Host species and geographic location shape microbial diversity and functional potential in the conifer needle microbiome

(2025) 13:222 Bowers et al. Microbiome https://doi.org/10.1186/s40168-025-02271-y Microbiome Open Access RESEARCH Host species and geographic location shape microbial diversity and functional potential in the conifer needle microbiome Robert M. Bowers1* , Shayna Bennett2, Robert Riley1, Juan C. Villada1, Iolanda Ramalho Da Silva2, Tanja Woyke1,2   and A. Carolin Frank2,3* Abstract Background The aerial surface of plants, known as the phyllosphere, hosts a complex and dynamic microbiome that plays essential roles in plant health and environmental processes. While research has focused on root-associated microbiomes, the phyllosphere remains comparatively understudied, especially in forest ecosystems. Despite the global ecological dominance and importance of conifers, no previous study has applied shotgun metagenomics to their phyllosphere microbiomes. Results This study uses metagenomic sequencing to explore the microbial phyllosphere communities of subalpine Western conifer needle surfaces from 67 trees at six sites spanning the Rocky Mountains, including 31 limber pine, 18 Douglas fir, and 18 Engelmann spruce. Sites span ~ 1,075 km and nearly 10° latitude, from Glacier National Park to Rocky Mountain Biological Laboratory, capturing broad environmental variation. Metagenomes were generated for each of the 67 samples, for which we produced individual assemblies, along with three large coassemblies specific to each conifer host. From these datasets, we reconstructed 447 metagenome-assembled genomes (MAGs), 417 of which are non-redundant at the species level. Beyond increasing the total number of extracted MAGs from 153 to 294, the three coassemblies yielded three large MAGs, representing partial sequences of host genomes. Phylogenomics of all microbial MAGs revealed communities predominantly composed of bacteria (n = 327) and fungi (n = 117). We show that both microbial community composition and metabolic potential differ significantly across host tree species and geographic sites, with site exerting a stronger influence than host. Conclusions This dataset offers new insights into the microbial communities inhabiting the conifer needle surface, laying the foundation for future research on needle microbiomes across temporal and spatial scales. Variation in functional capabilities, such as volatile organic compound (VOC) degradation and polysaccharide metabolism, closely tracks shifts in taxonomic composition, indicating that host-specific chemistry, local environmental factors, and regional microbial source pools jointly shape ecological roles. Moreover, the observed patterns of mobile genetic elements and horizontal gene transfer suggest that gene exchange predominantly occurs within microbial lineages, *Correspondence: Robert M. Bowers A. Carolin Frank Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Bowers et al. Microbiome (2025) 13:222 Page 2 of 21 with occasional broader transfers dispersing key functional genes (e.g., those involved in polysaccharide metabolism), which may facilitate microbiome adaptation. Keywords Microbial ecology, Phyllosphere, Conifer, Metagenome, Mobile genetic elements Background Coniferous forests, which dominate temperate and boreal ecosystems, provide critical ecosystem services such as carbon sequestration [97], regulation of water cycles [11], soil erosion control [99], and habitat for diverse plant and animal species. However, these forests are increasingly under threat from climate change [115]. Current perspectives on forest management often overlook the significance of forest tree microbiomes. These microbiomes comprise ectomycorrhizal (ECM) fungi [47] alongside other fungal and bacterial species adapted to forest vegetation and soil [4]. A better understanding of forest microbiomes is essential due to their role in maintaining forest health, resilience, and biodiversity. A significant portion of this biodiversity resides on the aerial surfaces of plants, also known as the phyllosphere, one of the largest microbial habitats on Earth [75, 124]. Microbes that reside on leaves are vital for nutrient cycling, plant growth, and mitigating both biotic and abiotic stress [75], yet while agricultural and model plant phyllospheres have been extensively studied [66, 72], the phyllospheres of forest trees remain understudied. The phyllosphere hosts a community of airborne generalists [14] that is shared among plants of different species [104, 124]. Leaf surface microbes are dispersed via aerosols and dust particles, and wind facilitates their movement over short and long distances. While the phyllosphere can act as a passive aerosol sampler [43], persistent microbial communities are maintained through plant–microbe interactions and adaptation to leaf environments [122]. The leaf community of a given plant is influenced by plants growing in the immediate vicinity but also with additional microbial signatures from more distant plant surfaces [81]. Conifer forests provide a vast and enduring reservoir of phyllosphere microbes. Unlike the ephemeral leaves of many agricultural plants, conifer needles offer a long-lived surface for microbial colonization, with individual needles persisting for years to decades [37]. The trees themselves can live for centuries or even millennia, sustaining these microbial communities over extended timescales. Studies of other evergreen plants, such as sagebrush [48], demonstrate that persistent communities of leaf-associated microbes, including at least 20 fungal genera, are maintained over time and are influenced by both weather and leaf age. Similarly, conifers likely provide stable habitats and significant reservoirs for phyllosphere-associated microbial communities, supporting interactions across both nearby and distant habitats and plant ecosystems. For example, in boreal forests, the composition of the microbial community living on nitrogen‑fixing mosses depends on both the moss species and the surrounding canopy structure [56]. A higher proportion of conifers in the overstory is associated with greater α‑diversity in (...truncated)