**2.1 Phylogenetic methods**

Amino acid sequences of ISPs were obtained from the NCBI database (*National Center for Biotechnology Information, U.S. National Library of Medicine*) using their BLAST search package using *E. coli* ecotin as a BLAST target [39]. Sequence alignment using the MUSCLE algorithm and phylogenetic maximum likelihood analysis was done in SeaView [40], with the selection of best-fit amino acid substitution matrices done with PROTTEST 3 [41]. Resulting tree files were manually edited to standardize terminal labels, and cladogram image files were exported using the iTol web tree tool (**Figure 1**) [42].

For the loci viewer project database input, complete genomic sequences for loci analysis were obtained from the same database using tBLASTn, a tool that takes amino acid sequences as input and searches for corresponding nucleotide sequences, with the search limited to RefSeq annotated genomes [39]. Data was input and manipulated into custom software developed for the author's master thesis [43]. The complete list of sequences with respective web links and NCBI GenBank IDs is listed in **Table 1**. After data input was completed, the database was manually manipulated using SQL queries to identify close genes to the left or right of the ecotin homologs in various species, aided by the visual map generated showing that ISPs occurred in two different loci around 50 kbp apart in most species. One of these genes is a putative katanin-encoding gene, and the other three are conserved hypothetical proteinencoding genes that were called CHP1, 2, and 3. Using the *L. braziliensis* genome as a reference, these four amino acid sequences were run through the NCBI tBLASTn tool using the same settings used for the ecotin homologs, and resulting CDSs were manually labeled *CHP1*, *CHP2*, *CHP3,* and *katanin-like* in the loci viewer database.

## **Figure 1.**

*Scheme on methodology employed in the phylogenetic reconstruction of ISPs from trypanosomatids.*


## **Table 1.**

*List of RefSeq genomic records inserted into the loci image generator database.*
