Unveiling the dark matter of riverine dissolved organic matter and its role in molecular chemodiversity

Chemodiversity of riverine dissolved organic matter (DOM) plays a crucial role in global elemental cycles and ecosystem function. However, DOM "dark matter"-molecules without assigned specific formulae-remain unexplored in the interpretation of chemodiversity. Here, we systematically investigated the characteristics and drivers of dark matter and its impacts on DOM chemodiversity using 551 river water and sediment samples. Compared to known matter with specific formulae, dark matter exhibited lower molecular weight and diversity, but higher compositional dissimilarity, with dissolved organic carbon (DOC) as the primary driver. Including dark matter into chemodiversity analysis changed mass, diversity, and compositional dissimilarity of DOM from -10.1 to -0.04 %, -0.7 to 4.9 %, and 0.4 to 28.8 % in waters, and from -17.5 to 5.7 %, -10.5 to 19.2 %, and -22.3 to 33.1 % in sediments, respectively. DOC mediated these ecosystem-dependent effects: lower DOC amplified the effects on chemodiversity in waters, whereas in sediments, the effects shifted from negative to positive with changing DOC. Furthermore, ignoring dark matter would overestimate the regulatory role of DOC on sediment DOM chemodiversity. Collectively, our findings clarify the influence of dark matter on DOM chemodiversity assessments across environmental gradients, improving understanding of riverine DOM and refining its analytical framework.