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Telomeric sequences are generally located at the ends of chromosomes; however, they can also be found in non-terminal chromosomal regions when they are known as interstitial telomeric sequences (ITSs). Distribution of ITSs across closely related and divergent species elucidates karyotype evolution and speciation as ITSs provide evolutionary evidence for chromosome fusion. In this study, we performed physical mapping of telomeric repeats by fluorescence  in situ  hybridisation (FISH) in seven Australian dragon lizards thought to represent derived karyotypes of squamate reptiles and a gecko lizard with considerably different karyotypic feature. Telomeric repeats were present at both ends of all chromosomes in all species, while varying numbers of ITSs were also found on microchromosomes and in pericentromeric or centromeric regions on macrochromosomes in five lizard species examined. This suggests that chromosomal rearrangements from ancestral squamate reptiles to Iguania occurred mainly by fusion between ancestral types of acrocentric chromosomes and/or between microchromosomes, leading to appearance of bi-armed macrochromosomes, and in the reduction of microchromosome numbers. These results support the previously proposed hypothesis of karyotype evolution in squamate reptiles. In addition, we observed the presence of telomeric sequences in the similar regions to heterochromatin of the W microchromosome in  Pogona barbata  and  Doporiphora nobbi , while sex chromosomes for the two species contained part of the nucleolar organiser regions (NORs). This likely implies that these ITSs are a part of the satellite DNA and not relics of chromosome fusions. Amplification of telomeric repeats may have involved heterochromatinisation of sex-specific W chromosomes and play a role in the organisation of the nucleolus.

Distribution and amplification of interstitial telomeric sequences (ITSs) in Australian dragon lizards support frequent chromosome fusions in Iguania