Different atmospheric methane-oxidizing communities in European beech and Norway spruce soils

Abstract

Norway spruce (Picea abies) forests exhibit lower annual atmospheric methane consumption rates than do European beech (Fagus sylvatica) forests. In the current study, pmoA (encoding a subunit of membrane-bound CH(4) monooxygenase) genes from three temperate forest ecosystems with both beech and spruce stands were analyzed to assess the potential effect of tree species on methanotrophic communities. A pmoA sequence difference of 7% at the derived protein level correlated with the species-level distance cutoff value of 3% based on the 16S rRNA gene. Applying this distance cutoff, higher numbers of species-level pmoA genotypes were detected in beech than in spruce soil samples, all affiliating with upland soil cluster alpha (USCalpha). Additionally, two deep-branching genotypes (named 6 and 7) were present in various soil samples not affiliating with pmoA or amoA. Abundance of USCalpha pmoA genes was higher in beech soils and reached up to (1.2 +/- 0.2) x 10(8) pmoA genes per g of dry weight. Calculated atmospheric methane oxidation rates per cell yielded the same trend. However, these values were below the theoretical threshold necessary for facilitating cell maintenance, suggesting that USCalpha species might require alternative carbon or energy sources to thrive in forest soils. These collective results indicate that the methanotrophic diversity and abundance in spruce soils are lower than those of beech soils, suggesting that tree species-related factors might influence the in situ activity of methanotrophs.

FIG. 1.

Correlation of DNA and protein sequence similarity of pmoA and PmoA versus 16S rRNA gene similarity (22 pairwise comparisons of methanotrophic strains). The dashed line represents the 97% species cutoff value based on the 16S rRNA gene (49). Percentage values indicate the similarity cutoff value for species-level OTUs based on pmoA and PmoA sequence corresponding to distance values of 13% and 7%, respectively (intersection of dashed and regression line). R² was 0.85 for the DNA panel and 0.79 for the protein panel. Accession numbers of analyzed sequences are given in Table S1 in the supplemental material.

FIG. 2.

Dendrogram of species-level OTUs within USCα (30); clusters 5 (31), MHP (peat bog) (7), and AC (aquatic environments; named in this study); and Methylocapsa acidiphila-related sequences. Analysis of species-level OTUs was performed with 406 PmoA sequences based on a 7% sequence distance cutoff value using DOTUR (46). Analysis was restricted to amino acids 59 to 206 according to the PmoA of Methylococcus capsulatus Bath (NC_002977) and resulted in 26 different species-level OTUs. Thirty-nine selected sequences out of these 26 OTUs were used to construct a dendrogram in MEGA4 (52) using the neighbor-joining method (18) without evolutionary correction to apply the distance cutoff concept (10,000 bootstrap steps) (45). Bootstrap values higher than 50% are shown. Bold names indicate sequences from the current study. Accession numbers are given in parentheses. Bold numbers on the right side (1 to 10) represent different species-level OTUs within the corresponding clusters. The PmoA sequence of M. capsulatus Bath (NC_002977) was used as an out-group (arrow). The scale bar represents 5% sequence difference. Env seq, environmental sequence.

FIG. 3.

Community composition based on pmoA clone libraries. OTUs were defined according to a methanotrophic species-level cutoff value of 7%. Gene libraries were retrieved from pmoA PCR products from beech and spruce soils (O_a horizon pooled with 0- to 5-cm mineral soil) from three forests, Solling, Steigerwald, and Unterlüß. The numbers of analyzed sequences per clone library were as follows: Solling beech, 44; Solling spruce, 45; Steigerwald beech, 85; Steigerwald spruce, 75; Unterlüß beech, 75; and Unterlüß spruce, 41. Analysis was performed with 406 PmoA sequences using amino acids 59 to 206 according to the PmoA sequence of M. capsulatus Bath (NC_002977). Gray, USCα-1; dark green, USCα-7; purple, USCα-8; light green, USCα-2; orange, USCα-4; light blue, USCα-9; yellow, USCα-6; red, USCα-10; black, cluster 6; dark blue, cluster 7; white, ammonia-oxidizing bacteria within the Betaproteobacteria.

FIG. 4.

Phylogenetic tree showing the relationship of PmoA sequences obtained from forest soils in this study to known groups of PmoA and AmoA sequences. USCα (21), USCγ (30), cluster 1 (33), JR3 (2), and Methylocystaceae (16) represent putative atmospheric methane oxidizing genotypes. The tree was calculated based on amino acids 59 to 206 according to the PmoA sequence of M. capsulatus Bath (NC_002977) using the ARB-implemented PROML method (36) with the evolutionary model JTT (25). The scale bar represents 10% sequence difference. Accession numbers of sequences used for tree reconstruction are as follows: AF148521, AF148522, AF200729, AJ278727, AJ579669, AJ868245, AJ868259, AJ868265, AJ868278, AJ868281, AJ868409, AY550736, DQ295899, EF591085, EF644409, FJ970601, FN564735, FN564878, FN564924, FN564930, U76553, and NC_002977.

FIG. 5.

pmoA, amoA, and 16S rRNA gene numbers. Bars show the number of pmoA, amoA, and 16S rRNA genes measured by qPCR in beech and spruce soils from Solling, Steigerwald, and Unterlüß. Error bars indicate standard deviations of three replicate DNA extractions of five soil cores, i.e., from a total of 15 values. The asterisk indicates significant differences between beech and spruce soil (Mann-Whitney U test).