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.

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.

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.

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.

Repetitive DNA represents an important driver of sex chromosome differentiation. Yet, repetitive sequences tend to be misrepresented or overlooked in genomic studies. We analysed repetitive DNA landscape of sex chromosomes in several populations of a turquoise killifish Nothobranchius furzeri and its sister species N. kadleci (Teleostei: Nothobranchiidae), representatives of African annual killifishes with high rate of karyotype and sex chromosome evolution. We combined bioinformatic analyses of repeatome with molecular cytogenetic techniques such as comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, genes for ribosomal RNAs (rDNA) and bacterial artificial chromosomes (BACs) and immunostaining of SYCP3 and MLH1 proteins, which marked lateral elements of synaptonemal complexes and recombination sites, respectively. We revealed that N. furzeri and N. kadleci share the XY sex chromosome system, which is thus much older than previously assumed. Sex chromosomes are mostly heteromorphic as evidenced by distinct distribution of satellite DNAs and major rDNA. Yet, the heteromorphic XY sex chromosomes pair almost exclusively regularly in meiosis, which implies synaptic adjustment. Physical mapping of BACs identified inversions on Y chromosomes of the N. kadleci populations, akin to the pattern previously reported in N. furzeri. Yet, repetitive DNA landscape of X and Y sex chromosomes either diverged in parallel in populations of both species or it evolved in their common ancestor and thus predates the inversions. The observed differentiation via repeat repatterning thus cannot be explained by the classical sexually antagonistic model. Rather, we hypothesized that relaxed meiotic drive and recombination reduced by neutral processes could drive changes in repeatome and secondary inversions could be maintained by sexually antagonistic regulatory effects resulting from early evolution of dosage compensation. Author summary Early differentiation of sex chromosomes is not yet satisfactorily understood despite intensive research effort. Homomorphic sex chromosomes and their rapid turnover are common in teleost fishes, which makes them excellent models for studying evolution of nascent sex chromosomes. We investigated sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, particularly their repetitive landscape, which was misrepresented in previous genomic studies. Combination of cytogenetic and genomic approaches revealed that both species share heteromorphic XY sex chromosome system. The N. furzeri XY sex chromosomes thus evolved earlier than previously expected. In N. kadleci, Y-linked inversions analogous to those reported in N. furzeri were detected. Changes in repetitive DNA distribution on sex chromosomes are either convergent or occurred in a common ancestor of both species, prior to the inversion events. The observed sex chromosome differentiation on repetitive DNA level thus cannot be reconciled with the classical theoretical model of sex chromosome evolution driven by sexually antagonistic selection. We invoke alternative explanations such as relaxed meiotic drive and recombination reduced by neutral processes, and we hypothesize that secondary inversions could be maintained by early evolution of dosage compensation resulting in sexually antagonistic regulatory effects.

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species3,4 that have often been considered as selfish genetic elements that behave as genome parasites.5,6 The observation that in some species Bs can be either restricted or predominant in one sex7-14 raised the interesting hypothesis that Bs could play a role in sex determination.15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.