*2.2. Clustering Based on Secondary Structures*

For each existing domain of ACE2 of the nineteen species, the secondary structure was predicted (Figure 4). For each domain, species are grouped into several subgroups.

Concerning the D1 domain:


These individual eight clusters show six different secondary structures in D1 shared by sixteen species, which shows high similarities in their secondary structures, while the remaining two have a unique secondary structure for D1 domain. Thus, these eight clusters have similar secondary structures indicating that the species in the eight clusters are closely related.

**Figure 2.** Multiple sequence alignments of D1, D2, and D3 domains of ACE2 of nineteen species (**A**) and respective phylogenies (**B**).

**Figure 3.** Clusters of species based on domain-based sequence homology.

With respect to the D2 domain:


Similarly, for the D2 domain, we found four clusters with the same secondary structure, indicating that they are closely related.

With respect to the D3 domain:


Again for the D3 domain, four different clusters were bearing similar secondary structures; therefore, these species are also closely related.

Based on the similarity among the three domains, all eighteen species were clustered (Figure 5). From the clusters (Figure 5) based on the secondary structure of the three domains of ACE2, it was observed that the species S4 was clustered uniquely, though S4 is clustered with S9 and S19 based on ACE2 full-length sequence homology. Furthermore, S6 and S13 were found to be similar based on ACE2 homology, but they got clustered into two different clusters when the secondary structure of three domains was concerned. In contrast, the group of species {*S*1, *S*2, *S*3}, {*S*9, *S*12}, {*S*16, *S*18}, and {*S*5, *S*7, *S*11} remained in the same clusters concerning ACE2 homology as well as individual secondary structures of the domains.


**Figure 4.** Predicted secondary structures of D1, D2, and D3 domains for 18 species and only D3 domain for *Salmo salar*.

**Figure 5.** Clusters of species based on the secondary structures of the D1, D2, and D3 domains.
