Comprehensive phylogenomics of Methylobacterium reveals four evolutionary distinct groups and underappreciated phyllosphere diversity

Abstract

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well-studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere, and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfers. We showed that Methylobacterium contains four evolutionary distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition of recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), which early branching in Methylobacterium history was heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandon of Methylorubrum.

Lanthanide-dependent isolation of phyllosphere methylotrophs selects for a phylogenetically conserved but metabolically diverse community

“…Yet, studies investigating the physiology, biodiversity, and ecology of methylotrophic communities of the phyllosphere have only emerged within the last decade (3,31) and do not take into account the role of lanthanides in influencing their community composition. Furthermore, despite the abundance of methylotrophs in the phyllosphere, the bulk of phylogenomic studies on Methylobacterium species use genomes isolated from non-phyllosphere environments (24). Here, we describe the first community-level characterization of the role of lanthanides during the metabolisms of natural methylotrophic populations in soybean plants, revealing novel insights into methylotrophic biodiversity, metabolic capabilities, and lanthanide-dependent metabolisms.…”

“…Marker gene sequencing for assigning taxonomic groups has proven difficult in Methylobacterium, as16s rRNA has poor phylogenetic resolution in Methylobacterium (24). We characterized our soybean methylotroph community composition using rpoB (Figure 1A), a highly polymorphic single-copy gene with sub-species resolution with Methylobacterium (24).…”

“…Using NCBI BLAST, rpoB sequences from each isolate were compared against the Joint Genome Institute's (JGI) Integrated Microbial Genomes (IMG) database to putatively assign species-level taxonomic classifications to each isolate. All isolates matched to Methylobacterium species within group B (24) , with the closest species being Methylobacterium extorquens AM1, Methylobacterium extorquens PA1, Methylobacterium extorquens TK001, and Methylobacterium zatmanii 135. Untrimmed rpoB sequences from each isolate, as well as from reference Methylobacterium and related species, were aligned using MUSCLE, and Maximum-Likelihood trees with 100 bootstrap replications were constructed using MEGA (Figure 1A).…”