**3. Results**

## *3.1. Genome-Wide Analysis of SOD Gene Family in Four Water Lily Species*

In this comparative study, we discovered 43 *SOD* genes in the genomes of four water lily species. Protein sequences of eight *A. thaliana* (*AtSODs*) were used as queries and removed the repetitive redundant sequences (Table S2), and 9–15 genes were obtained for each species, for example three diploid water lilies *N. colorata* (9 *SODs*), *N. thermarum* (10 *SODs*), *N. minuta* (9 *SODs*), and a tetraploid water lily *N. mexicana* (15 *SODs*) (Table 1; Table S3). After conducting domain scrutiny, we identified 15 proteins with a *Cu*/*Zn-SODs* domain (Pfam; 00080), 19 with a *Fe-SODs* domain (Pfam; 00081), and 9 with a *Mn-SODs* domain (Pfam; 02777) in water lily species. These results are consistent among all species and contain all *SOD* genes and domains with sub-families.

**Table 1.** Characteristics of the *SOD* genes from four water lily species.


The biochemical and physiological characteristics of all *SODs* were investigated by calculating various parameters (Table 1). Protein length in the four representative water lily species ranged from 117 to 362 amino acids. Consequently, the molecular weight was found to be from 12.166 to 40.905 kDa. Most of the species investigated in this study displayed acidic properties, exhibiting pI values among 4.68 to 10.15. Furthermore, the predicted GRAVY values of *SOD* proteins were negative, showing that they are hydrophilic.

According to the subcellular localization prediction, the majority of water lily *CSDs* were found in the cytoplasm, while only a few localized in the chloroplast. Water lily *FSDs* and *MnSDs* were specifically located in the chloroplast and mitochondria, respectively (Table 1).

Furthermore, an NCBI domain search was conducted to perform domain-based analysis on all *SOD* proteins to acquire data and TBtools was then used to make the domain structures. As a result of this analysis, the presence of the *SOD* family was verified on all chosen protein sequences (Figure 1).

**Figure 1.** Symbolic *SOD* domain structures of four water lily species: (**a**) *Nymphaea colorata*; (**b**) *Nymphaea minuta*; (**c**) *Nymphaea mexicana*; and (**d**) *Nymphaea thermarum*. Among four species only *Nymphaea minuta* contains Mn and Cu/Zn, while others constitute all three subfamilies of *SOD*.

#### *3.2. Phylogenetic Relationships and Conserved Motif Analysis in Representative Water Lily Species*

To uncover the evolutionary relations among *SODs* in various plant species, a phylogenetic tree was created using the complete protein sequences. The present research investigated the evolutionary relationships among genes of *NcSODs*, *NtSODs*, *NminSODs*, *NMSODs*, and *AmtSODs*. By considering the domains (*Fe-SODs*, *Cu*/*Zn-SODs*, and *Mn-SODs*) and analyzing a phylogenetic tree, a total of 50 *SODs* were classified into two main groups (Figure 2a). To assess the structural diversity of water lily *SOD* proteins and predict their functions, we utilized the MEME software to analyze their full-length protein sequences and identify conserved motifs. By examining conserved motifs in the *SOD* family, this analysis confirmed the categorization and evolutionary connections among *SOD* genes within the water lily species. The investigation revealed that 10 conserved motifs were present in all species (Figure 2b). The detailed information about the identified motifs, including their names, sequences, and widths is displayed in Table S4. The number of conserved motifs in *SOD* proteins ranged from two to six, and their distribution aligned with the groups. The *Cu-SOD* and *Fe-SOD* groups had only one motif in two genes (Figure 2b). Furthermore, *MnSODs* and *FeSODs* were classified in the similar group and subcluster, whereas *Cu*/*ZnSODs* were placed in a distinct group. Interestingly, motifs 1, 2, 4, and 9 were estimated to be particular to *Cu*/*Zn-SOD* whereas motifs 3, 5, 6, and 10 were exclusive to the *MnSODs* and *FeSODs* groups. In conclusion, the reliability of group arrangements was strongly supported by analyzing conserved motif patterns and phylogenetic relationships between water lily species. This suggests that water lily *SODs* proteins possess highly conserved amino acid residues within groups. Consequently, it is reasonable to infer that proteins with analogous structures and motifs may have similar efficient roles.

**Figure 2.** Classification of *SOD* genes according to their subfamilies: (**a**) A neighbor-joining phylogenetic tree; (**b**) Conserved motif analysis. The motifs supported the two subfamilies which are mentioned in tree. Reliability of group arrangements was strongly supported by analyzing conserved motif patterns and phylogenetic relationships between water lily species. Different types of motifs represented by differently colored boxes.
