ZZ/ZW Sex Determination with Multiple Neo-Sex Chromosomes is Common in Madagascan Chameleons of the Genus Furcifer (Reptilia: Chamaeleonidae)

Chameleons are well-known, highly distinctive lizards characterized by unique morphological and physiological traits, but their karyotypes and sex determination system have remained poorly studied. We studied karyotypes in six species of Madagascan chameleons of the genus Furcifer by classical (conventional stain, C-banding) and molecular (comparative genomic hybridization, in situ hybridization with rDNA, microsatellite, and telomeric sequences) cytogenetic approaches. In contrast to most sauropsid lineages, the chameleons of the genus Furcifer show chromosomal variability even among closely related species, with diploid chromosome numbers varying from 2n = 22 to 2n = 28. We identified female heterogamety with cytogenetically distinct Z and W sex chromosomes in all studied species. Notably, multiple neo-sex chromosomes in the form Z1Z1Z2Z2/Z1Z2W were uncovered in four species of the genus (F. bifidus, F. verrucosus, F. willsii, and previously studied F. pardalis). Phylogenetic distribution and morphology of sex chromosomes suggest that multiple sex chromosomes, which are generally very rare among vertebrates with female heterogamety, possibly evolved several times within the genus Furcifer. Although acrodontan lizards (chameleons and dragon lizards) demonstrate otherwise notable variability in sex determination, it seems that female heterogamety with differentiated sex chromosomes remained stable in the chameleons of the genus Furcifer for about 30 million years.


Introduction
Sex determination systems are variable in amniotes; however, this variability is unequally distributed across clades. Several clades such as viviparous mammals, caenophidian snakes, iguanas, softshell turtles, lacertids, and varanids and their relatives exhibited notable stability of sex chromosomes for several dozens of millions of years [1][2][3][4][5][6][7]. As far as is known, the variability in sex determination is concentrated to a few clades such as emydid and geoemydid turtles, geckos, and acrodontan lizards [8][9][10]. The reasons for the differences in the stability of sex determination systems among clades are still not fully understood. Some models suggest that the variability in sex determination is a consequence of the ancestral environmental sex determination (ESD) in a group and multiple independent transitions from ESD to genotypic sex determination (GSD), which is then largely resistant to transitions to ESD [9][10][11][12]. Other scenarios assume that transitions between ESD and GSD are possible in both directions [13,14]. In any case, it seems that highly differentiated sex chromosomes tend to be evolutionary stable and recent molecular evidence disputed earlier reported transitions from GSD with highly differentiated sex chromosomes to ESD [15,16]. On the other hand, poorly differentiated sex chromosomes in reptiles, e.g., in boas and pythons [17,18], tend to be prone to sex chromosome turnovers, but it is not yet clear whether they can be replaced only with other GSD systems or with ESD as well. More accurate testing of these scenarios is currently prevented by the lack of primary data on sex determination in most reptilian lineages.
Iguania is an important squamate group with approximately 1935 species [19]. The two iguanian sublineages, Pleurodonta and Acrodonta, differ in the stability of sex determination. As far as is known, all pleurodontan lizards share male heterogamety, with sex chromosomes being homologous across all iguana families with the exception of basilisks [4,20]. In contrast, species with either ESD, female heterogamety, or male heterogamety were previously reported in Acrodonta (chameleons and agamids). Our knowledge of sex determination in chameleons has, until now, been scarce. Earlier reports on ESD in this group appear questionable [21][22][23]. Male heterogamety with poorly differentiated XX/XY sex chromosomes was found in Chamaeleo calyptratus [10], while female heterogamety was reported in Furcifer oustaleti and Furcifer pardalis [24]. In both species of the genus Furcifer, the sex chromosomes are heteromorphic and the W chromosomes are heterochromatic. Notably, F. pardalis has multiple neo-sex chromosomes of the type Z 1 Z 1 Z 2 Z 2 /Z 1 Z 2 W [24].
Here, we present the results of our cytogenetic examination based on classical (conventional stain, C-banding) and molecular (comparative genomic hybridization, in situ hybridization with rDNA, microsatellites, and telomeric sequences) cytogenetic approaches in six species of the genus Furcifer with the aim of expanding our knowledge of the evolution of karyotypes and sex determination in this group.

Samples and Species Verification
Blood samples were collected from six species of chameleons from the genus Furcifer, to prepare mitotic chromosome suspensions and DNA isolation (

DNA Isolation and Species Identification
Total DNA was isolated from all examined individuals using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). The standard DNA barcoding region of the mitochondrial gene cytochrome c oxidase I (COI) gene was amplified by PCR with primers optimized for reptiles [25] and following standard protocol [26]. The PCR products were purified and sequenced bi-directionally by Macrogen (Seoul, Korea). The obtained COI sequences were trimmed in FinchTV [27], aligned in BioEdit v5.0.9 [28], analysed in MEGA v10 [29] and DnaSP v6 [30], and compared to sequences deposited in public databases by BLASTn [31] for accurate taxon identification. All sequences were deposited in GenBank under the accession numbers MN757875-MN757882.

Chromosome Preparation and Staining
The mitotic chromosomal suspensions were prepared by leucocyte cultivation from fresh peripheral blood. Our protocol was previously described in Mazzoleni et al. [32]. Briefly, the cultivation medium Chromosomal preparations were dropped to slides and stained by Giemsa for karyotype reconstruction and C-banding for visualization of constitutive heterochromatin, following the protocol of Sumner [33] modified according to Mazzoleni et al. [32].

Fluorescence In Situ Hybridization (FISH) with Telomeric Probe
We applied in situ hybridization with telomeric probe with the aim of uncovering the distribution of telomeric-like sequence (TTAGGG) n in the genomes of the chameleons of the genus Furcifer. The probe was prepared and labelled by PCR according to the protocols of Ijdo et al. [34] and Rovatsos et al. [35]. The probe was diluted in hybridization buffer (50% formamide in 2 × saline-sodium citrate (SSC), pH 7.0).
Slides with chromosomal spreads were washed in 2 × SSC solution and subsequently treated with RNase A (100 µg/mL) for 1 h at 37 • C. The slides were again washed in 2 × SSC and treated by 0.01% pepsin for 10 min at 37 • C. Subsequently, the slides were washed in phosphate buffered saline (PBS) solution, post-fixed in 1% formaldehyde solution for 10 min, washed again in PBS, dehydrated in ethanol series and air-dried. The slides were treated in 70% formamide for 4 min at 75 • C, washed in 2 × SSC and again dehydrated in ethanol series. Once the slides were dried, we added the telomeric probe and incubated them overnight at 37 • C. The second day started with washing the slides in 2 × SSC and then three times in 50% formamide for 5 min at 37 • C. In the next step, the slides were washed twice in 2 × SSC and once in 4 × SSC/0.

Fluorescence In Situ Hybridization with rDNA Probe
The probe for FISH with rDNA loci was prepared from a plasmid (pDmr.a 51#1) with an 11.5-kb insert encoding the 18S and 28S ribosomal units of Drosophila melanogaster [36] and labelled by dUTP-biotin using nick translation (Abbott Laboratories, Lake Bluff, IL, USA). The rDNA probe was hybridized to chromosome suspensions and detected according to the protocol for FISH with telomeric probes, described above.

Fluorescence In Situ Hybridization with Probes for Repetitive Elements
We tested the distribution of repetitive elements in sex chromosomes of three microsatellite motifs: GATA, AG, and TAC in the species where we had female individuals. Those microsatellite motifs often accumulate in the heterochromatic regions of sex chromosomes in reptiles, including caenophidian snakes [37], chameleons [24], geckos [38], and turtles [39]. For the FISH experiment, we used commercially-synthesized and biotin-labelled probes with sequences (GATA) 8 , (AG) 15

Comparative Genome Hybridization (CGH)
The probes for CGH were prepared by nick translation (Abbott Laboratories, Lake Bluff, IL, USA), according to the manufacturer's protocol. The probe specific for male genome was labeled by dUTP-biotin (Roche, Basel, Switzerland), while the probe specific for female genome by dUTP-digoxigenin (Roche, Basel, Switzerland). Both probes were mixed in equal concentration and diluted in hybridization buffer (50% formamide in 2 × SSC; 10% dextran sulfate; 10% sodium dodecyl sulfate; and Denhard's buffer, pH 7.0). The FISH protocol follows the protocol of the FISH with telomeric probe for the slide preparation. The slides were incubated for 48 h at 37 • C. The probe was washed in 50% formamide/2 × SSC three times for 5 min at 37 • C. In the next step, the slides were washed in 2 × SSC at room temperature and 4 × SSC/0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). After the washes, we incubated the slides in 4 × SSC/5% blocking reagent for 30 min at

Furcifer antimena
The karyotype consists of 2n = 24 chromosomes (Figure 1a), in accordance with the results of Bourgat [40]. C-banding revealed heterochromatin in the centromeric and pericentromeric chromosome regions of many chromosomes (Figure 2a). Only males were examined and no sign of sex chromosomes were identified. The telomeric sequences were detected in terminal positions of all chromosomes and in the centromeric, pericentromeric, and interstitial regions of the sixth largest chromosome pairs (Figure 3a). rDNA loci were located in terminal position of the q arm of the third chromosome pair (Figure 4a).

Furcifer bifidus
The karyotype consists of 2n = 24 chromosomes in males, but 2n = 23 chromosomes in females (Figure 1c,d). C-banding revealed distribution of heterochromatin in the centromeric and pericentromeric regions in all autosomes with a notable accumulation on the W chromosome (Figure 2c,d). The sex chromosomes are of Z 1 Z 1 Z 2 Z 2 /Z 1 Z 2 W constitution. Both Z 1 and Z 2 chromosomes are microchromosomes, while the W chromosome is medium-sized, subtelocentric, and partially heterochromatic, with a prominent C-positive band in the centromere (Figure 1c,d and Figure 2c,d). The telomeric sequences were detected in the terminal positions of all chromosomes and in the centromeric regions of all chromosomes, except the first pair and the W chromosome (Figure 3c). rDNA loci were located in the terminal position of the q arm of the second chromosome pair (Figure 4c). GATA motif accumulates in three pairs of autosomes, but not in the sex chromosomes (Figure 5a). AG motif accumulates in three pairs of autosomes and in the centromeric region of the W chromosome ( Figure 5e). TAC motif accumulates in three pairs of autosomes and in the interstitial regions of the W chromosome (Figure 5i). CGH revealed strong sex-specific differences only in the female in the short arm and/or pericentromeric region of the W chromosome (Figure 5m,q).

Furcifer lateralis
The karyotype consists of 2n = 24 chromosomes in both sexes (Figure 1e,f). C-banding revealed distribution of heterochromatin in (peri)centromeric regions in most autosomes and across the whole W chromosome (Figure 2e,f). The sex chromosomes are of the ZZ/ZW type. Both Z and W chromosomes are microchromosomes and the W chromosome is heterochromatic (Figure 2e). The telomeric sequences were detected in the terminal positions of all chromosomes and in the centromeric, pericentromeric and interstitial regions of five chromosome pairs (Figure 3d). rDNA loci were located in the terminal position of the q arm of the third chromosome pair (Figure 4d). GATA motif accumulates in two pairs of microchromosomes and in both sex chromosomes (Figure 5b). AG and TAC motifs accumulate in all autosomes and in both sex chromosomes (Figure 5f,j). CGH revealed strong sex-specific differences only in the female across the whole W chromosome (Figure 5n,r).

Furcifer minor
The karyotype consists of 2n = 22 chromosomes (Figure 1b). C-banding revealed distribution of heterochromatin in the (peri)centromeric chromosome regions (Figure 2b). Sex chromosomes were not identified; however, only males were examined in this species. The telomeric sequences were detected in the terminal positions of all chromosomes, and in the centromeric, pericentromeric, and interstitial regions of five chromosome pairs (Figure 3b). rDNA loci were located in terminal position of the q arm of the second chromosome pair (Figure 4b).

Furcifer verrucosus
The karyotype consists of 2n = 22 chromosomes in males, but 2n = 21 chromosomes in females (Figure 1g,h). Karyotype with 2n = 22 chromosomes was previously reported for this species from Bourgat [41]. C-banding revealed distribution of heterochromatin in the centromeric and pericentromeric regions in many autosomes and notable block on the W chromosome (Figure 2g,h). The sex chromosomes are of the Z 1 Z 1 Z 2 Z 2 /Z 1 Z 2 W type. Both Z 1 and Z 2 chromosomes are microchromosomes, while the W chromosome is small-sized, subtelocentric and partially heterochromatic (Figures 1g and 2g). The telomeric sequences were detected in the terminal positions of all chromosomes and in the centromeric, pericentromeric and interstitial regions of six chromosome pairs (Figure 3e). rDNA loci were located in the terminal position of the q arm of the 4 th chromosome pair (Figure 4e). GATA motif accumulates in the W chromosome (Figure 5c). AG motif accumulates in all chromosomes, including the sex chromosomes (Figure 5g). TAC motif accumulates in the W chromosome (Figure 5k). CGH revealed strong sex-specific differences only in females in the short arm and part of the long arm of the W chromosome (Figure 5o,s).

Furcifer willsii
The karyotype consists of 2n = 28 chromosomes in males, but 2n = 27 chromosomes in females (Figure 1i,j). A karyotype with 2n = 28 chromosomes was previously reported for this species [42]. C-banding revealed distribution of heterochromatin in the (peri)centromeric regions in all autosomes and a large accumulation in the W chromosome (Figure 2i,j). The sex chromosomes are of the Z 1 Z 1 Z 2 Z 2 /Z 1 Z 2 W type. Both Z 1 and Z 2 chromosomes are microchromosomes, while the W chromosome is large, subtelocentric, and partially heterochromatic (Figures 1i and 2i). The telomeric sequences were detected in terminal positions of all chromosomes and in the centromeric regions of the first and second chromosome pair and in the W chromosome (Figure 3e). rDNA loci are located in the terminal positions of four chromosome pairs in five clusters (Figure 4f). GATA, AG, and TAC motifs accumulate in the centromeric regions of the first and second chromosome pair and in the p arm of the W chromosome (Figure 5d,h,l). CGH revealed strong sex-specific differences only in the central part of the long arm of the W chromosome (Figure 5p,t).

Discussion
Chromosome numbers revealed by us in F. antimena, F. verrucosus, and F. willsii agree with the pioneering descriptions of their male karyotypes performed before the onset of advanced cytogenetic techniques [40][41][42]. According to our knowledge, karyotypes of three species (F. bifidus, F. lateralis, and F. minor) are described here for the first time. Our study increased the number of the chameleons of the genus Furcifer with described karyotypes to 14 (reviewed in [43]) and confirmed that the members of this genus have rather variable karyotypes, with diploid chromosome numbers (2n) in males being 2n = 22, 24, 26, and 28. Notably, chameleons range in diploid chromosome numbers from 2n = 20 to 2n = 62 [43] and have generally higher variability in comparison to other closely-related squamate lineages, such as iguanas (2n = 19-48) [44][45][46], snakes (2n = 24-56) [44,47], and varanids (2n = 40 in all studied species) [48]. This variability is visible not only in the chromosomal numbers, but also in differences in size and shape of chromosomes as can be exemplified in the variation of the position of interstitial telomeric repeats ( Figure 3) and rDNA loci (Figure 4). Such variation could be the result of multiple inter-and intra-chromosomal rearrangements.
Before our present study, a chromosome system of sex determination was known in only three species of chameleons (F. oustaleti, F. pardalis, and C. calyptratus) [10,24]. Our data show that female heterogamety is common in the genus Furcifer, as we detected cytogenetically distinguishable sex chromosomes in all four species (F. bifidus, F. lateralis, F. verrucosus, and F. willsii), with available female specimens. The W chromosomes in all these species contain notable heterochromatic blocks ( Figure 2) and CGH revealed that their genetic content is at least partially female-specific ( Figure 5) in accordance with the previously published results in F. oustaleti and F. pardalis [24]. The female-specific signals in CGH co-localize with heterochromatin in the W chromosomes. On the contrary, the XX/XY sex chromosomes in C. calyptratus are euchromatic and not detectable by CGH [43,49]. C-banding, CGH, and FISH with telomeric repeats did not detect any clear sex-specific differences in karyotypes of several other species of chameleons from the genera Calumma, Rieppeleon, and Trioceros [43]. It seems that female heterogamety with a distinct W sex chromosome is an apomorphy of the clade containing the studied members of the genus Furcifer [50][51][52][53]. Only limited (AG and TAC) or even lack (GATA) of accumulations of microsatellite motifs were observed on the W chromosome in F. bifidus, but notable accumulation of all three motifs was observed in F. verrucosus, F. lateralis, and F. willsii, suggesting that the W chromosomes are homologous in these three species, with the accumulations emerging in their common ancestor after their split from F. bifidus. Despite this variability in the W chromosomes, the Z chromosomes seem to have similar morphology in all examined species. Therefore, we assume that female heterogamety was already present in the common ancestor of the studied members of the genus Furcifer living approximately 30 million years ago (dating follows [51]).
In vertebrates, multiple neo-sex chromosomes evolve frequently under male heterogamety, but rarely under female heterogamety as documented in mammals, birds, reptiles, and fishes [54,55]. Several hypotheses were suggested to explain this pattern, including stronger sexual selection in males reducing effective population size for the Y, male-biased mutation rates [55], and the preferential segregation against neo-sex chromosomes from meiotic drive in female meiosis [54]. The latter hypothesis suggests that rearrangements connected with the emergence of multiple neo-sex chromosomes under female heterogamety could result in a biased sex ratio due to the female meiotic drive, which should be penalized by selection for equal sex ratio. According to this hypothesis, the fixation of the rearrangements of the Y chromosome leading to multiple neo-sex chromosomes can be fixed in a population more frequently, as this male-specific chromosome is not involved in female meiosis and is therefore sheltered against the effects of the female meiotic drive affecting the X, Z, and W chromosomes and autosomes. Interestingly, four species of the genus Furcifer (F. bifidus, F. verrucosus, F. willsii, and F. pardalis) with female heterogamety possess multiple sex chromosomes. Multiple sex chromosomes can arise either by fusion of the ancestral W chromosome with an autosome or by fission of the ancestral Z chromosome. In the members of the genus Furcifer with multiple sex chromosomes, the W is always a large chromosome, therefore, we assume that their multiple sex chromosomes were formed by fusions. Even more interestingly, four species with multiple neo-sex chromosomes do not form a monophyletic clade in respect to the two species with simple ZZ/ZW sex chromosomes (F. lateralis and F. oustaleti) in the reconstructed phylogeny of the genus ( Figure 6). We assume that the Z 1 Z 1 Z 2 Z 2 /Z 1 Z 2 W neo-sex chromosomes were formed independently several, possibly even four times, from the ancestral simple ZZ/ZW system. However, we cannot exclude a scenario that simple ZZ/ZW sex chromosome systems are derived and emerged within the genus via chromosomal fissions from the ancestral multiple neo-sex chromosomes. These alternatives should be investigated in future by identification of the gene content of the sex/neo-sex chromosomes, allowing the assessment of their homology. The genus Furcifer together with elapid sea snakes, the other reptilian lineage with multiple emergences of neo-sex chromosomes under female heterogamety [54], can serve as excellent models to investigate evolutionary hypotheses on the origin of multiple neo-sex chromosomes.