*2.4. Phylogeny and Clustering Based on Polarity*

In the D1 domain, it was observed that the polarity of thirteen amino acids among nineteen (24–42 aa) amino acids were found to be conserved across eighteen species. Based on the amino acids' polarity and non-polarity nature, the species were arranged in a phylogenetic tree (Figure 11).

It was found that *Homo sapiens, Pan troglodyte, Macaca mulatta*, and *Danio rerio* closer according to this analysis. *Pteropus alecto, Pteropus vampyrus*, and *Sus scrofa* occurred in parallel along with the above three and formed a different clade indicating the closeness based on polarity. Again, the case for *Gallus gallus, Rhinolophus ferrumequinum, Mustela putorius furo, Equus caballus*, and *Felis catus* is similar. Two separate groups, *Mesocricetus auratus, Manis javanica*, and *Capra hircus, Bos taurus*, were similarly placed nearby, indicating that the polarity of amino acids of the proteins for these species was similar. *Pelodiscus sinensis* and *Rattus norvegicus* occurred separately and were not grouped with any other species but bears similarity with both the groups containing species *Mesocricetus auratus, Manis javanica, Pteropus alecto, Pteropus vampyrus, and Sus scrofa*.

In the D2 domain, out of six amino acid long sequences, the polarity of three amino acids was conserved across eighteen species, and among them, one amino acid was a binding residue. *Homo sapiens, Pan troglodytes, Macaca mulatta, Pteropus vampyrus*, and *Pteropus alecto* were grouped together since the overall polarity of their amino acid chain was found to be similar, and, simultaneously, *Danio rerio, Mustela putorius furo, Gallus gallus*, and *Pelodiscus sinensis* were placed together. In addition, three groups comprising *Manis javanica, Capra hircus*, *Bos taurus, Sus scrofa*, *Rattus norvegicus, and Rhinolophus ferrumequinum*, respectively, were placed in close proximity based on their polarity and non-polarity of the amino acids in the protein sequence. However, *Equus caballus, Felis cattus*, and *Mesocricetus auratus* were placed separately since they did not show much resemblance based on polarity.

In the D3 domain of ACE2 sequences of *Salmo salar* and *Danio rerio*, there was an insertion of a polar amino acid into one of the binding residue positions that may affect the binding of ACE2 to that of RBD of SARS-CoV2 negatively. A total of three binding residues were already reported in the D3 domain, of which one of them remained conserved concerning polarity across the nineteen species. *Rattus norvegicus, Mustela putorius furo, Mesocricetus auratus*, and *Felis catus* were grouped under a single clade based on the polarity of their protein sequence. It was a similar case for *Danio rerio, Pelodiscus sinensis, Salmo salar*, and *Gallus gallus*. Due to the sequence similarity between *Pteropus vampyrus* and *Pteropus alecto*, their polarity of the protein sequence was also similar and thus grouped. Sequence similarity was also observed for *Homo sapiens, Pan troglodytes*, and *Macaca mulatta*, so again these were categorized together. Two groups comprised of *Rhinolophus ferrumequinum*, *Capra hircus*, and *Bos taurus, Sus scrofa*, respectively, were sorted together indicating their similar nature

of polarity and non-polarity of protein sequence. Lastly, *Manis javanica* and *Equus caballus* were placed separately signifying that the sequences of both species were quite distinct.

The individual groups of species based on the polarity of individual D1, D2, and D3 domains have emerged into six disjoint clusters (Figure 12).

Here, the clusters {*S*1, *S*2, *S*3}, {*S*16, *S*17, *S*18}, {*S*8, *S*14}, {*S*6, *S*13}, and {*S*10, *S*12} remained invariant with regard to the homology of full length ACE2 as well as polarity sequence of the D1, D2, and D3 domains.

**Figure 11.** Polarity sequence of the D1, D2, and D3 domains across all species alignment and associated phylogenetic relationships.

**Figure 12.** Clusters based on groups of species based on domain-wise polarity.
