Replication banding and FISH analysis reveal the origin of the Hyla femoralis karyotype and XY/XX sex chromosomes

The pine woods treefrog, Hyla femoralis, is unique among North American hylid frogs in having a metacentric chromosome 6 and heteromorphic sex chromosomes of the XY/XX type. The X chromosome is distinguished by having a nucleolar organizing region (NOR) in the short arm. The Y chromosome does not possess an NOR. Until the present study, it was not known if the NOR was not present on the Y chromosome or inactive and therefore not detectable by conventional cytogenetic methods like silver staining. Exclusive of its unique features the karyotype of H. femoralis closely resembles those of North American frogs with karyotypes like H. chrysoscelis. We used replication banding and fluorescence in situ hybridization (FISH) with a DNA probe to the 18S + 28S ribosomal genes, which are located at the NOR, to characterize the H. femoralis karyotype. Our analysis revealed that the 18S + 28S ribosomal genes are not present on the Y chromosome, and that the karyotype of H. femoralis was derived from an H. chrysoscelis-like karyotype by relocation of the NOR to the X chromosome from chromosome 6 and either a concurrent or subsequent pericentric inversion of chromosome 6.