# Phylosymbiosis in the Rhizosphere Microbiome Extends to Nitrogen Cycle Functional Potential
## 4 Discussion
The nif nitrogenases are the best described and their sequences are abundant in reference databases. Indeed, as of July 2020, the KEGG gene database identified >800 nif, ~50 anf, and only 30 vnf nitrogenases [38; 40]. We thus focused on nif nitrogenases but also identified sequences for anfG (K00531, 117 aa) as a proxy to investigate the presence of alternative nitrogenases. However, as only a few sequences for anfG were detected in some samples, their distribution will not be further discussed here. Regarding the relative abundance of nif nitrogenase, the sequences for nifH (K02588, 293 aa), nifK (K02591, 458 aa), _nifD_ (K02586, 477 aa), and _nifW_ (K02595, 124 aa) are unevenly distributed within and across samples. This reflects the complex evolution of _nif_ nitrogenase. Indeed, among the others, _nifH_ has a distinct evolutionary history relative to the other _nif_ genes and is not always located within the nif operon [61]. In addition, several genomes contain multiple copies of _nifH_ (e.g., _Rhodospseudomonas palustris_) [58; 62; 63; 61]. Finally, the potential for N fixation in sequenced bacterial genomes has shallow phylogenetic conservatism (an average clade depth of ~0.018 165 rRNA distance) [51]. Together, these points suggest that N-fixers have discrete signature sets of sequences supporting N fixation. In consequence, although one could expect to find a consistent and stable potential for N fixation, the variation in the distribution of _nif_ sequences identified here suggests that plants select for N-fixing lineages endowed with specific sets of genes supporting N fixation. The distribution of the more abundant traits for the conversion of nitrate to nitrite and nitrite to ammonia across samples mirrors their abundance in sequenced genomes, with KEGG genes identifying >3000 _nirB_ nitrite reductase (K00362) and >300 nitrate reductase (K10534), among others [38; 40]. Despite being abundant and potentially beneficial to both the rhizosphere's microbial community and thus to the plant host, the distribution of these sequences does not correlate with the plant phylogeny (Figure 5, Table 1). Next, the distribution of sequences for ammonia assimilation was less variable across samples than the sequences for less abundant pathways (Figure 5, Table 1). However, even within the ammonia assimilation pathway, sequence abundance was variable. This suggests some variation in gene content in the microbial lineages inhabiting the rhizosphere and highlights how essential the N recycling function is for the microbiome and the plant. Globally, the functional potential for ammonia assimilation, although correlated with the microbial community composition, is only marginally affected by the plant phylogeny (Figure 5, Table 1). Next, the distribution of traits for nitrification and denitrification fluctuated as the microbial taxonomic community changed, but this was not correlated with the plant phylogeny (Table 1). Denitrification ultimately causes the loss of bioavailable N, which has an adverse effect on plant growth in N-limited environments. Although our reductive approach provided new information supporting the phylosylmbiosis in plant root-microbiome associations, we recognize that the ability of the plants to control their root-associated microbial community, as described in this common garden experiment, might deviate from the natural environment. Among other reasons, the homogenized standard soil mixture used in our experiment likely comes with a distinct microbial community. However, the plant's ability to promote a specific microbial community remains and we believe our experimental design is useful for studying the plant root-microbiome association in plants sampled globally. Indeed, at the global scale, a reciprocal transplant experiment would introduce many additional variables, such as soil, season, precipitation, and temperature variability.