# Phylosymbiosis in the Rhizosphere Microbiome Extends to Nitrogen Cycle Functional Potential
## 4 Discussion
Interestingly, in 2018, [PERSON] et al. analyzed the composition of the rhizosphere and endosphere microbiome in 30 distantly related angiosperms and identified no correspondence between the rhizosphere and the plant phylogeny. Only the endosphere's microbiome composition was strongly affected by the plant phylogeny, although the endosphere's and the rhizosphere's microbiome compositions were correlated [3]. These previous approaches highlighted the importance of the microbial community for plant health and revealed plants' genetic determinants supporting the microbial community assembly. However, as genetic material and functional potential vary greatly among microbial lineages, it is essential to investigate how these structural variations relate to change in functions. Although this can be achieved through the targeted amplification of specific genes (e.g., _nifi_) [49; 50], whole-metagenome sequencing provides a much broader picture, allows for the combined analysis of the composition and the functional potential, and allows us to compare multiple traits. We found that changes in the microbiome composition mirrored the variation in the overall distribution of functional traits supporting N cycling (Figure 4). The functional potential for N utilization and N cycling is not randomly distributed in microbial communities, just as there is a non-random pattern in carbon utilization across ecosystems more broadly [20]. This is further supported by the clumped distribution of traits for N fixation in the sequenced bacterial lineages [51], and suggests that the overall distribution of the other traits for N cycling are, to some extent, also phylogenetically constrained in microbes. Since analyses of the relationship between overall microbiome composition and overall functional potential are biased towards the most abundant traits (i.e., ammonia assimilation), we analyzed each N cycling pathway independently. Pathways producing bioavailable N (e.g., N fixation and sequential conversion of nitrate to nitrite and ammonia), processes contributing to the recycling of N (e.g., ammonia assimilation), and processes causing the loss of bioavailable N (e.g., denitrification) have different impacts on the microbiome, on the plant, and thus on the whole ecosystem's functioning. The distribution of all the traits, except processes supporting the production of nitrite, showed some degree of microbial phylogenetic constraint, as the variation in each functional potential correlated with the microbial community composition. However, only a few N pathways were also affected by the plant phylogeny (Figure 5). Not all the investigated N processes are under selection or resulted in a phylosymbiotic pattern. Sequences for pathways supporting the production of bioavailable N are essential for the whole ecosystem yet are among the least abundant sequences in chloroidoid grass rhizosphere. This is consistent with previous findings [4; 5]. This also suggests that the abundance and distribution of N-fixing bacteria are restricted. Among other reasons, the amount of unavailable N and the availability of other nutrients (e.g., phosphorus) are essential factors affecting the development of N-fixers [52; 53]. In addition, predatory invertebrates [54] and microbial antibiotic production [55; 56] also have adverse effects on N-fixers. At the molecular level, N fixation is supported by highly conserved molybdenum-nif nitrogenase multiprotein complexes encoded in the _nif_ operon [52; 57]. In addition, two alternative nitrogenase systems are known, namely vanadium-vnf nitrogenase and the alternative-anf nitrogenase. These nitrogenases share similar proteins; however, the two alternative complexes contain an additional protein (i.e., vnfG and anfG). Nitrogenase complexes are not consistently distributed across microbial lineages, with most N-fixers having one or two of these operons [58]. The alternative nitrogenases likely evolved in response to environmental variations in metal availability [59] and are activated under Mo limitation [60].