**3. Results**

We generated a total of 142 sequences of 64 species of Squamata from Paraguay, including one exotic species: *Hemidactylus mabouia*. In the Table S2, we present a list of specimens used for genetic analyses based on the field work for this project. For comparison we added 107 sequences from GenBank (Table S2). The final alignment constituted of a dataset of 249 samples of 615 bp length. Sequences are available in GenBank.

The best substitution model for the Barcoding dataset was GTR+G, according to the BIC. The sample of *Sphenodon punctatus* was retrieved as the sister clade to the Squamata (Figure 4). Deep nodes have low bootstrap values, meaning that the phylogenetic relationships are weakly supported. Nevertheless, the shallowest divergences have higher support values, recovering most of the genera included in the analysis as monophyletic, with the exception of *Manciola* (Scincidae) and the tribe Xenodontini (Colubridae).

The tribe Xenodontini (Figure S1) of the Subfamily Dipsadinae (Colubridae) contains the samples of *Erythrolamprus aesculapii* in a monophyletic clade, whereas *E. poecilogyrus* appears as paraphyletic. *Erythrolamprus reginae* clusters sister to the above-mentioned taxa. The genus *Xenodon* seems to be paraphyletic, given that two samples of *Xenodon pulcher* are sister to *Erythrolamprus*, whereas *Xenodon merremi* is sister to *Xenodon pulcher* + *Erythrolamprus*. Finally, in this clade a sample of *Erythrolamprus sagittifer* is sister to a sample of *Lygophis dilepis*.

Sister to Xenodintini is a clade composed of *Phalotris* + *Philodryas* (Figure S2). Both genera are monophyletic in the tree. The genera *Psomophis* and *Dipsas* are clustered together, and nested as sister to the above-mentioned snakes (Figure S3). Other genera of Dipsadinae that are rendered as monophyletic are *Hydrodynastes*, *Helicops*, and *Thamnodynastes* (Figure S4). A clade grouping members of the Colubrinae subfamily is composed of three genera (*Chironius*, *Leptophis*, and *Palusophis*) that also show monophyly (Figure S5). The *Pseudoboini* (Dipsadinae: Colubridae) is shown in its own clade (Figure S6) with four of the five genera used in the analysis being monophyletic, whereas the two species of the genus *Phimophis* appear in di fferent positions of the gene tree.

The genus *Micrurus* (Elapidae) is the sister clade of the Colubridae, whereas the genus *Bothrops* (Viperidae) seems to be the sister to Elapidae + Colubridae (Figure S7). Located in a most basal position among snakes are the two species of *Epicrates* (Boidae), with *Amerotyphlops* (Typhlopidae) as the sister clade of the remaining snakes (Figure S8). The genus *Amphisbaena* (Amphisbaenidae) is monophyletic, where *A. alba* and *A. bolivica* are in their own clades, and *A. mertensii* shows also monophyly (Figure S9). *Amphisbaena angustifrons* is the sister taxon of the other *Amphisbaena*, with a sample of *Amphisbaena* sp. (PCS 314) as the most basal taxon of the clade (Figure S9). In our analysis, *Amphisbaena* is sister to Teiidae + Gymnophthalmidae. Gymnophthalmidae appears as a monophyletic clade, and the four genera show monophyly as well (Figure S10).

**Figure 4.** General view of the barcoding tree performed with a Maximum Likelihood approach. Clades are collapsed (in genera or tribes) for a better visualization. Numbers on nodes represent Bootstrap values. The scale bar represents branch length (substitutions/site).

The Family Teiidae is shown as paraphyletic. The only Tupinambinae in our samples was *Salvator*, which clusters as sister to Gymnophthalmidae (Figure S11). Samples of Teiinae are clustered together showing monophyly, where *Ameivula* and *Kentropyx* are sister clades (Figure S11), as are *Teius* and *Ameiva* (Figure S12). The Family Scincidae is sister to the all above-mentioned clades (Figure S13). Samples of *Manciola* show paraphyly (Figure S13). The remaining cluster contains members of the Anguidae, Gekkonidae, Phyllodactylidae, Liolaemidae, Polychrotidae, and Tropiduridae families. The clade composed by geckos shows monophyly in the genera, but not in the families given that *Phyllopezus* and *Homonota* are currently placed in Phyllodactylidae, whereas *Hemidactylus* and *Lygodactylus* are Gekkonidae (Figure S14). Sister to the Gekkota (Gekkonidae + Phyllodactylidae) is *Liolaemus*, and *Stenocercus* is rendered as sister to the Gekkota + *Liolaemus*.

The last cluster, sister to Gekkota + *Liolaemus* + *Stenocercus*, is represented by *Ophiodes* (Anguidae), *Polychrus*(Polychrotidae) (Figure S15), and *Tropidurus*(Tropiduridae) (Figure S16). In this case, the Family Tropiduridae is polyphyletic since the other member of the family (*Stenocercus*) is sister clade to Gekkota + *Liolaemuus*.
