Cytogenetically Elusive Sex Chromosomes in Scincoidean Lizards

The lizards of the species-rich clade Scincoidea including cordylids, gerrhosaurids, skinks, and xantusiids, show an almost cosmopolitan geographical distribution and a remarkable ecological and morphological divergence. However, previous studies revealed limited variability in cytogenetic traits. The sex determination mode was revealed only in a handful of gerrhosaurid, skink, and xantusiid species, which demonstrated either ZZ/ZW or XX/XY sex chromosomes. In this study, we explored the karyotypes of six species of skinks, two species of cordylids, and one gerrhosaurid. We applied conventional and molecular cytogenetic methods, including C-banding, fluorescence in situ hybridization with probes specific for telomeric motifs and rDNA loci, and comparative genomic hybridization. The diploid chromosome numbers are rather conserved among these species, but the chromosome morphology, the presence of interstitial telomeric sequences, and the topology of rDNA loci vary significantly. Notably, XX/XY sex chromosomes were identified only in Tiliqua scincoides, where, in contrast to the X chromosome, the Y chromosome lacks accumulations of rDNA loci. We confirm that within the lizards of the scincoidean clade, sex chromosomes remained in a generally poor stage of differentiation.

The scincoidean lizards have been examined cytogenetically for decades, but sex chromosomes were reported only rarely. XX/XY sex chromosomes have been identified in a few skink species by C-banding, silver-staining, fluorescence in situ hybridization (FISH) with repetitive elements and rDNA loci, or by size differences between X and Y [7,[13][14][15]. Heteromorphic XX/XY sex chromosomes were documented in Oligosoma oliveri [13; reported there as Cyclodina oliveri], Saproscincus czechurai, Lampropholis sp. [16], Ctenotus rawlinsoni, Pseudemoia entrecasteauxii, and Pseudemoia pagenstecheri [17]. In Acritoscincus (formery Bassiana) duperreyi, Shine et al. [18] identified XX/XY chromosomes by size differences between X and Y, which was later confirmed by C-banding, CGH and FISH with telomeric motifs [14], and isolation of a Y-specific molecular marker [19]. An interesting situation was found in the genus Scincella. Heteromorphic XX/XY sex chromosomes were identified in three American species [20,21] and X 1 X 1 X 2 X 2 /X 1 X 2 Y multiple sex chromosomes were found in a population of Scincella lateralis. Hedin et al. [22], based on the analysis of synaptonemal complexes, concluded that the X 1 X 1 X 2 X 2 /X 1 X 2 Y is derived from XX/XY by fusion of the Y chromosome with a macrochromosome. On the other hand, heteromorphic ZZ/ZW sex chromosomes were reported in the congeneric Scincella melanosticta from Thailand [23], representing the only reported case of female heterogamety in skinks.
Recently, two studies based on whole-genome sequencing and subsequent genomic coverage analysis identified shared X-specific gene content in two phylogenetically distant skink species, Scincus scincus [24] and Eulamprus heatwolei [25]. The X-specific exonic sequences of S. scincus were used to develop a qPCR-based method, which allowed to test the homology of sex chromosomes across 13 additional species of skinks covering most of the phylogenetic spectrum of the family Scincidae [24]. The same study revealed that the XX/XY sex chromosomes of skinks are poorly differentiated, but evolutionary stable for at least 85 million years [24]. The high age of the XX/XY sex determination system in skinks was independently supported by the analysis of the divergence of gametologs [25]. Recently, a Y-specific probe was designed from Illumina reads, derived from double-digest restriction-site associated DNA sequencing (ddRADseq), which accurately revealed the Y chromosome in the skink Carinascincus ocellatus, but not in the closely related Liopholis whitii [15]. Notably, heteromorphic ZZ/ZW sex chromosomes have been identified by conventional staining in the skink Scincella melanosticta [23], which would represent a rare case of sex chromosome turnover among skinks. ZZ/ZW sex chromosomes have been identified in the xantusiid Xantusia henshawi by restriction site-associated DNA sequencing (RADseq) [26]. A ZZ/ZW sex determination is also in accordance with the production of both sexes by facultative parthenogenesis in the xantusiid Lepidophyma smithii [27].
Sex determination is highly understudied in the families Gerrhosauridae and Cordylidae, and so far, ZZ/ZW sex chromosomes were identified only in the gerrhosaurid Tracheloptychus petersi by FISH with a probe for the rDNA loci, but not with comparative genome hybridization (CGH) [28]. The W chromosome of this species lacks the accumulation of rDNA loci linked to Z chromosomes.
In this study, we applied conventional (C-banding, karyogram preparation) and molecular cytogenetic (FISH with rDNA and telomeric probes, comparative genomic hybridization) methods to metaphases of, wherever possible, male and female specimens of nine species of the Scincoidea radiation (Table 1), in order to analyze their karyotype and to explore the presence of sex chromosomes.

Acontias percivali
The karyotype consists of 2n = 22 chromosomes (Figure 1a), which is in line with the observations of Gordon [10]. We were not able to identify the sex of the studied specimen. Heterochromatic blocks are present in the centromeric region of a pair of macrochromosomes and a pair of microchromosomes (Figure 2a). FISH with the rDNA probe revealed signals at the terminal position of a pair of macrochromosomes, corresponding to the chromosome pair 3 or 4 (but we cannot safely conclude since both pairs have similar morphology) (Figure 3a). Telomeric sequences are visible at the terminal position of all chromosomes (Figure 4a).

Scincopus fasciatus
The karyotype consists of 2n = 32 chromosomes with several telocentric and acrocentric pairs (Figure 1h,i). Both male and female specimens were examined. C-banding did not reveal any heterochromatic block. Signals of rDNA were visible on a submetacentric pair of macrochromosomes, corresponding to chromosome pair 5 or 6 (but we cannot safely conclude since both pairs have similar morphology) (Figure 3h,i). Telomeric sequences were found at terminal positions of all chromosomes and interstitial telomeric repeats (ITRs) were absent (Figure 4h,i). Sex-specific differences were not detected by CGH (Figure 5c,d).

Tiliqua gigas
The karyotype consists of 2n = 32 chromosomes (Figure 1j,k), as previously reported by Donnellan [16]. Both male and female specimens were examined. Heterochromatic regions are visible at two pairs of macrochromosomes as well as several microchromosomes (Figure 2h

Tiliqua scincoides
The karyotype consists of 2n = 32 chromosomes (Figure 1l,m), as previously described by de Smet [29]. Both male and female specimens were examined. Heterochromatic regions are present in several microchromosomes (Figure 2j,k). FISH with the rDNA probe showed signals on two microchromosomes in females, but only on a single microchromosome in the male (Figure 3l,m). Telomeric sequences are present at the terminal position of all chromosomes (Figure 4l,m).

Tracheloptychus madagascariensis
The karyotype consists of 2n = 34 chromosomes (Figure 1n,o), with 12 of them being macrochromosomes, as previously described by Odierna et al. [2]. Both male and female specimens were examined. Heterochromatin is present in the centromeric region of four pairs of macrochromosomes as well as in several microchromosomes (Figure 2l

Discussion
Except for Acontias percivali with 2n = 22 chromosomes (Figure 1a), all other studied species showed a low variation in the diploid chromosome numbers, having karyotypes with 2n = 32 or 2n = 34 chromosomes (Figure 1). Our results are following previous cytogenetic studies, which showed that karyotypes with 2n = 28 to 2n = 34 are mostly reported in skinks, cordylids, and gerrhosaurids [3]. Nevertheless, chromosome morphology (mainly centromere position and chromosome size) varied significantly among species with the same chromosome number (Figure 1). We speculate that chromosomal rearrangements (including inversions) have commonly occurred during the karyotype evolution in Scincoidea, in contrast to other reptilian lineages such as turtles, where chromosome morphology seems to be rather unchanged in the long term [30].
The comparative coverage analysis of Illumina DNAseq reads revealed homologous XX/XY sex chromosomes in the distantly related skinks Scincus scincus [24], Eulamprus heatwolei, and Eulamprus tympanum [25]. A qPCR-based method, which is based on the comparison of copies of X-specific genes between sexes supported the homology of XX/XY sex chromosomes across 13 species of skinks. The same technique was used in all skinks in the current paper, except for Acontias percivali, to validate the sex of each species. Therefore, we are aware that all the skinks studied here, except Acontias percivali, have homologous XX/XY sex chromosomes. However, the sex chromosomes were detected by cytogenetic methods only in Tiliqua scincoides (Figure 3l,m). It seems that these highly evolutionary stable sex chromosomes are poorly differentiated and mostly cryptic in the majority of the skink species. In Tiliqua scincoides, the probe specific for the rDNA loci hybridized in a single microchromosome in males, but in two microchromosomes in females (Figure 3l,m). We conclude that the rDNA loci seem to be located on the X chromosome, but missing on the Y, as was previously revealed in the distantly related Scincus scincus [24]. Scincopus fasciatus, which belongs to the same subfamily as S. scincus (Scincinae), is missing this sex-specific distribution of rDNA loci. Ribosomal DNA loci show sex-specific accumulations in many taxa, including the gerrhosaurid Tracheloptychus petersi, where they are located on a pair of autosomes, and additionally, on the Z chromosome, but missing on the W chromosome [28]. In the closely related Tracheloptychus madagascariensis, rDNA loci are present in a pair of microchromosomes in both sexes (Figure 3o,p), which could be homologous to the sex chromosomes of T. petersi. Odierna et al. [2] observed in T. madagascariensis the same distribution of active rDNA loci, visualized by silver staining. Notably, we detected rDNA loci in a single macrochromosome in the male of Platysaurus sp. 2, but we could not distinguish if the single signal reflects an autosomal polymorphism or a sex chromosome. Additional specimens from both sexes are needed to further investigate the sex determination system in this species.
Interspecific differences in the sex-linkage of rDNA loci are common in reptiles. In Australasian side-necked turtles, sex-specific differences are present in Chelodina novaeguineae, Chelodina reimanni, Elseya novaeguineae, and the hybrid Emydura subglobosa × Elseya novaeguineae, but not in Chelodina expansa, Chelodina rugosa, Chelodina mccordi, and Emydura macquarii krefftii [31]. In the spiny softshell turtle Apalone spinifera, accumulations of rDNA loci are present on both the Z and W, but the block is much bigger on the W [32]. This difference in copy number of rDNA loci was further demonstrated by qPCR in nine tested trionychid species, but in Lissemys punctata punctata, male and female had equal number of copies of rDNA loci, and in Lissemys punctata andersoni, females even had less copies of rDNA loci than males [33]. Not only sex-specificity, but also the overall distribution of rDNA loci in the karyotype vary greatly among species. rDNA loci are present on macrochromosomes in Acontias percivali, Eutropis multifasciata, Scincopus fasciatus, Platysaurus sp. 1, and Platysaurus sp. 2, but on microchromosomes in Cyclodomorphus gerrardii, Tiliqua gigas, Tiliqua scincoides, and Tracheloptychus madagascariensis. Currently, we cannot conclude if the variability in the position of rDNA loci between species correspond to homologous genomic regions reshuffled by chromosomal rearrangements (e.g., fusions/fissions), or independent emergence of amplification of rDNA loci. The variability in chromosome position and number of repeats has been previously reported in other vertebrate lineages and it is often linked to the high mobility and intrinsic recombinational instability of rDNA loci [34,35].
Comparative genomic hybridization is another molecular cytogenetic tool commonly used to identify sex chromosomes. This method uncovered the W chromosome in several reptiles, including the agamid Pogona vitticeps, the acrochordid snake Acrochordus javanicus, several species of the chameleon genus Furcifer and of monitor lizards, as well as the Y chromosome in the Australasian side-necked turtles Chelodina expansa, Chelodina novaeguineae, and Elseya novaeguineae, in the legless geckos of the genus Lialis and in the Cuvier's Madagascar swift Oplurus cuvieri [31,[36][37][38][39][40][41]. On the contrary, comparative genomic hybridization did not identify the W chromosome in Tracheloptychus petersi [28] and the Y chromosomes in Scincus scincus, Tropidophorus baconi [24], Eutropis multifasciata, Scincopus fasciatus, and Tiliqua gigas ( Figure 5). Genome sequencing revealed that the X-specific region in the skinks Scincus scincus [24] and Eulamprus heatwolei [25] makes up only a small portion of the X chromosome, and it is probably beyond the detection efficiency of the method. Despite the long-term stability of their XX/XY sex determination system (estimated to 79-116 My) [24,25], skinks seem to have poorly differentiated sex chromosomes. Although the canonical model of sex chromosome evolution [42,43] predicts that sex chromosomes should differentiate over-time, which leads to the loss of functional genes, accumulation of repetitive elements and heterochromatinization of Y/W, skinks consist a rare example among vertebrates, where sex chromosomes remained in a poorly differentiated stage in the long term. In addition, poorly differentiated sex chromosomes tend to be prone to sex chromosome turnovers [44,45], but skinks constitute a notable case that even poorly differentiated sex chromosomes can be stable for a long evolutionary time. We believe that skinks are a magnificent group to enrich our knowledge and deserve further attention to explore the drivers of sex chromosome evolution.
Despite the fact that karyograms revealed extensive differences in chromosome morphology between species, interstitial telomeric repeats were documented only in the skink Eutropis multifasciata, the gerrhosaurid Tracheloptychus madagascariensis, and the cordylids Platysaurus sp. 1 and Platysaurus sp. 2 ( Figure 4). Interstitial telomeric repeats are common in squamate reptiles [46], and can be remnants of interchromosomal rearrangements, or the activity of transposable elements [47][48][49][50][51]. ITRs can be part of satellite motifs, and occasionally are present in the heterochromatic regions of sex chromosomes [31,[52][53][54]. Data on the distribution of ITRs are still scarce, but ITRs were not detected in the sex chromosomes of the studied species from the scincoidean clade. Notably, the variability in the topology of ITRs has been detected in the closely related species Tracheloptychus madagascariensis (present study) and Tracheloptychus petersi [28], despite the fact that both species share similar karyotypes.
In summary, the lizards of the clade Scincoidea have karyotypes with little variability in diploid chromosome number and chromosome variability. Nevertheless, molecular cytogenetic methods revealed extensive variability in the topology of rDNA loci and interstitial telomeric repeats among species. Despite the fact that previous studies revealed an extreme homology of sex chromosomes across skinks based on genomic and molecular methods [24,25], by cytogenetic methods we identified XX/XY sex chromosomes only in Tiliqua scincoides indicating that the sex chromosomes remained at a generally poor stage of differentiation in skinks, and perhaps in other lineages of the clade Scincodea. Future studies should focus on revealing the sex chromosome gene content by genomic methods in phylogenetically informative species of the scincoidean megadiversity. Prominent topics of research should be the validation of the ZZ/ZW sex determination in the skink Scincella melanosticta, the identification of the sex determination mode in cordylids, and the test of homology of ZZ/ZW sex determination systems between gerrhosaurids and xantusiids.

Fluorescence In Situ Hybridization with Probes for rDNA Loci (18S/28S) and Telomeric Motifs
We prepared the probe for the rDNA loci from a plasmid (pDmr.a 51#1) with an 11.5-kb insert encoding the 18S and 28S ribosomal units of Drosophila melanogaster [56], labelled by nick-translation with dUTP-biotin. The telomeric probe was prepared through PCR, using the primers (TTAGGG) 5 and (CCCTAA) 5 , without a DNA template, according to Ijdo et al. [57]. Both probes were precipitated with salmon sperm and sodium acetate in ethanol at −20 • C overnight. After centrifugation and drying, the pellet was dissolved in a hybridization solution (50% formamide in 2 × SSC). For the fluorescence in situ hybridization, the chromosomal spreads were hydrated in 2 × SSC for 5 min at room temperature, followed by RNase treatment for 1 h at 37 • C, washing in 2 × SSC for 5 min at room temperature (3 times), pepsin treatment for 10 min at 37 • C, washing in PBS for 5 min at room temperature (2 times) and 1% formaldehyde in PBS for 10 min. Dehydration through an ethanol series (70%, 85%, 100%), denaturation in 70% formamide for 4 min at 75 • C, followed by dehydration in ethanol series. Before hybridization, the probes were incubated at 75 • C for 6 min, followed by 10 min at −20 • C. The probe was incubated on the chromosomal spread at 37 • C overnight. Post-hybridization washes were performed in 50% formamide for 5 min at 37 • C (3 times), 2 × SSC for 5 min at room temperature (2 times), and washed in 4 × SSC/0.05% Tween20 (Sigma). Chromosomal spreads were treated with 4 × SSC/5% blocking reagent (Roche, Basel, Switzerland) for 45 min at 37 • C. Incubation with 4 × SSC/5% blocking reagent with avidin-FITC (Vector Laboratories, Burlingame, CA, USA) for 30 min at 37 • C was followed by two consecutive treatments with a modified system of avidin-FITC/biotinylated anti-avidin signal enhancement (Vector Laboratories, Burlingame, CA, USA). Chromosomal spreads were stained by Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, USA).

Comparative Genomic Hybridization
Male DNA was labeled with dUTP-biotin and female DNA was labeled with dUTPdigoxigenin using a Nick Translation Kit (Abbott Laboratories, Lake Bluff, IL, USA). The labelled DNA from both sexes was co-precipitated with salmon sperm and sodium acetate (3M) in ethanol for 1 h at −80 • C. After centrifuging and drying, the probe was dissolved in hybridization mix (50% formamide in 2 × SSC). Before hybridization, the probe was heated to 75 • C for 6 min, followed by 10 min at −20 • C. Chromosomal spreads before hybridization were treated in the same way as described above for the FISH experiments, but hybridization time was increased to two nights. Post-hybridization washes were done in 50% formamide at 37 • C for 5 min, repeated three times. The slides were washed three times in 2 × SSC for 5 min and once in 4 × SSC/0.1% Tween20 for 30 s, followed by 4 × SSC/5% blocking reagent for 30 min at 37 • C. The slides were incubated with 4 × SSC/5% blocking reagent, containing avidin-FITC and anti-digoxigenin-rhodamine for 30 min at 37 • C, they were washed three times with 4 × SSC/0.1% Tween20, dehydrated through an ethanol series, and stained with Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Comparative genome hybridization in Eutropis multifasciata, Scincopus fasciatus, and Tiliqua gigas ( Figure 5).

Microscopy and Image Analyses
For each specimen and method, we captured at least 10 images by either a Zeiss Axio Imager Z2 (Zeiss, Oberkochen, Germany) equipped with an automatic Metafer-MSearch scanning platform and a CoolCube 1 b/w digital camera (MetaSystems, Altlussheim, Germany) or an Olympus Provis AX70 fluorescence microscope equipped with a DP30BW digital camera (Olympus, Tokyo, Japan).