Shared Origin of Y and Z Chromosomes in the Turnover of XY and ZW Systems in the Frog Glandirana rugosa

TL;DR

This study explores how the Japanese frog Glandirana rugosa uses different sex determination systems and finds that its Y and Z chromosomes share a common origin.

Contribution

The study reveals the shared origin of Y and Z chromosomes in a species with multiple sex determination systems.

Findings

All sex chromosomes in G. rugosa originated from chromosome 7.

The Y and Z chromosomes show high sequence similarity, suggesting a shared ancestral origin.

The findings support transitions between XY and ZW systems via homologous chromosome reuse.

Abstract

The Japanese frog Glandirana rugosa, endemic to Japan, exhibits both XY and ZW sex determination systems in different populations, representing a rare example of sex chromosome turnover within a single species. To explore the genetic basis of this phenomenon, we analyzed the X, Y, Z, and W chromosomes using microdissection followed by next-generation sequencing. All chromosomes originated from chromosome 7, and the sex chromosomal sequences were homologous. Comparative analyses revealed a high degree of sequence similarity between the Y and Z chromosomes. This suggests that the Y and Z chromosomes may have originated from the same ancestral chromosome and remained highly homologous at the genomic sequence level. This relationship supports the idea that transitions between the XY and ZW systems can occur through the reuse of homologous chromosomes. Our findings indicate that G. rugosa provides an informative model for studying the early stages of sex chromosome differentiation and turnover. Understanding these processes in G. rugosa may enhance our understanding of the evolutionary dynamics of sex chromosomes across vertebrates.

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1. Introduction

Sex chromosomes are generally thought to have originated from a pair of ordinary autosomes and play a crucial role in genetic sex determination [1,2,3]. However, sex chromosomes show remarkable diversity in their structure, gene content, and modes of differentiation across eukaryotes, reflecting a wide range of evolutionary trajectories from homomorphic to highly differentiated systems [4,5,6,7,8,9]. Despite this diversity, sex chromosomes are frequently replaced during evolution, a process known as sex chromosome turnover [10]. The evolution of sex chromosomes, particularly sex chromosome turnover, represents one of the most intriguing and complex processes in genetics and evolutionary biology. Sex chromosome turnover refers to the replacement or transition of one sex chromosome system by another, such as a change from an XX/XY (male heterogamety) to a ZZ/ZW chromosome (female heterogamety) system or vice versa, or the independent emergence of sex chromosomes with distinct evolutionary origins (e.g., a shift between two different XY systems) [11]. Among vertebrates, amphibians and fishes are particularly notable for their frequent and dynamic sex chromosome turnover, offering exceptional opportunities to investigate the mechanisms that drive these transitions [12,13,14,15].

To explore the molecular and evolutionary mechanisms underlying sex chromosome turnover, we focused on the Japanese endemic frog Glandirana rugosa, known as the Japanese wrinkled frog, which uniquely exhibits both XY and ZW sex chromosome systems within the same species [14]. This species provides a rare natural model for studying the evolutionary dynamics of sex chromosome transitions, and the transition between the XY and ZW chromosome systems still occurs. So far G. rugosa consists of at least six geographically distinct populations: East Japan (homomorphic XY; recently redefined as G. reliquia), West Japan (homomorphic XY), North-Western Japan (heteromorphic ZW), Eastern Central Japan (heteromorphic XY), Western Central Japan (heteromorphic ZW, referred to as Neo-ZW), and Neo-West Japan (homomorphic XY) (Figure 1) [14]. This species possesses 13 pairs of chromosomes and a large genome of approximately 7 Gb [15,16]. In these populations, the North-Western and Western Central Japan groups exhibit heteromorphic ZW chromosomes, whereas the Eastern Central Japan group has heteromorphic XY chromosomes. Interestingly, all three populations share chromosome 7 as their sex chromosomes [15].

Previously, we sequenced the genome of a ZZ male individual from the Western Central population (classified as Neo-ZW) [17] and identified sex-linked genes on chromosomes 3 and 1 in two homomorphic XY populations in East and West Japan, respectively [18]. These studies suggest that sex chromosomes derived from at least three distinct ancestral autosomes have independently evolved within this single species. In particular, chromosome 7 has independently differentiated into both heteromorphic XY and ZW systems in the Eastern Central and North-Western/Western Central populations (Figure 1) [16], whereas other populations retain homomorphic undifferentiated sex chromosomes. This remarkable diversity makes G. rugosa an exceptional species for investigating the molecular processes and evolutionary pathways involved in sex chromosome turnover.

Despite these extensive characterizations, the molecular mechanisms underlying the frequent sex chromosome turnover in G. rugosa remain poorly understood. In particular, it is unclear how the XY and ZW chromosome systems, both derived from chromosome 7, have diverged and to what extent they share homologous regions and evolutionary features. To address these questions, we performed a comparative genomic analysis of the X, Y, Z, and W chromosomes in G. rugosa. The aim of this study is to clarify the genomic origin and homology of the X, Y, Z, and W chromosomes in G. rugosa using microdissected chromosome sequences. Our analysis provides new insights into how homologous chromosomes evolve into distinct sex chromosome systems, thereby shedding light on the dynamic nature of vertebrate sex chromosome evolution.

2. Materials and Methods

2.1. Animal Collection, Chromosome Preparations, and Microdissection of Sex Chromosomes

Specimens of XY male and ZW female G. rugosa used for the experiments were collected from the Eastern Central Japan population, where they were at Hamamatsu City in Shizuoka Prefecture, and the North-Western Japan population, where they were at Niigata City in Niigata Prefecture, respectively, and mitotic metaphase chromosomes were prepared from blood cell cultures. We performed microdissection using an inverted phase Zeiss Axio contrast microscope Vert.A1 (Zeiss, Oberkochen, Germany) equipped with Eppendorf TransferMan NK 2 micromanipulator (Eppendorf, Hamburg, Germany). Glass needles were made from 1.0 mm diameter capillary glass using a glass capillary puller, Sutter P-30 Micropipette Puller (Sutter Instrument, Novato, CA, USA) and sterilized by irradiation of ultra violet. We scratched candidates of the X, Y, Z, and W chromosomes from the metaphase spreads of two individuals (XY male and ZW female) using the size and morphological differences of chromosomes previously reported [16]. We amplified sex chromosome DNA using the GenomePlex single-cell whole-genome amplification kit (Sigma, Ronkonkoma, NY, USA), following the manufacturer’s instructions.

2.2. Probe Preparations, and FISH Analysis

We generated X, Y, Z, and W chromosome probes from the amplified DNA of each single chromosome. The sex chromosome probes were labeled by nick translation incorporating SpectrumGreen-dUTP (Abbott, Abbott Park, IL, USA) or SpectrumOrange-dUTP (Abbott, USA) and precipitated with 20 µg of glycogen as a carrier. After decantation, the labeled probe pellets were resuspended in 15 µL of hybridization buffer (50% formamide, 10% dextran sulfate, 2 × SSC, 40 mmol/L sodium phosphate pH7.0 and 1× Denhardt’s solution). The resuspended probe mixture was placed on a chromosome slide, covered with a coverslip, and sealed using rubber cement. The slides were then denatured on a hot plate at 68.5 °C for 5 min and hybridized overnight in a humid chamber at 37 °C for two days. The slides were then washed first with 0.4 × SSC, 0.3% IGEPAL (Sigma-Aldrich, St. Louis, MO, USA) at 55 °C for 2 min, followed by 2 × SSC, 0.1% IGEPAL for 1 min at room temperature. The slides were dehydrated using an ethanol series, air-dried, and mounted with anti-fade medium Vectashield (Vector Laboratories, Newark, CA, USA) containing 20 µg/mL 4′,6-diamidino-2-phenylindole (DAPI). FISH images were captured using a Zeiss Axioplan epifluorescence microscope equipped with a charge-coupled device (CCD) camera (Zeiss, Concord, NC, USA). AxioVision 4.8 (Zeiss) was used for microphotography and image analyses. We prepared four X chromosome probes, 10 Y chromosome probes, eight Z chromosome probes, and four W chromosome probes of the amplified DNA from each single chromosome.

2.3. Sequencing of Chromosome DNAs

We sequenced the amplified chromosome DNAs of single microdissected X, Y, Z, and W chromosomes, which were validated by FISH analyses. Next-generation sequencing of the DNA library was performed at the Beijing Genome Institute (BGI) using an HiSeq2000 (Illumina, San Diego, CA, USA). We sequenced a 500 bp insert library, and all reads were trimmed to 90 bp for both ends to obtain 2 GB of clean reads.

2.4. Mapping, Generating Consensus Sequences and Phylogeny

Reads from the X, Y, Z, and W chromosomes were mapped onto the contigs of the reference genome of a male ZZ individual from the Western Central population, which is located in Kyoto city in Kyoto prefecture, using BWA-MEM2 [17,19]. Each consensus sequences of the X, Y, Z, and W chromosomes were created from the mapped reads using the GATK pipeline [20]. Among the consensus sequences, 38 were common to all. Multiple sequence alignment was performed on these common sequences using ClustalW [21], and Mega11 [22] was used to construct a phylogenetic tree using the neighbor-joining method with a bootstrap test (1000 replicates). Pairwise alignment of consensus sequences of each chromosome pair was performed by EMBOSS software 6.5.7 [23], and nucleotide divergence was calculated using Mega11 [22].

3. Results

We sequenced the X, Y, Z, and W chromosomes to directly compare their differences. Single X and Y chromosomes were microdissected from metaphase spreads of a male individual from Hamamatsu (the Eastern Central Japan population), and Z and W chromosomes were obtained from a female individual from Niigata (the North-Western Japan population) (Figure 1). The microdissected DNA was subjected to FISH analysis to confirm that the target chromosomes were accurately microdissected (Figure 2). Each chromosomal DNA was amplified, and the amplified DNA was used for chromosome painting as probes to validate their origin and quality. Figure 2 shows the staining pattern using probes with the highest specificity, which stained the entire target chromosome. Owing to the homology between the X and Y chromosomes or between the Z and W chromosomes, the results of chromosome painting showed that the probe from one sex chromosome weakly stained the other sex chromosome (Figure 2b,d,f,h). Furthermore, the probe from the Y chromosome stained the end of the long arm of chromosome 2, indicating potentially shared homologous sequences (Figure 2d). These DNA were then sequenced using an Illumina HiSeq2000 (~2 GB reads per chromosome, read length of 90 bp; Table 1). The estimated size of haploid chromosome 7 was approximately 400 Mb, and the sequencing depth achieved in this study was approximately five-fold coverage.

Reads from the X, Y, Z, and W chromosomes were mapped onto the reference genome, and the Z chromosome of the Kyoto population (Western Central Japan) was not identical to that of the Niigata population (North-Western Japan) used in this study. Most Z and Y reads were successfully mapped, whereas approximately 80% of the W reads and 30% of the X reads were mapped (Table 1). Of the 1,440,004 contigs in the reference genome, all X, Y, Z, and W chromosomal reads were on 37,678 contigs, covering a total length of 613 Mb. These contigs ranged from 503 to 463,040 bp in length, with a median length of 1182 bp. The mapped lengths of the X, Y, Z, and W chromosomes were 80, 335, 226, and 113 Mb, respectively, corresponding to 20–84% of the estimated length of each chromosome, indicating that only partial sequences were obtained. The contigs that the Z and Y chromosomal reads mapped were the largest number of contigs (3958 contigs in total; 67 Mb; Figure 3a).

Consensus sequences of the X, Y, Z, and W chromosomes were generated and aligned for pairwise comparisons. Although most sequences were highly similar, the Z and Y chromosome sequences were more similar to each other than to the other chromosomes (Table 2). Among all the sex chromosomal sequences, 38 contigs were common, with an average divergence of 0.019 ± 0.006%. Phylogenetic analysis using concatenated sequences of common contigs revealed that the Y and Z chromosomes were closely related (Figure 3b). Separate phylogenetic analyses of the 38 contigs also supported this observation. Among them, 20 contigs exhibited identical sequences across all four sex chromosomes, 12 showed identical sequences between two or three chromosomes, and five indicated a close relationship between the Y and Z chromosomes. In addition, one contig exhibited sequence similarity between the Y and W chromosomes. Although only partial sex chromosomal sequences were obtained, this result demonstrated a close relationship between the Y and Z chromosomes, suggesting that they originated from homologous chromosomes that still share similar allelic sequences.

4. Discussion

Our comparative analysis of the X, Y, Z, and W chromosomes in G. rugosa revealed a high degree of similarity between the Y and Z chromosomes, suggesting that they originated from the same ancestral chromosome (Figure 3 and Figure 4). This finding is consistent with the hypotheses proposed in previous studies [16,24]. Because the Y and Z chromosomes show strong similarity and the other sex chromosomes tend to resemble each other, our results partially support the idea that the Y and Z chromosomes originated from the same allelic homologous chromosome, whereas the X and W chromosomes were derived from the opposite allelic homologous chromosome (Figure 4). However, our comparative analysis did not show a strong pairwise sequence similarity between the X and W chromosomes compared to the Y and Z chromosome pairs. This does not mean that the X and W chromosomes originate from completely different chromosomes, and there is no doubt that the X and W chromosomes originate from chromosome 7. Note that the Y and Z chromosomes maintain high similarity as allelic chromosomes, whereas the X and W chromosomes do not maintain such high similarity as allelic chromosomes (Figure 4). One reason for this is that sequence differentiation and transposon insertion have progressed on the X and W chromosomes due to chromosomal rearrangements, and the similarity between the X and W chromosomes has become lower than that between the Y and Z chromosomes (Figure 4). Although it may be possible to identify more homologous regions among sex chromosomes if the sequence depth and quality are improved, this study currently suggests that their relationship is more complex or modified than previously thought and that the evolution of sex chromosomes in G. rugosa cannot be explained by a simple linear model, such as “W chromosome to X chromosome” or vice versa. In addition, only 38 contigs were shared among all four sex chromosomes, despite the large number of contigs identified for each chromosome (Figure 3a). This likely reflects both biological differences among the four chromosomes and sequencing bias caused by limited DNA quantity and whole-genome amplification after chromosome microdissection.

Our results imply that during turnover events, the same ancestral chromosome can shift its role from the Z chromosome to the Y chromosome or from the Y chromosome to the Z chromosome. Previous single-locus phylogenetic studies have proposed possible transitions between these systems; however, our study is the first to provide direct genome sequence-level evidence supporting such relationships. From an evolutionary perspective, the transition from a ZW chromosome system to an XY chromosome system (or vice versa) could occur through the emergence of a new male- or female-determining gene on a homologous chromosome. It is possible that a new male-determining factor appeared on the proto-Y chromosome, transforming the ancestral Z chromosome into a Y chromosome in the case of the ZW to XY chromosome transition. However, our current sequencing data were insufficient to identify candidate sex-determining genes.

Previous studies have shown that chromosome 7 includes three genes involved in sex determination and differentiation, such as SRY-box transcription factor 3 (SOX3), androgen receptor (AR), and Ad4-binding protein/steroidogenic factor 1 (Ad4BP/SF1), in G. rugosa [16]. SOX3 is a transcription factor belonging to the SOX family that plays essential roles in early neural development and gonadal differentiation [25,26]. SOX3 is also known as an ancestral gene of the mammalian sex-determining gene, sex-determining region Y (SRY) [2]. In amphibians, SOX3 has been implicated in sex differentiation processes and has been proposed as a candidate gene for sex determination (African bullfrog, Pyxicephalus adspersus) [27]. In G. rugosa, Miura et al. [28] recently proposed that SOX3 may play a role in female sex determination in the ZW system, which corresponds to the same population examined in this study. AR is a nuclear hormone receptor that mediates the biological effects of androgens by regulating the transcription of target genes. Androgen signaling pathways, rather than single master genes, often modulate sex differentiation in vertebrates [29]. In amphibians, androgen signaling has been shown to influences gonadal differentiation and sexual phenotype development through hormone-dependent regulatory mechanisms [30]. However, in G. rugosa, the direct involvement of AR in sex determination was not supported, suggesting that AR is unlikely to function as a primary sex-determining factor in this species [28]. Ad4BP/SF1 (nuclear receptor subfamily 5 group A member 1, NR5A1) is a key transcriptional factor of steroidogenic enzymes and plays a fundamental role in the development and function of gonads and adrenal glands [31]. Ad4BP/SF1 is required for proper steroid hormone biosynthesis and is involved in both male and female pathways of gonadal development, acting upstream of multiple genes in the sex determination network [29]. The roles of these genes in sex chromosome turnover remain hypothetical and require future functional and expression analyses. Uno et al. [16] suggested that a large-scale inversion involving the centromeric region may have contributed to heteromorphic sex chromosome turnover between XY and ZW, based on differences in the order of these three genes and the position of the centromere. This inversion may be one of the events that led to sex chromosome differentiation on the X and W chromosomes. These genes were not identified in our genome data, probably because the current assemblies are still incomplete and require further improvement to achieve full gene-level resolution.

Mawaribuchi et al. [24] estimated the coding regions of the X, Y, Z, and W chromosomes by comparing transcriptomes between the sexes and found that all identified sex chromosome linked genes were homologous across all sex chromosomes, whereas Y or W chromosome specific genes have not yet been clearly identified. In addition, the coding sequences of SOX3, which has been suggested to be associated with sex determination in this frog, were found to be identical among all the sex chromosomes. These observations are consistent with our finding that the genomic sequences of sex chromosomes are highly conserved across species. This conservation likely reflects the relatively recent origin of sex chromosomes in G. rugosa before substantial differentiation and degeneration occur. In such species, it is plausible that structural changes, such as chromosomal rearrangements and regulatory modifications, rather than changes in coding sequences, play a major role in driving the early stages of sex chromosome turnover.

Sex chromosome turnover has been documented across a wide range of vertebrate taxa, including fish, reptiles, and amphibians, and is increasingly recognized as a dynamic and recurrent evolutionary process rather than an exceptional event [13]. Similar patterns of repeated sex chromosome turnover and reuse of homologous chromosomes have also been reported in invertebrates, particularly in Diptera, where numerous independent transitions among sex-determining systems have occurred over relatively short evolutionary timescales [32]. Comparative studies in teleost fishes and amphibians have demonstrated that transitions between sex-determining systems can occur rapidly and repeatedly, often involving chromosomes that are only weakly differentiated or entirely homomorphic [33,34,35]. Recent transcriptomic studies on closely related fish species have shown that XY and ZW sex chromosome systems can drive distinct patterns of sex-biased gene expression [36]. Identifying sex-biased genes on the sex chromosomes is an important next step in G. rugosa. These observations suggest that sex chromosomes retain a high degree of evolutionary flexibility during the early stages of differentiation. In this context, the high sequence similarity observed in G. rugosa provides a striking example of how homologous chromosomes can repeatedly assume different sex-determining roles. Rather than representing independent origins from distinct autosomes, our data support a scenario in which allelic homologs of chromosome 7 have alternately evolved into Y or Z chromosomes. This finding aligns with theoretical predictions that turnover is facilitated when sex chromosomes remain largely undifferentiated and recombination suppression is incomplete [37,38,39]. This evolutionary flexibility in G. rugosa is consistent with the “hot-potato” model of sex chromosome turnover, which proposes that sex-determining roles are repeatedly transferred among chromosomes to avoid the long-term accumulation of deleterious mutations [40].

Interestingly, it has been suggested that evolution through the replacement of sex chromosome systems with the same ancestral homologous chromosome may have also occurred during the transition from avian to mammalian ancestors. This hypothesis is based on the observation that the genomes of extant monotremes contain multiple pairs of XY chromosomes that are partially homologous to the ZW chromosomes of modern birds [41]. Repeated chromosomal translocations and large-scale genome rearrangements are hypothesized to have driven the evolutionary transition from the ZW to XY chromosome systems in early mammalian ancestors before the divergence of monotremes and Theria [42]. Although this hypothesis is intriguing, it remains difficult to test directly because it involves ancient evolutionary events that occurred hundreds of millions of years ago, during which recurrent mutations and repeated chromosomal rearrangements may have obscured ancestral genomic signatures.

In contrast, G. rugosa provides a unique opportunity to study similar processes in contemporary species, as sex chromosome turnover may occur within a timescale of hundreds of thousands to millions of years according to the divergence time from relatives of the Glandirana genus [43]. Therefore, this species could serve as a valuable model for elucidating the general principles of sex chromosome turnover in vertebrates. Future studies should aim to obtain high-quality chromosome-level genome assemblies to test these hypotheses in greater detail. Such data will enable comprehensive analyses of gene content, synteny, and structural variations, including inversions and translocations, which may underlie species-specific turnover events in G. rugosa. Integrating genomic, transcriptomic, and epigenetic information is essential for identifying sex-determining genes and reconstructing the evolutionary pathways that led to the coexistence and transition of the XY and ZW chromosome systems within this single species.

5. Conclusions

In this study, we conducted a comparative genomic analysis of the X, Y, Z, and W chromosomes in the Japanese frog G. rugosa, a unique species with both XY and ZW sex chromosome systems. Our sequence-based comparisons revealed a strong similarity between the Y and Z chromosomes, suggesting that they share a common ancestral origin. This finding provides direct molecular evidence of the dynamic nature of sex chromosome turnover, in which homologous chromosomes can repeatedly change their roles in determining sex. Although our current data were limited to partial chromosome sequences, they highlight the structural relationships that may underlie the ongoing transitions between the XY and ZW chromosome systems in this species. Future studies employing chromosome-level assemblies and functional analyses of candidate genes are essential to clarify the molecular mechanisms driving sex chromosome evolution in G. rugosa. Together, our findings emphasize the exceptional value of this species as a model for understanding the plasticity and repeated renewal of vertebrate sex-determination systems.