We examined the phylogenetic relationships and evolutionary divergence of Phrynocephalus versicolorStrauch 1876, as well as the genetic diversity and population structure in Mongolia using mtDNA variation. Phrynocephalus is one of the most diverse genera within the Agamidae family, with confusing or controversial taxonomy. This resulted in several independent suggestions for subspecies designations within Phrynocephalus versicolor. As the most dominant and abundant reptilian species in the Mongolian Gobi Desert, Phrynocephalus versicolor is ecologically important within the food chain of the ecological system it inhabits. We used 77 samples from 9 populations of P. versicolor in central and southern Mongolia. We amplified and sequenced 522–base pair (bp) –long fragment of the ND2 gene and recorded moderate levels of sequence diversity (Hd = 0.74 ± 0.023, π = 0.035 defined by 97 polymorphic sites). We conducted phylogenetic analyses using of Bayesian inference, maximum likelihood, and median-joining (MJ) network analysis that resulted in four well-supported clades (A, B, C, D) among our Mongolian samples. All of these clades, except Clade A, were population specific. The genetic distance values (mean Fst = 0.768), and analysis of molecular variance (AMOVA) also showed greater genetic distances between these clades. We ran a time-calibrated phylogenetic analysis using a strict molecular clock model with a single fossil calibration. The estimated divergence times in this study were comparable with previous findings, for example, 1.8 million years ago (mya) (1.31–2.94) for the node (Bayesian posterior probability [BPP] > 1.0 and bootstrap > 98) from which Clade A and B originated. In conclusion, our findings showed that there are at least three genetically distinct subspecies, namely P. v. versicolor, P. v. kulagini, and P. v. hispidus, with one previously unknown clade/lineage.

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