Otolith microchemistry analysis accurately reconstructed the spawning ground utilization of fish in the upper Nu-Salween River

1. Freshwater habitats face substantial degradation worldwide, threatening critical ecosystem functioning and services. Fish spawning grounds play a vital role in sustaining fish populations, biodiversity, and aquatic ecosystem health. However, high-resolution empirical data on fish habitat use in large river systems remain scarce due to limitations in tagging methodologies. This study reconstructs spawning ground utilization of a fish (Clupisoma yunnanensis) in the upper Nu-Salween River by matching otolith microchemistry signatures with high-resolution water chemistry maps (chemoscapes).2. We analyzed otolith and water element ratios (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca) using electron probe microanalysis (EPMA) and inductively coupled plasma mass spectrometry (ICP-MS), respectively, and established their quantitative relationships. These data were then applied in a random forest (RF) model to reconstruct fish spawning ground distribution, with validation performed through environmental niche analysis assessing the overlap between microchemistry-derived spawning grounds and traditionally observed sites.3. EPMA revealed significant core-to-edge otolith microchemistry variations, paralleled by spatiotemporal water chemistry heterogeneity, demonstrating the species experienced diverse habitats. The RF model achieved 98% accuracy in identifying downstream areas as primary spawning grounds. Environmental niche analyses showed 83% concordance between microchemistry-derived spawning grounds and traditionally observed sites.4. This study establishes a novel biogeochemical framework combining otolith microchemistry, chemoscape, and machine learning to accurately reconstruct critical fish habitats. Our approach not only resolves the spawning ecology of C. yunnanensis in the Nu-Salween system but also provides a transferable methodology for conservation planning in data-scarce river basins worldwide.