The patterns of isolation by distance (IBD) entailing increased genetic differentiation among populations have aroused extensive concerns for evolutionary biologists. Although the IBD may act on spatial processes contributing to the genetic differentiation among populations in anuran species, the factors shaping the IBD of frogs among populations in natural systems are largely unknown. Here, we studied the genetic differentiation among six populations with 24 individuals of the spotted-leg treefrog along a latitudinal gradient (1860.31 km) based on 1020 single nucleotide polymorphisms from restriction site-associated DNA sequencing. The results showed that the genetic diversity differed significantly among populations and that the insular populations had higher genetic diversity than the mainland populations. Furthermore, we also found a significant genetic differentiation among populations (F-ST = 0.277) and no sign of inbreeding (F-UNI = -0.145). The IBD was detected for all populations, and a higher degree of the IBD was indicated when controlling for the effects of the isolation between Hainan and mainland populations caused by the Qiongzhou Strait. Our findings suggest that the form of the Qiongzhou Strait plays a key role in shaping the genetic diversity and population differentiation in treefrogs. |