Ubiquity of methanogenic archaea in the trunk of coniferous and broadleaved tree species in a mountain forest

Wetwood of living trees is a habitat of methanogenic archaea, but the ubiquity of methanogenic archaea in the trunk of various trees has not been revealed. The present study analysed methanogenic archaeal communities inside coniferous and broadleaved trees in a cold temperate mountain forest by culture-dependent or independent techniques. Heartwood and sapwood segments were obtained from the trunk of seven tree species, Cryptomeria japonica, Quercus crispula, Fraxinus mandshurica, Acer pictum, Aesculus turbinata, Magnolia obovata, and Populus tremula. Amplicon sequencing analysis of 16S rRNA genes showed that Methanobacteriaceae predominated the archaeal communities and Methanomassiliicoccaceae also inhabited some trees. Real-time PCR analysis detected methanogenic archaeal mcrA genes from all the tree species, with a maximum of 10⁷ copies g⁻¹ dry wood. Digital PCR analysis also detected mcrA genes derived from Methanobacterium spp. and Methanobrevibacter spp. from several samples, with a maximum of 10⁵ and 10⁴ copies g⁻¹ dry wood. The enumeration by the most probable number method demonstrated the inhabitation of viable methanogenic archaea inside the trees; 10⁶ cells g⁻¹ dry wood was enumerated from a heartwood sample of C. japonica. Methanogenic archaea related to Methanobacterium beijingense were cultivated from a heartwood sample of Q. crispula and F. mandshurica. The present study demonstrated that the inside of various trees is a common habitat for methanogenic archaeal communities and a potential source of methane in forest ecosystems.