Abstract: Rivers are hotspots of global methane (CH₄) emissions. We collected sediments from the upstream, midstream, and downstream reaches of the Wuxijiang River. Combined with anaerobic slurry incubation experiments and molecular biological techniques, we examined the spatial distribution of methanogenic pathways, as well as the abundance and community composition of methanogenic archaea. Results showed that hydrogenotrophic pathway (with H₂ and CO₂ as substrates) predominated across all reaches. The potential of this pathway reached its maximum in the middle reach. Quantitative PCR showed that mcrA gene abundance in the middle reach (1.44×10⁸ copies·g^-1) was significantly higher than that in the upper (9.18×10⁷ copies·g^-1) and lower reaches (1.01×10⁸ copies·g^-1). High-throughput sequencing indicated that the diversity of methanogenic archaea was significantly higher in the upper reach than in the middle and lower reaches, with community structure varying significantly among reaches. The methanogenic community in the upper and middle reaches was dominated by Methanoregula, Methanobacterium and Methanosarcina, whereas that in the lower reach was dominated by Methanoregula and Methanolinea. The relative abundance of Methanoregula showed an increasing trend from upstream (36.2%) to midstream (54.9%) and downstream (61.0%). Correlation analyses indicated that sediment organic carbon contents, NH₄⁺-N contents, and pH were significantly associated with the spatial heterogeneity of methanogenic pathways and methanogenic archaeal community composition. Collectively, the midstream reach represented a potential hotspot of CH₄ production in the Wuxijiang River, and these findings would provide new insights into the spatial heterogeneity of methane emissions in mountainous river system.

Key words: mountainous river, Methanoregula, hydrogenotrophic methanogenesis, organic carbon, NH₄⁺-N