Abstract

Arctic warming is leading to permafrost thawing which modifies, in cascade, hydrosystems at diverse levels. This study aimed to compare prokaryotic community structure and methane (CH4) dynamics across 16 sub-Arctic waterbodies, and to assess how these features are shaped by permafrost thaw. The sampled waterbodies, located in an ice-rich discontinuous permafrost region (Southwestern Yukon, Canada) differed in size, depth, stratification and degree of thaw influence. Prokaryotic communities were characterised through 16S rRNA gene sequencing and qPCR targeting mcrA (methanogenesis) and pmoA (methanotrophy) genes. Community structures differed significantly between shallow stratified, deep stratified and non-stratified waterbodies. Methanogens, predominantly represented by the Methanobacterium genus, were mostly detected in shallow non-stratified waterbodies. Methanotrophs, primarily represented by the Methylacidiphilaceae family, were more abundant in oxic layers whereas bacteria of Crenothrix and Methylobacter genera dominated in anoxic conditions. Our results showed that non-stratified waterbodies directly affected by permafrost thaw harboured distinct prokaryotic communities, including specific methanogens and methanotrophs. The two sites with the highest CH4 emissions were affected by permafrost thaw, with fluxes reaching up to 1.7 × 10−1 mg m−2 s−1. Future investigations should address gaps in CH4-related processes in thaw-affected systems, as they are hotspots for methane emissions and harbour different microbial communities. © 2025 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.