Chromosome evolution in the genus litoria (Anura, Hylidae)

The mitotic chromosomes of 19 species of Litoria and the 4 new species (L.barringtonensis, L.genimaculata, L.nyakalensis and L.personata) studied here prepared from bone marrow after in vivo colchicines treatment and analyzed by conventional staining, C-banding, Ag-NOR staining, DAPI/Distamycin A, DAPI/Mithramycin, Q-banding and Telomere FISH. All species were 2n=26, fundamental number (FN) = 52 chromosomes, except Litoria infrafrenata, which was 2n=24, FN=48. In tern of arm ratios and centromere positions, the chromosome morphology of Litoria species was very characteristic. Pairs 1 and 4 were metacentric, pair 2 and 6 were submetacentric and pairs 3 and 5 were subtelocentric. Species-specific chromosome markers were determined and included secondary constriction, location of NORs and heterochromatin distribution. Sex chromosomes could not be identified in the Litoria species studied. The secondary constriction showed major despiralization in L.barringtonensis, which is regarded as the nucleolar organizer. The C-banding studied revealed substantial differentiation in the heterochromatic component of the complement including the possession of whole arm C-blocks, some of which had evolved by chromosome addition; pair 12 of L.meiriana, while others involved a process of euchromation information. None of the species analyzed shared the same C-banding pattern, although certain closely related species had very similar and highly derived karyotypes. All species examined had only one pair of the nucleolar organizer regions in their chromosomes; the four Litoria species exhibited on the short arm of the large chromosome pair and the other species shown on the long arm of the small chromosome pair. Fluorescence banding in the distamycin A/DAPI counter stained the chromosomes of all species and showed a uniform fluorescence, the mithramycin-stained chromosome of all species exhibited the brightes mithramycin fluorescence on the NOR regions, which can be used to verify the position of nucleolar organizer regions in each species and the quinacrine mustard showed negative fluorescence of the centromeric regions of all species of Litoria. In situ hybridization with the (GGGTTA)7 and (TAACCC)7 oligomers revealed, as expected, distinct hybridization signals at the telomeres of all chromosomes of Litoria species. Moreover, it was found that four species of Litoria showed signals not only at the telomeres of chromosomes, but also at the centromere of chromosomes (pair 4 in L.eucnemis, L.genimaculata and L.verreauxii and pair 1, 2 and 3 in L.fallax). The karyotype of most species in the genus Litoria shown quite stable, characterized by a similar macrostructure, this seems to be evidence for a low chromosome evolution rate. Chromosome morphology, banding pattern and position of the nucleolar organizer regions (NORs) provide relevant characters for the understanding of the phylogeny and systemics of these Litoria tree frogs in Australia.