# Phylosymbiosis in the Rhizosphere Microbiome Extends to Nitrogen Cycle Functional Potential
## 3 Results
### Functional Potential for Nitrogen Cycling  
Across the sequenced microbiomes, variation in the microbial community composition (at the genus level) and the overall distribution of sequences supporting N cycling were highly correlated (r\({}_{\text{Pearson}}\) = 0.72, \(p\) < 0.001; r\({}_{\text{Spearman}}\) = 0.83, \(p\) < 0.001), thus confirming that a change in microbial community composition affects the functional potential for N cycling (Figure 2B). The plant origin had no significant effect on the relationship between the microbial community composition and the functional potential for N cycling (Supplementary Figure S2B). Sequences for N cycling pathways accounted for 3.4 to 3.9% of the annotated reads. After rarefaction (\(n\) = 1,207,202), identified sequences involved in N cycling were not evenly distributed (Supplementary Figures S4 and S5). The most prevalent sequences were for the ammonia assimilation and assimilatory nitrite to ammonia pathways. Specifically, short-read sequences matching long glutamine synthase (K01915, 529 aa), glutamate synthase (K00266, 610 aa), asparagine synthase (K01953, 529 aa), and glutamate dehydrogenase (K00261, 534 aa) together accounted for ~43% of the identified sequences for N cycling. Conversely, sequences for N fixation and denitrification accounted for ~3.25% and 0.43% of the identified sequences, respectively. As the frequency of target sequences in the short-read metagenome reflected both the abundance of the sequence of interest in the sample and their length, we normalized the sequence count according to the corresponding domain length in the KEGG reference database. Accounting for the size of the targeted sequences revealed that the frequency of short sequences for the nitrite reductase small subunit (K00363, 175 aa), nrfC (K04014, 334 aa) and nitrogenase nifH (K02588, 293 aa) tended to be underestimated relative to the longer sequences, including the nitrite reductase large subunit (K00362, 1096 aa), among others (Supplementary Figure S5). Figure 3: Microbial community structure across plants. NMDS ordination of the grass samples (colored by continent; see Supplementary Table S1) according to their microbial community composition. The identified bacterial genera are colored by phylum and overlaid with a 2D kernel density plot to highlight the phyla. The bacterial genera most affected by the plant phylogeny are labeled. Numbers in the colored boxes represent the plants (see Supplementary Table S1). See Supplementary Figure S3 for a high-resolution figure with all bacteria genera labeled. Within samples, sequences for the same pathway generally displayed a consistent distribution (Figure 4). For example, the frequency of sequences for assimilatory nitrite to ammonia correlated with each other. This pattern was also identified for sequences in less abundant pathways such as the dissimilatory nitrate to nitrite and the denitrification pathways. We next investigated sample clustering based on the distribution of the sequences for N cycling pathways (Figure 4). The functional clustering produced two major clusters. In Cluster I, we found traits for assimilatory nitrate to nitrite, assimilatory nitrate to ammonia and dissimilatory nitrate to nitrite were overabundant relative to Cluster II. Conversely, Cluster II was enriched in traits for ammonia assimilation. Finally, the distribution of traits supporting the denitrification, dissimilatory nitrate to ammonia, and nitrification pathways were more evenly distributed between the two clusters. Replicates tended to cluster together (\(p<0.001\)). Combining the replicate samples and performing the NMD  
Figure 4: Functional potential for N cycling pathways across sequenced microbiomes. The microbiomes are identified by the plant number (see Figure 1 and Supplementary Table S1) and replicate (A or B; see Table S2). The traits involved in N cycling are grouped by pathways and are identified by the reaction number and KO ID (see Supplementary Table S3).