Fig. 6.

A limited range of cultured core symbiont strains recycle N in vitro. Results summarize findings from symbionts of five cephalotine ant species, including four from the genus Cephalotes and one from its sister genus Procryptocerus. a Genes and pathways used by specialized gut symbionts to recycle the N-wastes uric acid and urea. The conserved architecture for clusters of symbiont N-recycling genes is illustrated. Red boxes within these pathways represent the metabolic steps assayed for (b). b Shown at left are phylogenies of cultured symbionts subject to metabolic assays in vitro and their closest relatives in the NCBI database, which are highlighted with taxon-specific, colored boxes. Most cultured isolates had 16S rRNA sequences that were identical or highly related to at least one sequence obtained from a Cephalotes ant through culture-independent means. Genomes from such isolates also showed high similarity to abundantly represented scaffolds from our metagenomes (Supplementary Fig. 15). Nodes for cultured symbionts are connected to relevant rows within data tables, where the results of assays for urea production and urea degradation assays are illustrated. Asterisks highlight isolates with a sequenced genome; for each of these, in vitro results matched expectations derived from genome content. Additional symbols used in the urea production table indicate whether allantoin boosted urea production and whether urea production was completely allantoin dependent, and hence likely dependent on uric acid metabolism (e.g., urea can separately be produced through arginine metabolism). Three biological replicates were run as a single trial for 13 symbionts subjected to the urea production assay. For four strains we ran one or two additional trials (Supplementary Data 5). Statistical analyses included a two-way, repeated measures ANOVA to examine an effect of allantoin on urea production, an effect of time, and an interaction between these two factors. Holm–Sidak tests were then used for pairwise comparisons between treatments (e.g., allantoin presence vs. absence) at particular time points, for all trials with a significant effect of treatment or a treatment by time interaction effect