Figure 4. Alignment and tree of nifHDKT and of dreped MAGs for reconcilation and ancestral reconstruction

For the reconstruction of ancestral states and the phylogenetic reconciliation, all genomes and MAGs of the order Chromatiales that were available in GTDB were included in the analysis. MAGs classified as Desulfuromonas in GTDB were also included as outgroup. The diversity of MAGs was reduced by following the dRep v3.2.2 dereplicate workflow with the S_ANI parameter set to 0.995 and a phylogenomic tree was inferred from the dereplicated MAGs. Sequences of the nifH, nifD, nifK and nifT genes were chosen to infer the evolutionary history of nitrogen fixation because they form an operon with the minimum catalytic gene set for the reaction. Sequences were extracted from the anvi’o annotation (described in the section MAG annotation and metabolic reconstruction) of the dereplicated MAGs. MAGs that did not contain the full set of genes were excluded from the analysis. Each gene extraction was aligned independently with FSA v1.15.9 using the --fast flag. A tree was inferred from each alignment to ensure that the genes have evolved together. Subsequently, the alignments were concatenated using SeqKit v2.3.0 and a tree inferred from it. Both trees were inferred with IQ-Tree v2.2.2.1 with auto substitution model detection, 1,000 ultrafast bootstrap (UFB) replicates and 1,000 samples for SH-aLRT branch testing. Nodes with values of UFB greater or equal to 95% and of SH-aLRT greater or equal to 80% were considered to be strongly supported. The nifHDKT best-fit model was GTR+F+I+R6, and the one for the dreplicated tree was Q.insect+F+R9