Global meta-analysis reveals core Hyphomicrobiales in the phyllosphere.

Lichenihabitantaceae and Methylobacterium dominate the surface of tree leaves.

Novel phyllosphere clades RH-AL1 and JAJXWB01 were identified by phylogenomics.

16S rRNA gene limits taxonomic resolution in the phyllosphere; metagenomics refines taxonomy.

The phyllosphere, or above-ground part of plants, hosts diverse bacterial communities that play critical ecological roles and provide beneficial functions for the plant. The Hyphomicrobiales (Alphaproteobacteria) are a highly diverse and ecologically important clade known to be key members of the plant microbiome, in particular in association with plant roots, but their diversity remains largely uncharacterized in the phyllosphere. Using a meta-analysis combining metabarcoding, metagenomics and phylogenomics, we explored the diversity of leaf-associated Hyphomicrobiales. We confirmed Methylobacterium was ubiquitous in the phyllosphere and revealed the dominance of two under-characterized Hyphomicrobiales taxa: Lichenihabitantaceae, a lichen-associated family previously identified as “1174–901-12” in taxonomic databases, and RH-AL1, an undescribed lineage of bacteria related to Beijerinckiaceae. Despite their abundance in the phyllosphere, Lichenihabitantaceae and RH_AL1 could not be properly identified by 16S rRNA gene barcoding, due in part to limitations of short read sequencing leading to a lack of recognition of certain Hyphomicrobiales genera, and to incongruencies in the assignment of genera to families among existing taxonomic databases. A significant proportion of Lichenihabitantaceae were detected in association with lichens and in environments with harsh conditions like exposed surfaces, air and snow. Overall, our study stresses the need to agree on a common systematic framework to properly classify and identify key leaf-associated Hyphomicrobiales taxa, and to move toward metagenomics and culturomics to increase their representation in reference databases, to provide a better understanding of the evolutionary and functional mechanisms underpinning bacteria adaptations to living on plants.

Phyllosphere

Hyphomicrobiales

Methylobacterium

Lichenihabitantaceae

Metagenomics

Phylogenomics

© 2026 The Authors. Published by Elsevier GmbH.