*2.3. Sequence Analysis*

*N. furzeri* NUCB2 gene structure was recovered from the *Nothobranchius furzeri* Genome Browser–NFINgb [34], while human, mouse, and zebrafish sequences were recovered from the Ensembl Genome Browser [35]. The gene structure analysis was based on sequences retrieved by the Ensembl Genome Browser (Table S1). The evolutionary history was inferred using the Minimum Evolution method [36]. The optimal tree with the sum of branch length = 3.06014219 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [37] and are in the units of the number of amino acid substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm [38] at a search level of 1. The Neighbor-joining algorithm [39] was used to generate the initial tree. This analysis involved 5 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 496 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [40]. NUCB2 aminoacidic sequences were recovered from the National Center for Biotechnology Information–NCBI [41] and the alignment was performed using Clustal

Omega [42]. Identity percentage among sequences was calculated with the Basic local alignment search tool—Blast [43].
