DNA-SIP reveals salinity-associated niche differentiation of potentially active methanogens in mangrove soils

Mangrove forests are major blue carbon ecosystems but are often characterized by low surface methane (CH4) emissions. Such low emissions, however, do not necessarily indicate weak methanogenesis, because CH4 production may be offset by internal CH4 consumption before reaching the atmosphere. Although previous community, genomic, and transcriptomic studies have implicated methylotrophic methanogenesis in mangrove sediments, direct taxon-resolved evidence linking methylated carbon assimilation to potentially active methanogens remains limited. Here, we combined methanogenic activity assays, DNA stable isotope probing (DNA-SIP), mcrA and 16S rRNA gene analyses, and phylogenetic comparisons to identify potentially active methanogens across saline-influenced mangrove soils. The results showed that CH4 production potentials were consistently dominated by methylotrophic pathways (1.86-2.78 g CH4 g-1 soil hr-1) across all sites. DNA-SIP, together with consistent community patterns in fresh soils, indicated the potential activity of methylotrophic and mixotrophic methanogens under saline conditions. Methanolobus-affiliated methanogens were associated with salinity, Na+, Cl-, and NH4+, whereas Methanosarcina and unclassified Methanosarcinaceae were linked to soil soluble organic carbon availability and water content, indicating niche differentiation among active methanogenic groups. Phylogenetic analyses incorporating reference sequences from diverse environments further showed that potentially active mangrove methanogens were dominated by saline-associated lineages. Together with our previous methanotrophic evidence from the same sites, these findings suggest that low CH4 emissions from mangrove blue carbon ecosystems can mask substantial internal CH4 cycling sustained by active methanogenesis and CH4 consumption.