. The four modules were largely independent through time, and the two strongest concordances linked climate to tissue composition and to somatic indices. Conclusions: These results reveal a temporal response hierarchy in which seasonal climate acts first on whole-organism energy balance and then propagates to tissue architecture and intracellular processes with module-specific delays, showing that the direction of the hierarchy depends on the nature of the environmental driver. We also introduce a general analytical framework for testing temporal precedence in short multivariate biological time series.</jats:p>