# Phylosymbiosis in the Rhizosphere Microbiome Extends to Nitrogen Cycle Functional Potential
## 4 Discussion  
The plant's ability to control the root-associated microbial community is well established [44; 45]. However, we have not fully explored how plant evolutionary variation plays a role in the rhizosphere community composition. Here, we used a common garden experiment to link plant relatedness to their ability to control the root-associated microbial communities. To our knowledge, this is the first time that this approach has been used at this scale with plants sampled from around the world. We used chloroidoid grasses grown in a common garden experiment to reduce environmental heterogeneity and to investigate how the phylogeny of these cosmopolitan grasses affects the structure and function of the rhizosphere's bacterial microbiome. We allowed the microbial community to develop from the greenhouse environment, the soil mixture, and the seeds. Although all the grasses were maintained in unnatural greenhouse conditions and received the same treatment (e.g., water, soil), none displayed abnormal or delayed growth. The subfamily Chlorideae contains approximately 1700 species known to be adapted to mostly arid habitats [46]. This lineage of grasses seems to have originated in Asia or Africa and has its center of diversity in Africa. Chloroidoids have since spread to the other continents multiple times, with radiations in North America, South America, and Australia [46]. Although some of the taxa had been characterized before, many were sequenced for the purpose of this study. The resulting phylogeny of the 26 chloroidoid grasses and the outgroup, _Danthoniopsis ramosa_ (Figure 1A), corroborated the relationships  
Figure 5: Interactions among plant phylogeny, microbial community composition, and the functional potential for N cycling pathways. (**A**) Correlogram for the N cycling pathways, rhizosphere microbial community composition, and the plant phylogeny. (**B–F**) Scatterplots with linear regressions showing the relationships among plant phylogeny, microbial community composition, and the functional potential for N cycling. In all of the analyses, pairwise comparison among replicate samples were systematically excluded. found in previous studies that sampled more taxa and loci [46; 47; 48]. Importantly, species sampled in duplicate and species in the same genus were monophyletic. The rhizosphere microbiome was investigated using metagenome sequencing. Replicate samples (from the same plant) and samples from closely related plants (same species or genus) displayed a highly similar bacterial microbial community composition (Figure 2, Table 1). This validated our experimental design and suggested that the rhizosphere's microbiome assembly is not purely random. Moreover, dissimilarity in the taxonomic composition of the rhizosphere's bacterial microbiome was correlated with the plants' phylogenetic distance (Figure 2, Table 1), thus suggesting a phylosymbiosis between chloroidoid grasses and their rhizosphere' microbiomes. Thus, the grass species have inherited the tendency to associate with particular bacterial lineages. In the studied rhizosphere, some bacterial lineages remained abundant and formed a \"core microbiome\" (accounting for \(\sim\)30% of the reads; Figure 3). This core microbiome could reflect a signal conserved across all the studied plants, an external constraint imposed by our experimental design (e.g., soil type), or a combination of both. However, besides the core microbiome, each plant had a unique bacterial community with many rare microbial lineages and whose precise variation (composition and abundance) was correlated with the plant phylogeny. This confirmed that, even in unnatural conditions, chloroidoid grasses control the composition of their root-associated microbiome. Understanding the precise mechanism that plants use to select their rhizosphere remains a major challenge [22]. Some studies have investigated the microbial community composition using amplicon sequencing and inbred plants to explain the variation in plant phenotype (e.g., resistance to desiccation) [3; 26; 34].