Sex is determined by XX/XY sex chromosomes in Australasian side-necked turtles (Testudines: Chelidae)

Turtles demonstrate variability in sex determination and, hence, constitute an excellent model for the evolution of sex chromosomes. Notably, the sex determination of the freshwater turtles from the family Chelidae, a species-rich group with wide geographical distribution in the southern hemisphere, is still poorly explored. Here we documented the presence of an XX/XY sex determination system in seven species of the Australasian chelid genera Chelodina, Emydura, and Elseya by conventional (karyogram reconstruction, C-banding) and molecular cytogenetic methods (comparative genome hybridization, in situ hybridization with probes specific for GATA microsatellite motif, the rDNA loci, and the telomeric repeats). The sex chromosomes are microchromosomes in all examined species of the genus Chelodina. In contrast, the sex chromosomes are the 4th largest pair of macrochromosomes in the genera Emydura and Elseya. Their X chromosomes are submetacentric, while their Y chromosomes are metacentric. The chelid Y chromosomes contain a substantial male-specific genomic region with an accumulation of the GATA microsatellite motif, and occasionally, of the rDNA loci and telomeric repeats. Despite morphological differences between sex chromosomes, we conclude that male heterogamety was likely already present in the common ancestor of Chelodina, Emydura and Elseya in the Mesozoic period.

www.nature.com/scientificreports www.nature.com/scientificreports/ distinct clades, one distributed in Australasia and the other in South America [23][24][25][26][27] . Members of the family Chelidae have generally high diploid chromosome numbers ranging from 2n = 48 to 2n = 64 10,20 . Stable sex ratios of hatchlings incubated across a range of constant temperatures suggest the presence of GSD in at least three chelid species (Mesoclemmys gibba, Phrynops geoffroanus, Phrynops hilarii) 8 . Cytogenetic studies reported differentiated XX/XY sex chromosomes in three additional species, namely in Acanthochelys radiolata, Chelodina longicollis, and Emydura macquarii [20][21][22]28 . In A. radiolata, the pair of sex chromosomes consists of a medium-sized metacentric and a small acrocentric chromosome 20 . However, McBee et al. 20 examined just a single individual (male), and the authors could neither determine the X or the Y chromosome in the heteromorphic pair, nor test whether this heteromorphism is linked to sex. Therefore, we consider the report on sex chromosomes in A. radiolata dubious. The sex chromosomes in Chelodina longicollis were identified as a pair of small chromosomes with a subtelocentric X and a submetacentric Y chromosome 21 . However, based on the accumulation of the GATA microsatellite motif, Matsubara et al. 28 identified the Y chromosome in the same species as another, notably smaller microchromosome. The sex chromosomes in Emydura macquarii form the fourth-largest pair in the karyogram, consisting of a metacentric X and a submetacentric Y chromosome 22 , with a prominent C-positive band in the telomeric region of the short (p) chromosome arm 28 . In contrast to turtles from the family Chelidae, temperature-dependent sex determination was previously reported in species from the pleurodiran families Pelomedusidae (Pelomedusa subrufa, Pelusius castaneus) and Podocnemididae (Podocnemis unifilis, Podocnemis expansa, and Podocnemis erythrocephala) 8,29-32 . In the current study, we explored sex chromosomes and karyotypes in the side-necked turtles of the genera Chelodina (C. expansa, C. novaeguineae, C. mccordi, C. reimanni, C. rugosa), Emydura (Em. macquarii krefftii), Elseya (El. novaeguineae), and two sibling individuals of the intergeneric hybrid Em. subglobosa (♀) × El. novaeguineae (♂) by applying a combination of conventional and molecular cytogenetic methods. We reconstructed karyograms and examined the presence of differentiated sex chromosomes by C-banding, comparative genome hybridization (CGH), and fluorescence in situ hybridization (FISH) with probes specific for GATA motif, telomeric repeats and rDNA loci, i.e. repetitive elements which often accumulate on the sex chromosomes of reptiles 15,28 .

Results
Species verification. The 5′ end of the mitochondrial cytochrome c oxidase I gene (COI) and/or the mitochondrial cytochrome b gene (cytb) were successfully amplified and sequenced and whenever possible compared to sequences from type specimens recently published by Kehlmaier et al. 33 . All studied individuals showed distinctly less than 3% genetic p-distance from the respective type specimens of the species with which they were identified. However, C. novaeguineae and C. reimanni do not differ in their mitochondrial DNA and the validity of C. reimanni is doubtful 33 . Accordingly, the COI of our specimens of C. reimanni was identical with the type sequences of these two species and our material was identified based on morphology.

Karyotype reconstruction and heterochromatin distribution. All examined individuals of the genus
Chelodina had similar karyotypes with 2n = 54 chromosomes consisting of 12 pairs of macrochromosomes and 15 pairs of microchromosomes. All macrochromosomes were bi-armed, with the exception of the acrocentric chromosome pairs 5 and 8 in C. expansa, C. mccordi, and C. rugosa and of chromosome pair 5 in C. novaeguineae and C. reimanni (Fig. 1). C-banding stain revealed constitutive heterochromatin in the centromeric regions of all chromosomes. In addition, heterochromatic blocks were detected in up to four pairs of microchromosomes in all species as well as in the p-arms of the submetacentric chromosomes from the 4 th pair in C. novaeguineae (Fig. 1).
The individuals from the genera Emydura and Elseya possessed karyotypes with 2n = 50 chromosomes consisting of 12 pairs of macrochromosomes and 13 pairs of microchromosomes. All macrochromosomes were bi-armed. The 4 th largest chromosome pair consisted of two submetacentric chromosomes in females, but a metacentric chromosome and a submetacentric chromosome in males (El. novaeguineae, Em. macquarii krefftii, and the two male hybrids Em. subglobosa × El. novaeguineae; Fig. 2). C-banding revealed constitutive heterochromatin in the centromeric regions of all chromosomes. In addition, heterochromatic blocks were observed in four pairs of microchromosomes and in the 4 th largest pair (Fig. 2).
In situ hybridization with probes for GATA motif, telomeric repeats and rDNA loci. FISH with probes specific for the GATA microsatellite motif revealed a strong accumulation in a single microchromosome in the males of the genus Chelodina. Strong accumulations of this motif were revealed in all males of the genera Emydura and Elseya in the heterochromatic region in the terminal position of the p-arm of the metacentric chromosome from the 4 th pair. No accumulation of the GATA motif was detected in females ( Fig. 3) with the only exception of C. expansa, where the accumulation of the GATA microsatellite motif was identified in three microchromosomes in males but in only two microchromosomes in females.
The probe specific for the telomeric repeats revealed the expected terminal topology. In addition, strong accumulation of telomeric-like motifs was detected in microchromosomes in all studied individuals as well as in the terminal position of the p-arm of the metacentric chromosome from the 4 th pair in the hybrid Em. subglobosa × El. novaeguineae (Fig. 4).
FISH with probes specific for the rDNA loci showed strong accumulation in two chromosomes in both sexes of C. expansa and C. rugosa. rDNA loci were accumulated in two chromosomes in females of C. novaeguineae, but in three chromosomes in males of C. novaeguineae and C. reimanni. Notably, an accumulation of rDNA loci was detected in three microchromosomes in both sexes of C. mccordi. rDNA loci accumulated in two microchromosomes in females of El. novaeguineae and Em. macquarii krefftii. In addition, rDNA loci accumulated also in the telomeric position of the p-arm of the metacentric chromosome from the 4 th pair in male turtles of comparative genome hybridization. CGH revealed strong male-specific genomic content in a single microchromosome in metaphases from males of C. expansa and C. novaeguineae. Male-specific genomic content was detected at the terminal position of the p-arm of the metacentric chromosome from the 4 th pair in metaphases from males of El. novaeguineae. No sex-specific content was found in metaphases of females in C. expansa, C. novaeguineae and El. novaeguineae (Fig. 6).

Discussion
Freshwater turtles of the family Chelidae have karyotypes with 2n = 54 chromosomes in the genus Chelodina and 2n = 50 chromosomes in the genera Elseya and Emydura (Figs. 1 and 2). Our cytogenetic examination confirmed previously published karyotypes for C. expansa, C. novaeguineae, C. rugosa and Em. m. krefftii, with respect to chromosome numbers and morphology 10 , while karyotypes for C. mccordi, C. reimanni, and El. novaeguineae are www.nature.com/scientificreports www.nature.com/scientificreports/ presented here for the first time ( Figs. 1 and 2). Within the genus Chelodina there was an evolutionary change in the shape of the chromosome pair 8, which is metacentric in C. novaeguineae and C. reimanni, but acrocentric in C. expansa, C. mccordi and C. rugosa. The metacentric shape in C. novaeguineae and C. reimanni can be a synapomorphy of these closely related or synonymous species 33 . The transitions between acrocentric and metacentric shape in a chromosome pair together with the conservation in chromosome numbers are often caused by intrachromosomal rearrangements in reptiles [34][35][36][37] . This hypothesis should be tested in the genus Chelodina by comparative cytogenetics in future, using whole chromosome painting or comparative BAC-FISH.
In situ hybridization with probes specific for repetitive elements that often accumulate on vertebrate sex chromosomes revealed an extensive accumulation of the GATA microsatellite motif in odd numbers of chromosomes in the metaphases of all male chelids. The relevant chromosome corresponds to a dot-like microchromosome in the genus Chelodina but to a single chromosome of the 4 th pair of the complement in the genera Emydura and Elseya (Fig. 3). In addition, rDNA loci are amplified in odd numbers of chromosomes in males of C. novaeguineae, C. reimanni, El. novaeguineae and in the Em. subglobosa × El. novaeguineae hybrids (Fig. 4). Telomeric repeats seem to accumulate in a single chromosome of the 4 th pair of the complement in El. novaeguineae and in the Em. subglobosa × El. novaeguineae hybrids (Fig. 5). We suggest that the chromosome with the amplification of the GATA microsatellite motif and in some species also of the rDNA loci and telomeric-like repeats in males is the Y chromosome. This conclusion is further supported by the results of CGH performed here for three species (C. expansa, C. novaeguineae, and El. novaeguineae) visualizing a male-specific genomic content within these chromosomes (Fig. 6). A metacentric Y chromosome was previously described for Em. m. macquarii by Martinez et al. 22 with similar morphology as in El. novaeguineae, Em. macquarii krefftii and the two male Em. subglobosa × El. novaeguineae hybrids. In contrast to El. novaeguineae and the two male Em. subglobosa × El. novaeguineae hybrids, the accumulation of GATA microsatellite repeats was not detected here in the metacentric Y chromosome of Em. macquarii krefftii and previously also in Em. m. macquarii 28 . We assume that the GATA motif does not exist or accumulates in very low copy numbers in the Y chromosome of Em. macquarii krefftii and Em. m. macquarii, below the detection threshold of molecular cytogenetic methods. This situation likely reflects the extensive evolutionary dynamics of the heterochromatic content of degenerated sex chromosomes in sauropsids 28,38,39 .
As identified with our cytogenetic methods (i.e. karyotype reconstruction, C-banding, and FISH), the X chromosome is the submetacentric chromosome in the 4 th largest pair of the complement in Elseya and Emydura www.nature.com/scientificreports www.nature.com/scientificreports/ (Fig. 2). We were not able to visualize the X chromosome in the genus Chelodina by our cytogenetic methods, yet, the chromosome pairing in karyograms suggests that it should be a microchromosome (Fig. 1). Ezaz et al. 21 concluded that sex chromosomes in C. longicollis correspond to a pair of small-sized chromosomes with a prominent heterochromatic block. However, we assume that the sex chromosomes of C. longicollis were misidentified in the study of Ezaz et al. 21 . Our results agree with Matsubara et al. 28 who showed the Y chromosome in Chelodina is a different, tiny microchromosome with a prominent amplification of microsatellite repeats.
All Australasian chelid species studied to date possess an XX/XY sex determination system (this study 21,22,28 ). Homology between XX/XY sex chromosomes with dissimilar morphology in representatives from the genus Chelodina when compared with Elseya and Emydura might be supported by accumulation of the same repetitive motifs (GATA microsatellite, rDNA, telomeric-like sequences) in at least some members of these two clades (Figs. 3-5), but the accumulation of the same repetitive motifs in heterochromatic regions is generally a poor indicator of sex chromosome homology 28,39 . Matsubara et al. 28 suggested that the sex chromosomes in the ancestor of Australasian chelids were a pair of microchromosomes, similar to the recent Chelodina, and a rearrangement occurred in the common ancestor of Elseya and Emydura. According to Matsubara et al. 28 , the ancestral sex chromosomes either (i) fused with a medium-sized pair of autosomes or (ii) a part of the ancestral sex chromosomes, including the sex determining region and a surrounding repetitive content, was translocated www.nature.com/scientificreports www.nature.com/scientificreports/ to a medium-sized autosome. However, the clades of the genera Chelodina and Elseya/Emydura show a sister group relationship (Fig. 7). Therefore, we assume that another scenario for sex chromosome homology is equally parsimonious, i.e. that the ancestral sex chromosomes were of the Elseya/Emydura type and a chromosomal rearrangement in the ancestor of the genus Chelodina transferred the sex-determining locus to a microchromosome.
If sex determination is homologous between the two chelid clades, the XX/XY chromosomes in this group could date back to their last common ancestor living c. 50-120 million years ago 27,40,41 . In order to scrutinize the possible homology of sex chromosomes across Australasian chelids in future, it will be crucial to identify the gene content of their sex chromosomes using genomic methods, as recently applied in other reptilian lineages 9,42-46 .

Material and Methods
Studied material. We collected blood samples to establish cell cultures for chromosome preparations and for DNA isolation from side-necked turtles of the genera Chelodina (C. expansa, C. mccordi, C. novaeguineae, C. reimanni, C. rugosa), Emydura (Em. macquarii krefftii) and Elseya (El. novaeguineae), and two sibling hybrids Em. subglobosa (♀) × El. novaeguineae (♂). All turtles are either captive-bred or legally imported from the wild, and kept at Plzeň Zoo (Czech Republic), the Zoo Prague (Czech Republic), Turtle Island (Austria), or the Museum of Zoology, Senckenberg Dresden (Germany). A detailed list of specimens is provided in Table 1. Blood samples DNA isolation, chromosome preparation and staining. Genomic DNA was extracted from blood samples using a DNeasy Blood and Tissue Kit (Qiagen). Chromosome suspensions for cytogenetic analyses were obtained from whole-blood lymphocyte cultures following the protocol described in Mazzoleni et al. 19 . Chromosome spreads were stained with Giemsa for karyotype reconstruction (Figs. 1 and 2). The distribution of constitutive heterochromatin was detected by C-banding 47 , with slight modifications as described in Mazzoleni et al. 19 .

Species verification.
Species identification is challenging in the genus Chelodina, as taxonomy is complicated and not fully resolved yet (for review see Kehlmaier et al. 33 ). Therefore, we characterized our material and www.nature.com/scientificreports www.nature.com/scientificreports/ verified its taxonomy by sequencing the standard "DNA barcoding" region from the mitochondrial cytochrome c oxidase subunit I gene (COI) and/or the mitochondrial cytochrome b gene (cytb). This data is intended to genetically identify our cytogenetic material in future, regardless of potential taxonomic changes. The COI fragment was amplified by PCR using either the reptile-specific primers RepCOI-F and RepCOI-R 48 or the universal primers LCO1490 and HCO2198 49 . The cytb gene was amplified by PCR using the primers H16064 and L14919 50 . For both genes, we prepared the PCR reaction and cycling conditions according to Koubová et al. 51 . The PCR products were sequenced bi-directionally by Macrogen (Korea), and the obtained haplotype sequences were deposited in GenBank under the accession numbers MN757883-MN757886. The COI and cytb sequences were aligned using CLUSTALW 52 , as implemented in BioEdit v5.0.9 53 , and subsequently analyzed in DnaSP v5.10.1 54 . All sequences were compared with those from Kehlmaier et al. 33 , derived from type specimens, and Le et al. 55 . Genetic distances among haplotypes were calculated in MEGA v7 56 . fluorescence in situ hybridization. The distributions of the GATA microsatellite motif was examined, as well as of the TTAGGG telomeric repeat and the rDNA loci, using FISH. The (GATA) 8 probe was synthesized and labeled with biotin (Macrogen, Korea). The telomeric probe was synthesized and labeled with biotin by PCR according to a previously published protocol 57 . The probe for the rDNA loci was prepared from a plasmid (pDm r.a51#1) with an 11.5-kb insertion, encoding the 18S and 28S rRNA units of Drosophila melanogaster 58 ; for the labeling protocol see Rovatsos et al. 9 . Hybridization conditions, post-hybridization washes, signal amplification and detection are explained in detail in Rovatsos et al. 59 .
comparative genome hybridization. To detect sex-specific regions of the genome, CGH was performed using metaphase chromosomes of both male and female individuals of C. expansa, C. novaeguineae, and El. novaeguineae. The detailed protocol for probe and hybridization experiments is presented in Rovatsos et al. 59 .
Microscopy and image analysis. Giemsa-stained metaphase chromosomes were studied under a Carl Zeiss AxioImager.Z2 microscope, equipped with Metafer Scanning Platform (Metasystems) and a MetaSystems CoolCube digital camera. Images were processed for karyotype reconstruction with Ikaros karyotyping software www.nature.com/scientificreports www.nature.com/scientificreports/ (Metasystems). For C-banding, FISH and CGH methods, images from at least 20 metaphase chromosomes were analyzed using a Provis AX70 (Olympus) fluorescence microscope, equipped with a DP30BW digital camera (Olympus). All images were acquired in black and white, and later superimposed with colours in DP Manager imaging software (Olympus).   33 . Information on sex determination systems was compiled from this and previously published studies 8,21,22,[28][29][30][31][32] . Sex chromosomes are microchromosomes in turtles from the genus Chelodina, but macrochromosomes in turtles from the genera Elseya and Emydura. GSD: genotypic sex determination, TSD: temperature dependent sex determination.