Table_1_Species Boundaries and Molecular Markers for the Classification of 16SrI Phytoplasmas Inferred by Genome Analysis.xlsx

Phytoplasmas are plant-pathogenic bacteria that impact agriculture worldwide. The commonly adopted classification system for phytoplasmas is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains in the 16SrI group within the genus ‘Candidatus Phytoplasma’ and investigated the possible species boundaries. We confirmed that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units (OTUs) equivalent to species. Strains assigned to the same OTU share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different OTUs share < 94% ANI and < 75% of their genes. Reduction in homologous recombination between OTUs is one possible explanation for the discontinuity in genome similarities, and these findings supported the proposal that 95% ANI could serve as a cutoff for distinguishing species in bacteria. Additionally, critical examination of these results and the raw sequencing reads led to the identification of one genome that was presumably mis-assembled by combining two sequencing libraries built from phytoplasmas belonging to different OTUs. This finding provided a cautionary tale for working on uncultivated bacteria. Based on the new understanding of phytoplasma divergence and the current genome availability, we developed five molecular markers that could be used for multilocus sequence analysis (MLSA). By selecting markers that are short yet highly informative, and are distributed evenly across the chromosome, these markers provided a cost-effective system that is robust against recombination. Finally, examination of the effector gene distribution further confirmed the rapid gains and losses of these genes, as well as the involvement of potential mobile units (PMUs) in their molecular evolution. Future improvements on the taxon sampling of phytoplasma genomes will allow further expansions of similar analysis, and thus contribute to phytoplasma taxonomy and diagnostics.

植原体（Phytoplasmas）是一类侵染植物的病原细菌，对全球农业生产造成严重危害。当前主流的植原体分类系统基于其16S核糖体RNA（16S rRNA）基因的限制性片段长度多态性（RFLP）分析构建。随着植原体基因组序列数据的日益丰富，该分类系统得以进一步优化完善。本研究针对"候选植原体属（‘Candidatus Phytoplasma’）"中16SrI组的11个菌株展开分析，探究了其潜在的物种边界。研究证实，由于16S rRNA基因存在基因组内变异，且不同核苷酸位点的权重不均一，传统的RFLP分类方法存在局限性。值得注意的是，基于分子系统发育分析、染色体片段差异、基因组成差异以及同源序列分歧度的研究结果，均支持将这些菌株划分为多个等价于物种的操作分类单元（OTUs）。归属于同一操作分类单元的菌株，其全基因组平均核苷酸一致性（ANI）高于97%，且共有编码蛋白基因的比例超过78%；与之相对，不同操作分类单元的菌株ANI低于94%，共有编码蛋白基因的比例不足75%。操作分类单元间同源重组的减少，或许是基因组相似性出现间断分布的原因之一，上述研究结果也支持了"以95%平均核苷酸一致性作为细菌物种区分阈值"的提议。此外，通过对研究结果及原始测序读长数据的严格校验，我们发现有一个基因组是通过混合来自不同操作分类单元的植原体测序文库组装得到的，属于错误拼接的结果。这一发现为研究未培养细菌的科研人员敲响了警钟。基于对植原体进化分歧的新认知以及现有基因组数据资源，本研究开发了5个可用于多位点序列分析（MLSA）的分子标记。这些标记片段短小且信息含量高，同时在染色体上分布均匀，构建出了一套成本效益优异且抗重组干扰能力强的分型系统。进一步对效应因子基因分布的分析证实了这类基因的快速获得与丢失现象，同时也揭示了潜在移动单元（PMUs）在其分子进化过程中的作用。未来通过扩大植原体基因组的类群采样范围，可推动同类分析的进一步拓展，从而为植原体的分类鉴定工作提供更为坚实的支撑。