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

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.

The karyotype of iguanian lizards (Iguania) is highly conserved with a diploid complement of 36 including six pairs of macrochromosomes and twelve pairs of microchromosomes [2,14]. Karyotypes in Iguania is proposed to have evolved by repeated fusion between macrochromosomes and/or other microchromosomes, leading to the origin of bi-armed macrochromosomes and reduction of microchromosomes [6][7][8][9]12]. With few exceptions, most of the Australian dragon lizards exhibit 2n = 32, including six pairs of macrochromosomes and ten pairs of microchromosomes, implying that repeated fusion predominantly occurred in this group [2,14]. Dynamic transitions of sex determination are also found in the same evolutionary clade, although their karyotype is highly conserved [15][16][17][18]. Our previous chromosome mapping indicated the occurrence of simple translocation between macrochromosomes and sex microchromosomes or between macrochromosomes and microchromosomes in Australian dragon lizards ( [11], Matsubara et al. unpublished data). This suggests that change in chromosome structure affected the transition of sex determination mode and make this lineage an excellent research model for evolution of genome, chromosomes and sex chromosomes. Interestingly, change in chromosome structure, especially chromosome fusion often relates to telomeric repeats [19,20]; thus, it is extremely important to understand how telomeric repeats relate to chromosomal rearrangement in this lineage.
Vertebrate chromosome ends are capped by telomeres that physically protect them from DNA repair and recombination, maintain chromosome stability and integrity, and control replicative lifespan [21,22]. This structure comprises nucleoprotein complexes associated with tandem telomeric DNA repeats (TTAGGG), widely recognised as a remarkable evolutionary conservation feature [21,22]. However, the distribution of these sequences at non-telomeric sites, known as interstitial telomeric sequences (ITSs) or interstitial telomeric repeats (ITRs) was also observed in several lineages [22]. ITSs can be classified into two different types according to their organisation as short ITSs (s-ITSs) and heterochromatic ITSs (het-ITSs) [19]. The s-ITSs exist as short sized telomeric DNA involving mechanisms of DNA repair, while het-ITSs are large stretches of telomeric sequences located mainly in centromeric or pericentromeric heterochromatic regions or euchromatic regions within the chromosome arms. The het-ITSs are considered to be relics of chromosomal rearrangements in the evolutionary process involving fusion or inversion which occurred within derived karyotypes. In squamate reptiles, the presence of ITSs is frequently observed in Iguania, which is supposed to have a derived karyotype from other squamate reptiles [6,12,23,24]. To test this hypothesis and to better characterise Iguanian karyotype evolution, distribution of telomeric sequences in Australian dragon lizards was analysed together with a gecko lizard (Hemidactylus frenatus) with largely different karyotypes as the outgroup. The significance of telomeric distribution was discussed.

Animal and chromosome preparation
Seven dragon lizard species containing both male and female individuals and both male and female gecko lizard species were examined. Detailed information is presented in Table 1. Euthanasia and sexing were performed as described in Ezaz et al. [16]. Briefly, animals were euthanised by an intraperitoneal injection of sodium pentobarbitone at a concentration of 150 mg/g body weight. Phenotypic sex was determined on the basis of external morphology by hemipenes eversion [25] and by internal examination of gonadal morphology. All experiments were performed with the approval of the University of Canberra and Kasetsart University Animal Experimentation Ethics Committee (CEAE 11/07 and ACKU61-SCI-021). Metaphase chromosomes were prepared either from short-term culture of whole blood or from fibroblast cell lines as described by Ezaz et al. [15,16] and Srikulnath et al. [26]. Chromosome slides were treated with 100 μg/ml RNase for 1 h at 37˚C and then rinsed three times in 2 × SSC.

Results
Fluorescence hybridisation signals of the PNA telomeric probe Cy 3 -(CCCTAA) 3 were observed at the telomeric ends of all chromosomes in seven dragon lizards and the gecko lizard (Figs 1-3). The hybridisation signals were weak on macrochromosomes; by contrast, high intensity signals were observed on almost all microchromosomes in the seven dragon lizards.
ITSs were observed in all species examined. In Pogona barbata, ITSs were found on several microchromosomes and sex microchromosomes (Z and/or W chromosomes), while ITSs were localised to one microchromosome pair of Ctenophorous fordi and Physignathus leseuirii ( Table 1). ITSs were localised to the W microchromosome and pericentromeric regions of chromosome 1 in Diporiphora nobbi but only near the pericentromeric region of chromosome 1 in Hydrosaurus pustulatus. By contrast, ITSs were detected in the centromeric regions of all macrochromosomes and two microchromosome pairs in Amphibolurus norrissi and A. muricatus (Fig 2). No ITSs were found on the gecko lizard H. frenatus (Fig 3).

Discussion
Comparative chromosome maps of squamate reptiles suggest that chromosome fusion between macrochromosomes or other microchromosomes occurred in iguanian lizards, resulting in all macrochromosomes being bi-armed and in the reduction of microchromosome numbers [6][7][8][9]12], while cross-species chromosome painting analysis revealed several conserved syntenies among gecko lizard chromosomes (Gekko and Hemidactylus) [28]. In our study, highly intense signals were observed on almost all microchromosomes in the seven dragon lizards but not on macrochromosomes, suggesting that telomeric repeats were amplified site-specifically. We also observed ITSs in pericentromeric or centromeric regions of all bi-armed macrochromosomes in A. norrissi and A. muricatus, similar what has been reported in several other iguanian lizards, such as Polychrus acutirostris, P. marmoratus, Anolis distichus and A. equestris and [23,29,30]. Iguanian lizards and snake chromosome 1 correspond to two acrocentric chromosomes of lacertid lizards and two chromosome arms of different bi-armed chromosomes in gekkonid lizards [6][7][8][9]11,12,31]. ITSs observed in pericentromeric regions of chromosome 1 in D. nobbi, H. pustulatus, and two species of Amphibolurus also show cytogenetic evidence that chromosome 1 in iguanian lizard is derived from centric fusion between the ancestral acrocentric chromosomes. Several pericentric inversions have been discovered in macrochromosomes of Anolis carolinensis [32] which may represent remnants of ancestral  intrachromosomal rearrangements that occurred in the lineage. Additionally, ITSs were found in several microchromosomes within our study species with the exception of H. pustulatus, suggesting likely chromosome fusions between microchromosomes. However, several events involving chromosome fusion were not observed for ITSs in some iguanian lizard species. This suggests that the loss of telomeric repeats in interstitial regions have resulted from recombination events over evolutionary time [33], leading to a variety of ITS distribution in different iguanian lizards. Incidence of ITS distribution in iguanian lizards is higher than those of snakes and anguimorphid lizards (Toxicofera species) [23,30]. This suggests that these recently rearranged chromosomes in iguanian lizards have retained the relics of former telomeres on the segments when compared with those of snakes and anguimorphid lizards that probably  [39]. Distribution of ITSs and telomeric repeat amplifications of lizards and snakes were also obtained from Young et al. [11], Matsubara et al. [37], Augstenová et al. [34], Rovatsos et al. [23,30] and Srikulnath et al. [7,9]. occurred long evolutionary time ago (Fig 4). However, total number of species tested for the presence of ITSs remains small in snakes and anguimorphid lizards [7,23,34,35]. Therefore, chromosome mapping of telomeric repeats will be required in more snakes and anguimorphid lizards to clarify these finding as conclusive evidence. Telomeric DNA can also contain repeat elements similar to centromeric repeat elements [36]. This suggests that ITSs on the centromeres may result from extensive amplification of telomere-like satellite DNAs within centromere. Molecular cloning of centromeric satellite DNA in Australian dragon lizards will be required to test whether similar amplification has also occurred within this group. Additionally, the heterochromatic W microchromosome showed large amplification of telomeric repeats in P. barbata and D. nobbi. A similar pattern was observed in P. vitticeps as closely related species with P. barbata [11]. Such amplification of telomeric repeats in the heterochromatic region on W chromosomes has been reported in several caenophidian snakes and other squamate reptiles [11,34,35,37,38]. These telomeric repeats are probably considered as a regular component of satellite DNA in heterochromatin. This suggests that amplification of repeat sequences probably involved heterochromatinisation of sex-specific chromosomes, particularly W chromosomes in species D. nobbi, P. barbata and P. vitticeps. The W sex chromosomes of D. nobbi and P. barbata also contained part of the nucleolar organiser regions (NORs) in addition to chromosome 2 [Matsubara et al. unpublished data]. Such ITSs showing co-localisation with NOR have also been reported in butterfly lizard chromosomes [20,26]). This suggests the possibility that NORs are associated with telomeric sequences which may play a role in the organisation of the nucleolus [38].
The karyotypic variability in Australian dragon lizards allowed examination of ITS relationships with chromosomal rearrangements, heterochromatin and NORs. Different patterns of ITSs may be due to the age of chromosomal rearrangements. Further studies involving chromosome mapping of telomeric repeats as well as satellite DNA in more representative species from divergent taxa are required to better understand evolutionary origins and distributions of ITS in squamate reptiles.