Phylogenetic Analysis of Diploderma Lizards

Total genomic DNA for the new collected specimens was extracted from liver tissues with the standard extraction method (Sambrook et al. 1989 ). The mitochondrial gene NADH dehydrogenase subunit 2 (ND2) was amplified and sequenced by using published primers (Wang et al. 2019a (link)). PCR and sequencing methods followed Liu et al. (2020) (link). Sequences were edited and manually managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA 11 (Tamura et al. 2021 (link)). Representative species of Pseudocalotes Fitzinger were chosen as outgroups according to Wang et al. (2022) (link). Genetic data for 32 species of Diploderma and two species of outgroup taxa were obtained from GenBank (Table 1).
Sequences were aligned using MUSCLE (Edgar 2004 (link)) integrated in MEGA 11 (Tamura et al. 2021 (link)). The best substitution model GTR + Γ was selected using jModelTest 2.1.10 (Darriba et al. 2012 (link)). Bayesian Inference (BI) was performed in MrBayes 3.2.7 (Ronquist et al. 2012 (link)), based on the selected substitution model. Two runs were performed simultaneously with four Markov chains. The chains were run for 10,000,000 generations and sampled every 1,000 generations. The first 25% of the sampled trees was discarded as burn-in and then the remaining trees were used to estimate Bayesian posterior probabilities (BPP); nodes with BPP values of 0.95 and higher being considered well-supported (Huelsenbeck et al. 2001 (link); Wilcox et al. 2002 (link)). Maximum Likelihood (ML) analysis was performed in IQ-TREE 1.6.12 (Nguyen et al. 2015 (link)) using the selected substitution model. One thousand bootstrap pseudoreplicates via the ultrafast bootstrap (UFB) approximation algorithm were used to construct a final consensus tree, nodes with UFB values of 95 and above being considered significantly supported (Minh et al. 2013 (link)). Uncorrected genetic pairwise distances (p-distances) between species were calculated in MEGA 11 (Tamura et al. 2021 (link)) with the pairwise deletion option for handling alignment gaps and missing data.