*2.4. Data Analysis*

Chromatograms of forward and reverse sequences were assessed, and a consensus sequence for each sample generated in SeqTrace 0.9.0 [56]. For sequences alignment, we employed MAFFT2 [57,58] through the webserver [59], which includes a special search strategy (Q-INS-i) for the secondary structure of the rRNA 16S [60]. No later manual edition was introduced. Results of MAFFT2 were visualized in MSA Viewer [61] and exported as fasta files.

The best scheme for substitution model was explored in PartitionFinder 2.1.1 [62], using linked branch lengths (supported by most of the phylogenetic programs) using a PhyML 3.0 analysis [63]. Given that the analysis is based on Squamata, which can show highly divergent clades, we estimated the relative quality of the statistical models using the Bayesian Information Criterion (BIC) [64] since it penalizes more the number of parameters in the model and then is better for a large degree of heterogeneity [65].

Given that it is not recommended to use both +I (significant proportion of invariable sites) plus +G (rate of variation among sites follows a gamma distribution) together in the same substitution model, we chose the best suggested model using +I or +G in the partition schemes, but never both together [66].

The phylogenetic hypothesis was performed under a Maximum Likelihood (ML) approach, using IQ-Tree [67] through its webserver [68], setting 10,000 non-parametric bootstrap replicates plus 10,000 replicates of Shimodaira-Hasegawa approximate likelihood ratio (SH-aLRT) [69] and 10,000 ultrafast bootstrap (UFBoot) approximation replicates [70]. We used a sequence of *Sphenodon punctatus* to root the tree [71], which has been proposed as the sister clade to Squamata [72]. Here, it is important to highlight that the phylogenetic hypothesis is used to sorting groups, and we do not seek for a comprehensive evolutionary reconstruction.

For visualization and edition (branch arrangement, colors, font sizes, etc.) of the tree generated through ML analysis, we used FigTree 1. 4.3 [73]. The final alignment plus the ML tree are stored in TreeBASE repository (https://treebase.org/) under the submission number 24616. To do this, we first managed the alignments and trees in nexus format and combined them in a single file (containing one alignment and the ML tree) using Mesquite 3.31 [74].
