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

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 (Dos Santos et al. 2012). Sequences were extracted from the DRAM annotation of the dereplicated MAGs, including the genome of Vibrio natrigensis, which also served as the outgroup for the gene tree. MAGs that did not contain the full set of genes and were excluded from the analysis. Each renamed gene extraction (Rodriguez-R and Konstantinidis 2016) was aligned independently with FSA v1.15.9 (Bradley et al. 2009) 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 (Shen et al. 2016) and a tree inferred from it. For the reconstruction of ancestral states and the phylogenetic reconciliation we inferred a de-replicated version of the global symbiont phylogenomic tree. For this, the diversity of MAGs was reduced by following the dRep v3.2.2 (Olm et al. 2017) dereplicate workflow with the S_ANI parameter set to 0.99. To maintain the topology of the original tree, MAGs classified as Ca. Thiodiazotropha endolucinida Caribbean had to be dereplicated independently with the S_ANI flag set to 0.995 (to increase the represented diversity of the clade). The genome of Vibrio natrigensis genome (GCF_001456255.1), which was used as an outgroup, was downloaded from the NCBI database.Both trees wereinferred with IQ-Tree v2.2.2.1 (Minh et al. 2020; Hoang et al. 2018; Kalyaanamoorthy et al. 2017) 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 TN+F+I+G4, and the one for the dreped tree was Q.insect+F+I+G4