Atmospheric Methane Oxidizers Are Dominated by Upland Soil Cluster Alpha in 20 Forest Soils of China

Upland soil clusters alpha and gamma (USC alpha and USC gamma) are considered a major biological sink of atmospheric methane and are often detected in forest and grassland soils. These clusters are phylogenetically classified using the particulate methane monooxygenase genepmoAbecause of the difficulty of cultivation. Recent studies have established a direct link ofpmoAgenes to 16S rRNA genes based on their isolated strain or draft genomes. However, whether the results ofpmoA-based assays could be largely represented by 16S rRNA gene sequencing in upland soils remains unclear. In this study, we collected 20 forest soils across China and compared methane-oxidizing bacterial (MOB) communities by high-throughput sequencing of 16S rRNA andpmoAgenes using different primer sets. The results showed that 16S rRNA gene sequencing and the semi-nested polymerase chain reaction (PCR) of thepmoAgene (A189/A682r nested with a mixture of mb661 and A650) consistently revealed the dominance of USC alpha (accounting for more than 50% of the total MOB) in 12 forest soils. A189f/A682r successfully amplifiedpmoAgenes (mainly RA14 of USC alpha) in only three forest soils. A189f/mb661 could amplify USC alpha (mainly JR1) in several forest soils but showed a strong preferential amplification ofMethylocystisand many other type I MOB groups. A189f/A650 almost exclusively amplified USC alpha (mainly JR1) and largely discriminated againstMethylocystisand most of the other MOB groups. The semi-nested PCR approach weakened the bias of A189f/mb661 and A189f/A650 for JR1 and balanced the coverage of all USC alpha members. The canonical correspondence analysis indicated that soil NH4+-N and pH were the main environmental factors affecting the MOB community of Chinese forest soils. The RA14 of the USC alpha group prefers to live in soils with low pH, low temperature, low elevation, high precipitation, and rich in nitrogen. JR1's preferences for temperature and elevation were opposite to RA14. Our study suggests that combining the deep sequencing of 16S rRNA andpmoAgenes to characterize MOB in forest soils is the best choice.