（1）The phyllosphere, or above-ground part of plants, hosts diverse bacterial communities that play critical ecological roles and provide beneficial functions for the plant.叶际，即植物的地上部分，承载着多样化的细菌群落，这些群落发挥着关键的生态作用，并为植物提供有益功能。

（2）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 remain largely uncharacterized in the phyllosphere.丝杆菌目（ Hyphomicrobiales，属于α-变形菌纲）是一个高度多样且生态重要的分支，已知是植物微生物组的关键成员，尤其是在与植物根部结合时，但其在叶际中的多样性在很大程度上仍未得到表征。

A phylogenomic and metagenomic meta-analysis of bacterial diversity in the phyllosphere lifts a veil on Hyphomicrobiales dark matter

（1）The phyllosphere, or above-ground part of plants, hosts diverse bacterial communities that play critical ecological roles and provide beneficial functions for the plant.叶际，即植物的地上部分，承载着多样化的细菌群落，这些群落发挥着关键的生态作用，并为植物提供有益功能。

（2）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 remain largely uncharacterized in the phyllosphere.丝杆菌目（ Hyphomicrobiales，属于α-变形菌纲）是一个高度多样且生态重要的分支，已知是植物微生物组的关键成员，尤其是在与植物根部结合时，但其在叶际中的多样性在很大程度上仍未得到表征。

（3）Using a meta-analysis combining metabarcoding, metagenomics and phylogenomics, we explored the worldwide diversity of leaf-associated Hyphomicrobiales.通过一项结合了宏条形码、宏基因组学和系统发育基因组学的荟萃分析，我们探索了全球范围内与叶片相关的丝杆菌目的多样性。

（4）We confirmed Methylobacterium was ubiquitous in the phyllosphere and revealed the dominance of two under-characterized Hyphomicrobiales taxa: Lichenibacterium , a lichen-associated genus previously identified as “1174-901-12” in taxonomic databases, and RH-AL1, an undescribed lineage of bacteria related to Beijerinckiaceae, previously isolated from coal slag.我们证实了甲基杆菌属（Methylobacterium）在叶际中无处不在，并揭示了两个未被充分研究的丝杆菌目类群的优势地位：地衣杆菌属（Lichenibacterium），一个与地衣相关的属，之前在分类数据库中被标识为“1174-901-12”；以及 RH-AL1，一个与贝耶林克氏菌科（Beijerinckiaceae）相关、此前从煤渣中分离出来的未被描述过的细菌谱系。

（5）Despite their abundance in the phyllosphere, Lichenibacterium and RH_AL1 could not be properly detected by 16S rRNA gene barcoding, due in part to limitations of taxonomic resolution of the 16S rRNA gene and of representativeness in existing taxonomic databases, underlining limitations of this approach for their accurate identification in the phyllosphere.尽管地衣杆菌属和 RH-AL1 在叶际中含量丰富，但它们无法被 16S rRNA 基因条形码技术正确检测到，部分原因是 16S rRNA 基因的分类学分辨率有限，以及现有分类数据库的代表性不足，这凸显了该方法在叶际中准确识别它们的局限性。

（6）As for Methylobacterium, a significant proportion of Lichenibacterium and RH-AL1 were also detected in association with lichens and in environments with harsh conditions like exposed surfaces, air and snow, suggesting airborne or waterborne dispersal and high resilience in harsh environments.与甲基杆菌属类似，地衣杆菌属和 RH-AL1也有很大一部分被检测到与地衣相关，或在暴露的表面、空气和雪等恶劣环境条件下被发现，这表明它们通过空气或水进行传播，并且在恶劣环境中具有高适应性。

（7）Overall, our study stresses the need to move toward metagenomics and culturomics to increase the representativeness of leaf-associated bacterial taxa in reference databases, and to improve our understanding of the evolutionary and functional mechanisms underpinning bacteria adaptations to living on plants. 总的来说，我们的研究强调了转向宏基因组学和培养组学的必要性，以提高与叶片相关的细菌类群在参考数据库中的代表性，并加深我们对细菌适应植物生活方式的进化和功能机制的理解。

（8）Result

（9）Phyllosphere-associated Hyphomicrobiales MAGs mostly belong to Methylobacteriaceae, Lichenihabitantaceae and two undescribed taxa   叶际相关丝杆菌目的MAGs主要属于甲基杆菌科、地衣栖息菌科和两个未被描述的类群

基于 96 个参考基因组（NCBI RefSeq 基因组）和 73 个 MAGs（来自树叶、苔藓和地衣）构建的系统发育基因组树。核心发现是：叶际相关的丝杆菌目 MAGs 主要属于 Methylobacteriaceae（甲基杆菌科）、Lichenihabitantaceae（地衣栖息菌科）以及一个新类群 RH_AL1。

（10） Limits and improvements in the classification of phyllosphere-associated Hyphomicrobiales taxa with 16S rRNA metabarcoding and current databases   基于16S rRNA宏条形码和现有数据库对叶际丝杆菌目分类的局限性与改进

（11） Lichenihabitantaceae and Methylobacteriaceae are dominant phyllosphere-associated Hyphomicrobiales taxa worldwide according to 16S rRNA metabarcoding      根据16S rRNA宏条形码分析，地衣栖息菌科和甲基杆菌科是全球叶际中最主要的丝杆菌目类群

（12） Comparison of 16S rRNA and rpoB-based barcoding for the identification of phyllosphere-associated Hyphomicrobiales taxa  比较16S rRNA和基于rpoB的条形码在鉴定叶际丝杆菌目类群中的应用

（6）As for Methylobacterium, a significant proportion of Lichenibacterium and RH-AL1 were also detected in association with lichens and in environments with harsh conditions like exposed surfaces, air and snow, suggesting airborne or waterborne dispersal and high resilience in harsh environments.与甲基杆菌属类似，地衣杆菌属和 RH-AL1也有很大一部分被检测到与地衣相关，或在暴露的表面、空气和雪等恶劣环境条件下被发现，这表明它们通过空气或水进行传播，并且在恶劣环境中具有高适应性。

（7）Overall, our study stresses the need to move toward metagenomics and culturomics to increase the representativeness of leaf-associated bacterial taxa in reference databases, and to improve our understanding of the evolutionary and functional mechanisms underpinning bacteria adaptations to living on plants. 总的来说，我们的研究强调了转向宏基因组学和培养组学的必要性，以提高与叶片相关的细菌类群在参考数据库中的代表性，并加深我们对细菌适应植物生活方式的进化和功能机制的理解。