Meiosis typically generates genetic diversity but can be disrupted, leading to alternative reproductive modes such as hybridogenesis. Interspecific hybridization is often associated with such transitions, yet the underlying mechanisms in vertebrate systems remain poorly understood. We investigated this process by crossing Pelophylax ridibundus and P. lessonae, producing hybrid P. esculentus. Using low-coverage genome sequencing and repeat analysis, we developed a novel minisatellite marker (PlesSat01-48) specific to the P. lessonae genome and combined it with the previously described RrS1 marker of P. ridibundus to cytogenetically track both parental genomes. Fluorescent in situ hybridization revealed that newly formed P. esculentus hybrids undergo rapid modifications of gametogenesis. These include chromosome misalignment, lagging chromosomes, and micronucleus formation leading to premeiotic genome elimination of the P. lessonae genome, while the remaining genome is clonally propagated. This process establishes hemiclonal reproduction via hybridogenesis. Our findings support the recurrent origin of P. esculentus and provide direct cytogenetic insight into the evolution of hybridogenetic reproduction in water frogs.

This methodological study outlines effective and standardized procedures for obtaining high-quality chromosomal preparations in birds, which display complex karyotypes with a large number of microchromosomes. We describe every stage from sample obtaining and culture setup to the successful acquisition of metaphase chromosomes appropriate for in-depth analysis by combining three main methodologies (fibroblast, lymphocyte, and bone marrow cultures) into a coherent and methodical workflow. Furthermore, we offer thorough troubleshooting instructions to tackle typical problems, such as low mitotic indices, poor chromosome spreading, contamination, and suboptimal chromosomal morphology. We also focused on repeatability and adaptation across several bird taxa, taking into consideration species-specific differences in their body sizes, cell development, and chromosomal behavior, extending beyond standard procedure improvement. The incorporation of diverse methodologies into a unified framework offers avian cytogenetics researchers a readily available and practical workflow. Ultimately, our approach enhances the integration of cytogenetic data with novel genomic resources, facilitates the re-evaluation of chromosomal counts, and enables more precise karyotype characterization. This integration is crucial for advancing our understanding of chromosomal evolution, avian genome architecture, and the broader mechanisms underlying avian speciation and diversification.

The field of genetics of bird migration advances, driven by exponential refinements of sequencing and tracking technologies. In willow warblers (Phylloscopus trochilus), a complex repeat-rich region named MARB (Migration Associated Repeat Block) has recently been found to correlate with the routes taken by individual birds from Europe to their African wintering grounds. However, the genomic location of this region remains unknown. Here, we characterized MARB using a combination of approaches to understand how it evolved. We describe the region using long-read genome assemblies of two willow warbler subspecies (P. t. trochilus and P. t. acredula), two related species, the common chiffchaff (P. collybita) and the greenish warbler (P. trochiloides), and whole genome sequencing data from 76 willow warblers. Finally, we applied karyotyping and fluorescent in situ hybridization techniques on willow warbler spermatocytes to cytogenetically locate MARB. Due to the many repeats, we cannot order scaffolds in silico, but probe hybridization on the karyotype shows that MARB constitutes a single locus (~27.5 Mb) spanning most of the 11th largest chromosome in the willow warbler genome. Interestingly, the MARB regions of all species share several characteristics such as relatively high GC content (50%), a high density of specific repeat families and notably, more than 800 olfactory receptor sequences. Regions homologous to MARB may exist in several migrant bird genomes, though currently unassembled due to their complexity. Resolving these in species with similar migratory polymorphisms to willow warblers will be essential to determine whether MARB influences migratory behaviour across species.

Sex determination was once thought to be unstable in ectothermic vertebrates, but several highly diversified groups of non-avian reptiles, such as iguanas sensu lato (Pleurodonta), appear to have conserved sex chromosomes. However, this statement has been criticized as being based on a parsimonious conclusion from limited sampling. Here, we tested sex chromosome homology in a further 35 species of pleurodont lizards, mainly of the family Anolidae. We demonstrated sex chromosome homology based on the comparison of the gene dosage of X-specific genes by quantitative PCR. Including these new data, the homology of sex chromosomes has so far been reported in 92 out of over 1,200 recently recognized species of Pleurodonta. The very conserved sex chromosomes in all but one family are at least as old as the basal split of Pleurodonta (estimated over 90 million years). In the case of the family Corytophanidae, all genera share different XX/XY sex chromosomes, which are thus over 25 million years old. We discuss the efficiency and limitations of the approach used for tests of the homology of sex chromosomes, as well as the reasons for the evolutionary stability of sex chromosomes in some lineages. We argue that to obtain a complete picture, all extant species should be tested for homology; however, until we reach this ambitious goal, parsimonious estimates in lineages where fragmentary data suggest stability of sex chromosomes, as in Pleurodonta, are substantiated.

Reproduction entirely without males is exceptionally rare among vertebrates. Recently, an enigmatic all-female lineage of casquehead lizards, initially thought to be a captive anomaly, appeared in zoos. During a targeted expedition to Honduras, we discovered two wild, asexually reproducing populations near the type locality of Laemanctus julioi, a species originally described from a single female. Using a de novo phased long-read genome and cytogenetics, we reveal these lizards are clonal hybrids, originating from two geographically separated sexual species that diverged 12 million years ago. We confirmed two independent clonal lineages, one matching the zoo population. To form clonal eggs, these lizards duplicate their genome before meiosis. Experimental breeding with a male from a paternal species produced non-viable offspring with increased genome size, suggesting these clones established themselves by remaining isolated from parental species. As the largest-bodied known vertebrates reproducing exclusively without males, these lizards demonstrate that radically different reproductive strategies can remain undetected even in highly conspicuous animals.

Obligatory parthenogenesis in vertebrates is restricted to squamate reptiles and evolved through hybridisation. Parthenogens can hybridise with sexual species, resulting in individuals with increased ploidy levels. We describe two successive hybridisations of the parthenogenetic butterfly lizards (genus Leiolepis) in Vietnam with a parental sexual species. Contrary to previous proposals, we document that parthenogenetic L. guentherpetersi has mitochondrial DNA and two haploid sets from L. guttata and one from L. reevesii, suggesting that it is the result of a backcross of a parthenogenetic L. guttata × L. reevesii hybrid with a L. guttata male increasing ploidy from 2n to 3n. Within the range of L. guentherpetersi, we found an adult tetraploid male with three L. guttata and one L. reevesii haploid genomes. It probably originated from fertilisation of an unreduced triploid L. guentherpetersi egg by a L. guttata sperm. Although its external morphology resembles that of the maternal species, it possessed exceptionally large erythrocytes and was likely sterile. As increased ploidy level above triploidy or tetraploidy appears to be harmful for amniotes, all-female asexual lineages should evolve a strategy to prevent incorporation of other haploid genomes from a sexual species by avoiding fertilisation by sexual males.

BACKGROUND:Tandemly repeated satellite DNA sequences are an important part of animal genomes. They are involved in chromosome interactions and the maintenance of the integral structure of the nucleus, regulation of chromatin conformation and gene expression, and chromosome condensation and movement during cell division. Satellite DNAs located in the centromeric heterochromatin evolve rapidly and likely affect hybrid fertility and fitness. However, their studies are taxonomically highly biased. In lacertid lizards, satDNA has been extensively studied in the subfamily Lacertinae, but the subfamily Eremiadinae has been largely overlooked. RESULTS:In this work, we describe a novel 177-bp-long centromeric satDNA family EremSat177, which is present in all studied species of the genus Eremias, but not in related genera. EremSat177 is not homologous to any previously identified centromeric satellites. Using fluorescence in situ hybridization, we demonstrate its centromeric localization in E. velox and E. arguta. We also show its tandem organization and intra-genomic homogenization by in silico analysis in the genome of E. argus. The phylogenetic analysis of consensus EremSat177 sequences from 12 Eremias species demonstrates that the same monomer subfamily is the most abundant in all these species, and its evolution mainly follows the species phylogeny as revealed by the mtDNA sequences. CONCLUSION:The EremSat177 represents a novel, lineage-specific centromeric satellite DNA, and its role in centromere functioning should be revealed in further research.

Sex chromosomes have evolved repeatedly across eukaryotes. The emergence of a sex-determining (SD) locus is expected to progressively restrict recombination, driving convergent molecular differentiation. However, evidence from taxa like teleost fishes, representing over half of vertebrate species with unmatched diversity in SD systems, challenges this model. Teleost sex chromosomes are often difficult to detect as they experience frequent turnovers, resetting the differentiation process. Nothobranchius killifishes, which include the XY system shared by N. furzeri and N. kadleci and X1X2Y systems in six other species, offer a valuable model to study sex chromosome turnovers. We characterised X1X2Y systems in five killifish species and found that sex chromosomes evolved at least four times independently. Sex-determining regions resided near centromeres or predicted chromosome rearrangement breakpoints in N. brieni and N. guentheri, suggesting recombination cold spots may facilitate sex chromosome evolution. Chromosomes representing the XY system in N. furzeri/N. kadleci were sex-linked also in the outgroup Fundulosoma thierryi, with several genes, including gdf6, residing in the region of differentiation. Although the X1X2Y systems of N. guentheri, N. lourensi (both Coastal clade), and N. brieni (Kalahari clade) involved different chromosomes, they shared a potential SD region. We uncovered two sex-linked evolutionary strata of distinct age in N. guentheri. However, its potential SD gene amhr2 was located in the younger stratum and is hence unlikely to be the ancestral SD gene in this lineage. Our findings suggest recombination landscapes shape sex chromosome turnover and that certain synteny blocks are repeatedly co-opted as sex chromosomes in killifishes.

Sex chromosomes of teleost fishes often have low levels of differentiation and undergo frequent turnovers. Annual Nothobranchius killifishes comprise representatives with male-heterogametic XY or X1X2Y sex chromosome systems, scattered across their phylogeny, nested within species lacking cytologically detectable sex chromosomes. They thus provide a suitable system to study sex chromosome evolution and turnover. Here, we combined molecular cytogenetics and genomic analyses to examine several multiple sex chromosome systems in Nothobranchius spp. and their outgroup Fundulosoma thierryi . We used fluorescence in situ hybridization with three sex chromosome-specific painting probes and bacterial artificial chromosomes (BAC) bearing eight orthologues of genes found to be repeatedly co-opted as master sex determining (MSD) genes in fishes. Our results suggest at least four independent origins of sex chromosomes in the genus Nothobranchius . The synteny block carrying amhr2 gene was shared by X1X2Y systems of N. brieni , N. guentheri and N. lourensi , but the autosomal additions and the overall neo-Y chromosome structure differed among these species. On the other hand, gdf6 gene was localized to neo-Y of F. thierryi . None of the mapped MSD gene candidates seems to determine sex in N. ditte . We further sequenced genomes of F. thierryi female and N. guentheri male by long-read platforms and performed analyses of male and female Pool-seq data and coverage to delimit their non-recombining regions, determine degree of their differentiation, and thus complement the cytogenetic data in assessing potential MSD genes. We found low level of sex chromosomes differentiation in F. thierryi. In N. guentheri , however, we identified two distinct evolutionary strata on neo-Y. The amhr2 gene resides in the younger stratum and has low allelic variation, which questions its role in sex determination. ### Competing Interest Statement The authors have declared no competing interest.

Chameleons are well-known lizards with unique morphology and physiology, but their sex determination has remained poorly studied. Madagascan chameleons of the genus Furcifer have cytogenetically distinct Z and W sex chromosomes and occasionally Z1Z1Z2Z2/Z1Z2W multiple neo-sex chromosomes. To identify the gene content of their sex chromosomes, we microdissected and sequenced the sex chromosomes of F. oustaleti (ZZ/ZW) and F. pardalis (Z1Z1Z2Z2/Z1Z2W). In addition, we sequenced the genomes of a male and a female of F. lateralis (ZZ/ZW) and F. pardalis and performed a comparative coverage analysis between the sexes. Despite the notable heteromorphy and distinctiveness in heterochromatin content, the Z and W sex chromosomes share approximately 90% of their gene content. This finding demonstrates poor correlation of the degree of differentiation of sex chromosomes at the cytogenetic and gene level. The test of homology based on the comparison of gene copy number variation revealed that female heterogamety with differentiated sex chromosomes remained stable in the genus Furcifer for at least 20 million years. These chameleons co-opted for the role of sex chromosomes the same genomic region as viviparous mammals, lacertids and geckos of the genus Paroedura, which makes these groups excellent model for studies of convergent and divergent evolution of sex chromosomes.

Among vertebrates, obligate parthenogenesis occurs exclusively in squamate reptiles. Premeiotic endoreplication in a small subset of developing oocytes has been documented as the mechanism of production of unreduced eggs in minutely explored obligate parthenogenetic lineages, namely in teiids and geckos. The situation in the lacertid genus Darevskia has been discussed for decades. Certain observations suggested that the ploidy level is restored during egg formation through a fusion of egg and polar body nuclei in Darevskia unisexualis and D. armeniaca . In this study, we re-evaluated the fusion hypothesis by studying diplotene chromosomes in adult females of sexual species D. raddei nairensis and obligate parthenogens D. armeniaca, D. dahli and D. unisexualis . We revealed 19 bivalents in the sexual species and 38 bivalents in the diploid obligate parthenogens, which uncovers premeiotic endoreplication as the mechanism of the production of non-reduced eggs in parthenogenetic females. The earlier contradicting reports can likely be attributed to the difficulty in identifying mispairing of chromosomes in pachytene, and the fact that in parthenogenetic reptiles relying on premeiotic endoreplication only a small subset of developing oocytes undergo genome doubling and overcome the pachytene checkpoint. This study highlights co-option of premeiotic endoreplication for escape from sexual reproduction in all independent hybrid origins of obligate parthenogenesis in vertebrates studied to date.

BACKGROUND:Crocodilians are one of the oldest extant vertebrate lineages, exhibiting a combination of evolutionary success and morphological resilience that has persisted throughout the history of life on Earth. This ability to endure over such a long geological time span is of great evolutionary importance. Here, we have utilized the combination of genomic and chromosomal data to identify and compare the full catalogs of satellite DNA families (satDNAs, i.e., the satellitomes) of 5 out of the 8 extant Alligatoridae species. As crocodilian genomes reveal ancestral patterns of evolution, by employing this multispecies data collection, we can investigate and assess how satDNA families evolve over time.RESULTS:Alligators and caimans displayed a small number of satDNA families, ranging from 3 to 13 satDNAs in A. sinensis and C. latirostris, respectively. Together with little variation both within and between species it highlighted long-term conservation of satDNA elements throughout evolution. Furthermore, we traced the origin of the ancestral forms of all satDNAs belonging to the common ancestor of Caimaninae and Alligatorinae. Fluorescence in situ experiments showed distinct hybridization patterns for identical orthologous satDNAs, indicating their dynamic genomic placement.CONCLUSIONS:Alligators and caimans possess one of the smallest satDNA libraries ever reported, comprising only four sets of satDNAs that are shared by all species. Besides, our findings indicated limited intraspecific variation in satellite DNA, suggesting that the majority of new satellite sequences likely evolved from pre-existing ones.

Using African annual killifishes of the genus Nothobranchius from temporary savannah pools with rapid karyotype and sex chromosome evolution, we analysed the chromosomal distribution of telomeric (TTAGGG)n repeat and Nfu-SatC satellite DNA (satDNA; isolated from Nothobranchius furzeri) in 15 species across the Nothobranchius killifish phylogeny, and with Fundulosoma thierryi as an out-group. Our fluorescence in situ hybridization experiments revealed that all analysed taxa share the presence of Nfu-SatC repeat but with diverse organization and distribution on chromosomes. Nfu-SatC landscape was similar in conspecific populations of Nothobranchius guentheri and Nothobranchius melanospilus but slightly-to-moderately differed between populations of Nothobranchius pienaari, and between closely related Nothobranchius kuhntae and Nothobranchius orthonotus. Inter-individual variability in Nfu-SatC patterns was found in N. orthonotus and Nothobranchius krysanovi. We revealed mostly no sex-linked patterns of studied repetitive DNA distribution. Only in Nothobranchius brieni, possessing multiple sex chromosomes, Nfu-SatC repeat occupied a substantial portion of the neo-Y chromosome, similarly as formerly found in the XY sex chromosome system of turquoise killifish N. furzeri and its sister species Nothobranchius kadleci-representatives not closely related to N. brieni. All studied species further shared patterns of expected telomeric repeats at the ends of all chromosomes and no additional interstitial telomeric sites. In summary, we revealed (i) the presence of conserved satDNA class in Nothobranchius clades (a rare pattern among ray-finned fishes); (ii) independent trajectories of Nothobranchius sex chromosome differentiation, with recurrent and convergent accumulation of Nfu-SatC on the Y chromosome in some species; and (iii) genus-wide shared tendency to loss of telomeric repeats during interchromosomal rearrangements. Collectively, our findings advance our understanding of genome structure, mechanisms of karyotype reshuffling, and sex chromosome differentiation in Nothobranchius killifishes from the genus-wide perspective.

Satellite DNA (satDNA) is a rapidly evolving class of tandem repeats, with some monomers being involved in centromere organization and function. To identify repeats associated with (peri)centromeric regions, we investigated satDNA across Southern and Coastal clades of African annual killifishes of the genus Nothobranchius. Molecular cytogenetic and bioinformatic analyses revealed that two previously identified satellites, designated here as NkadSat01-77 and NfurSat01-348, are associated with (peri)centromeres only in one lineage of the Southern clade. NfurSat01-348 was, however, additionally detected outside centromeres in three members of the Coastal clade. We also identified a novel satDNA, NrubSat01-48, associated with (peri)centromeres in N. foerschi, N. guentheri, and N. rubripinnis. Our findings revealed fast turnover of satDNA associated with (peri)centromeres and different trends in their evolution in two clades of the genus Nothobranchius.

Crocodilians have maintained very similar karyotype structures and diploid chromosome numbers for around 100 million years, with only minor variations in collinearity. Why this karyotype structure has largely stayed unaltered for so long is unclear. In this study, we analyzed the karyotypes of six species belonging to the genera Crocodylus and Osteolaemus (Crocodylidae, true crocodiles), among which the Congolian endemic O. osborni was included and investigated. We utilized various techniques (differential staining, fluorescence in situ hybridization with repetitive DNA and rDNA probes, whole chromosome painting, and comparative genomic hybridization) to better understand how crocodile chromosomes evolved. We studied representatives of three of the four main diploid chromosome numbers found in crocodiles (2 n = 30/32/38). Our data provided new information about the species studied, including the identification of four major chromosomal rearrangements that occurred during the karyotype diversification process in crocodiles. These changes led to the current diploid chromosome numbers of 2 n = 30 (fusion) and 2 n = 38 (fissions), derived from the ancestral state of 2 n = 32. The conserved cytogenetic tendency in crocodilians, where extant species keep near-ancestral state, contrasts with the more dynamic karyotype evolution seen in other major reptile groups.

ABSTRACT Meiosis is a conservative process in all sexual organisms which ensures fertility and is central for producing genetic diversity by recombination and random segregation of parental chromosomes. Yet unexplored mechanisms may disrupt it and cause ‘loss of sex’ followed by the emergence of clonal modes of reproduction. Interspecific hybridization is the primary trigger for this process, but mechanistic basis of the transition to asexuality remains still unknown for most vertebrate animals. To study these processes in water frogs, we performed reciprocal mating between two sexual species, Pelophylax ridibundus and P. lessonae , and produced vital F1 progeny ( P. esculentus ). The RepeatExplorer2 analysis of low-coverage genomic data of the two parental species identified the P. lessonae -specific minisatellite marker PlesSat01-48 (44 bp), which hybridized to (peri)centromeric regions of two chromosome pairs in P. lessonae – the acrocentric chromosome 8 and the chromosome 10 (a carrier of nucleolar organizer region; NOR). Chromosomal mapping combining the novel hybridization probe with the previously designed marker for P. ridibundus -specific centromeric satellite DNA showed that the P. esculentus progeny do not reproduce sexually. Instead, the F1 generation of P. esculentus instantly modified its gametogenesis and established asexual reproduction via hybridogenesis. Gametogenic modifications included premeiotic elimination of one of the parental genomes and clonal propagation of the remaining genome via endoreplication followed by standard meiotic division. The origin of DNA elimination and hybridogenesis in laboratory-produced hybrids supports a hypothesis that P. esculentus arises recurrently in nature whenever parental species come into reproductive contact. Based on the observed pattern of DNA elimination in the F1 progeny we discuss the origin and evolution of population systems in water frogs and the applicability of a newly designed chromosomal probe for other Pelophylax taxa.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (similar to 3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Karyotype divergence may strongly affect the degree of hybridization between species. Western Palearctic slow worms (Anguis) are legless lizards forming different types of secondary contact zones. To identify the level of chromosomal variation in slow worms, we examined karyotype in multiple populations of all species except one and Pseudopus apodus as an outgroup. We applied conventional and molecular cytogenetic methods and whole-chromosome painting using macrochromosome probes from Varanus komodoensis to interpret results within the evolutionary framework of the common clade Anguiformes. All Anguis species and P. apodus have conserved karyotype structures composed of 44 chromosomes. Despite the conserved chromosome morphology, the phylogenetically oldest Anguis cephallonica living in partial sympatry with Anguis graeca, and parapatric Anguis colchica vs. Anguis fragilis exhibit distinct patterns of constitutive heterochromatin distribution and telomeric repeat accumulation. In contrast, the sister species A. colchica and A. graeca living in allopatry display highly similar karyotype features. Our findings thus indicate karyotype stasis in Anguis and Pseudopus for > 20 Myr, with fixed species-specific differences present in sympatric and parapatric species. These differences in repetitive DNA patterns may play a role as intrinsic factors co-maintaining species divergence. They may also be used as cytotaxonomic markers to identify slow worm species in practice.

Satellite DNA (satDNA) is rapidly evolving class of tandem repeats with some motifs being involved in centromere organization and function. Rapid co-evolution of centromeric satDNA and associated proteins has been mostly attributed to the so-called centromere drive. To identify repeats associated with centromeric regions and test for the role of meiotic drive in their evolution, we investigated satDNA across Southern and Coastal clades of African annual killifishes of the genus Nothobranchius. C-banding showed expansion of (peri)centromeric heterochromatin regions in the Southern-clade killifishes. Molecular cytogenetic and bioinformatic analyses further revealed that two previously identified satellites, Nfu-SatA and Nfu-SatB, are associated with centromeres only in one lineage of the Southern clade. Nfu-SatB was, however, detected outside centromeres also in other members of the Coastal clade, which is consistent with the "library" hypothesis of satDNA evolution. We also identified a novel satDNA, Cl-36, associated with (peri)centromeres in N. foerschi, N. guentheri and N. rubripinnis from the Coastal clade. Our findings could be explained by centromere drive shaping karyotype change and centromeric repeat turnover in Nothobranchius species with possible reversal of spindle polarity within the Southern clade.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.