Fine-Scale Adaptations to Environmental Variation and Growth Strategies Drive Phyllosphere Methylobacterium Diversity

Abstract

Methylobacterium is one of the most prevalent bacterial genera of the phyllosphere, present on the leaves of nearly every plant. Despite its ubiquity and its importance for host plant function, little is known about whether diversity over space and time within the genus reflects neutral processes like migration and drift, or environmental filtering of life history strategies and adaptations to temperature and host tree species. Here, we investigated how phylogenetic diversity within the genus Methylobacterium is structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we quantified seasonal shifts in Methylobacterium diversity over a year from early summer to fall in two temperate forests in Quebec, Canada. As an alternative to the 16S rRNA gene, we used rpoB, a highly polymorphic marker gene, which has not experienced detectable horizontal gene transfer nor copy number variation in Methylobacterium genomes. We cultured very different subsets of Methylobacterium diversity from the same leaf, depending upon the temperature of isolation and growth (20 C or 30 C). For instance, one the most abundant Methylobacterium lineages was almost exclusively isolated at 20 C, suggesting long-term adaptation to temperature. Both culture and barcoding approaches revealed that a considerable and previously underestimated diversity of Methylobacterium colonize the surface of leaves in temperate forests. This diversity was strongly structured according to both large (>100km; between forests) and small geographical scales (<1.2km within forests), and among host tree species, and was dynamic over seasonal time scales. To determine if these seasonal effects were driven by temperature, we measured growth of 79 isolates at different temperature treatments. Different lineages showed subtle and significant differences in growth performance when subject to temperature increase or decrease, but most have higher yield and slower growth rate at 20 than 30C, with strong lineage- and season-dependant variation in their overall growth strategies. We proposed that the homogenization of Methylobacterium community structure observed over a growing season resulted from the progressive replacement of isolates with high yield strategy typical of cooperative, structured communities, by isolates with rapid growth. Together our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This works paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus.

Aerobic methoxydotrophy: growth on methoxylated aromatic compounds by Methylobacterium

“…Locus tags are given in Supplementary Table 1. The clade structure generally agrees with previous Methylobacterium phylogenies based on 16S rRNA, rpoB, or core genes, though some analyses find clade A not to be monophyletic (Green and Ardley, 2018;Alessa et al, 2021;Leducq et al, 2021).…”

“…The phylogeny was composed using a concatenated alignment of four housekeeping genes (atpD, recA, rpob, and 16S rRNA), with Prochlorococcus marinus as an outgroup (not shown). Clade A/B/C designations are in accordance with(Green and Ardley, 2018; Alessa et al, 2021;Leducq et al, 2021). Branch labels indicate bootstrap percent; values <50 are not shown.…”

“…Two additional phylogenies were constructed as supplementary information: one containing all the Methylobacterium strains surveyed for this study, using the rpoB gene (Leducq et al, 2021) with Bradyrhizobium diazoefficiens USDA 110 as an outgroup; and one showing the relationships of the Rieske [2Fe-2S] oxygenase homologs. The rpoB tree includes five of the Methylobacterium strains carrying aromatic catabolism genes (sp.…”

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

“…We included 29 genomes from Methylobacterium type strains recently sequenced (Alessa et al ., 2021; Bijlani et al ., 2021), hence covering most Methylobacterium species described so far. We also included 38 genomes available from two large surveys of the Arabidopsis and wheat phyllospheres (Helfrich et al ., 2018; Zervas et al ., 2019), and sequenced 24 additional genomes of isolates from a large survey of the temperate forest phyllosphere (Leducq et al ., 2022), hence extending our dataset to the leaf-associated Methylobacterium diversity. The 24 newly assembled genomes had 41 to 405 scaffolds (depth: 188-304x) for a total size (5-7Mb) and average GC content (67-70%) in the expected range for Methylobacterium genomes (Dataset S2).…”

“…On the contrary, groups B and C included most Methylobacterium model species frequently used in the lab and isolated in anthropogenic environment. While group B is occasionally identified on and isolated from the surface of leaves (Lee et al, 2022;Leducq et al, 2022), group C is rarely, if ever, found in the phyllosphere, and seems to be more widespread in soil and in aquatic environments, often in association with plant roots (Lee et al, 2022). Interestingly, authors from a recent study estimated that Rhizobiales common ancestor likely had a free-living lifestyle, while Methylobacterium groups A, B and D's common ancestor likely had a plant-associated lifestyle (node 1 in Figure 1 from Wang et al study (Wang et al, 2020)).…”