Metagenomic analysis of fecal microbiomes reveals genetic potential for diverse hydrogen management strategies in marsupials.

Methane is an end product of plant biomass digestion by gut microbiota, though the amount produced and/or released varies between hosts. On a per-unit-of-feed basis, macropodid marsupials (e.g., kangaroos) have been reported to emit less methane than ruminant livestock, despite a similar diet, although measurements exist for only a subset of macropodid species. Competition for hydrogen within the gut microbiome, particularly through alternative hydrogen sinks to methanogenesis, influences methane production; therefore, characterizing hydrogen management strategies within a host system can provide insights into methane emission profiles. In this study, we analyzed 33 fecal microbiomes of 14 marsupial species (predominantly captive animals) to provide the first systematic characterization of methanogen types and hydrogen-cycling genetic capacity across marsupial gut microbiomes. We recovered 1,394 metagenome-assembled genomes and identified host-associated bacterial signatures that varied significantly between marsupial species. Comparative analysis with fecal microbiomes from high- and low-methane-emitting mammals revealed that marsupials display heterogeneous hydrogen management strategies: some harbor elevated methanogenesis genes (mcrA, methanogen-specific hydrogenases), while others show enrichment of bacterial hydrogen-uptake hydrogenases and alternative electron acceptor pathways (nitrate/nitrite reduction, sulfite reduction). This predicted functional variation occurs both between and within marsupial families and gut types, suggesting that hydrogen management capacity may differ within taxonomic and anatomical classifications. These results demonstrate that marsupial gut microbiomes cannot be treated as a functionally homogenous group regarding methane emissions and highlight the need for species-specific measurements to accurately assess their methanogenic potential and inform ecological models of greenhouse gas production.IMPORTANCEHerbivorous marsupials such as kangaroos and wallabies have been reported to produce significantly lower methane emissions than ruminant livestock despite eating a similar diet, yet the microbial mechanisms underlying this difference remain poorly understood. Here, we conduct a comparative study of fecal microbiomes of 14 marsupial species to provide the first investigation of hydrogen-cycling genetic capacity across these animals. Through comparative analysis with fecal microbiomes of high- and low-methane-producing animals, we identify enrichment of bacterial genes for alternative hydrogen uptake and disposal pathways in some marsupials, supporting competition for hydrogen playing a role in the level of methane production. These data also indicate variation in hydrogen management between marsupials, including within species, suggesting methane emission capacity may vary at the level of the individual.