Since the discovery of Sry in mammals [1, 2], few other master sex-determining genes have been identified in vertebrates [3-7]. To date, all of these genes have been characterized as well-known factors in the sex differentiation pathway, suggesting that the same subset of genes have been repeatedly and independently selected throughout evolution as master sex determinants [8, 9]. Here, we characterized in rainbow trout an unknown gene expressed only in the testis, with a predominant expression during testicular differentiation. This gene is a male-specific genomic sequence that is colocalized along with the sex-determining locus. This gene, named sdY for sexually dimorphic on the Y chromosome, encodes a protein that displays similarity to the C-terminal domain of interferon regulatory factor 9. The targeted inactivation of sdY in males using zinc-finger nuclease induces ovarian differentiation, and the overexpression of sdY in females using additive transgenesis induces testicular differentiation. Together, these results demonstrate that sdY is a novel vertebrate master sex-determining gene not related to any known sex-differentiating gene. These findings highlight an unexpected evolutionary plasticity in vertebrate sex determination through the demonstration that master sex determinants can arise from the de novo evolution of genes that have not been previously implicated in sex differentiation.

All salmonid species investigated to date have been characterized with a male heterogametic sex-determination system. However, as these species do not share any Y-chromosome conserved synteny, there remains a debate on whether they share a common master sex-determining gene. In this study, we investigated the extent of conservation and evolution of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene, sdY (sexually dimorphic on the Y-chromosome), in 15 different species of salmonids. We found that the sdY sequence is highly conserved in all salmonids and that sdY is a male-specific Y-chromosome gene in the majority of these species. These findings demonstrate that most salmonids share a conserved sex-determining locus and also strongly suggest that sdY may be this conserved master sex-determining gene. However, in two whitefish species (subfamily Coregoninae), sdY was found both in males and females, suggesting that alternative sex-determination systems may have also evolved in this family. Based on the wide conservation of sdY as a male-specific Y-chromosome gene, efficient and easy molecular sexing techniques can now be developed that will be of great interest for studying these economically and environmentally important species.

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Teleost fishes, thanks to their rapid evolution of sex determination mechanisms, provide remarkable opportunities to study the formation of sex chromosomes and the mechanisms driving the birth of new master sex determining (MSD) genes. However, the evolutionary interplay between the sex chromosomes and the MSD genes they harbor is rather unexplored. We characterized a male-specific duplicate of the anti-Müllerian hormone ( amh) as the MSD gene in Northern Pike ( Esox lucius ), using genomic and expression evidence as well as by loss-of-function and gain-of-function experiments. Using RAD-Sequencing from a family panel, we identified Linkage Group (LG) 24 as the sex chromosome and positioned the sex locus in its sub-telomeric region. Furthermore, we demonstrated that this MSD originated from an ancient duplication of the autosomal amh gene, which was subsequently translocated to LG24. Using sex-specific pooled genome sequencing and a new male genome sequence assembled using Nanopore long reads, we also characterized the differentiation of the X and Y chromosomes, revealing a small male-specific insertion containing the MSD gene and a limited region with reduced recombination. Our study reveals an unexpectedly low level of differentiation between a pair of sex chromosomes harboring an old MSD gene in a wild teleost fish population, and highlights both the pivotal role of genes from the amh pathway in sex determination, as well as the importance of gene duplication as a mechanism driving the turnover of sex chromosomes in this clade.

Evolutionary novelties require rewiring of transcriptional networks and/or the evolution of new gene functions. Sex determination (SD), one of the most plastic evolutionary processes, requires such novelties. Studies on the evolution of vertebrate SD revealed that new master SD genes are generally recruited from genes involved in the downstream SD regulatory genetic network. Only a single exception to this rule is currently known in vertebrates: the intriguing case of the salmonid master SD gene (sdY), which arose from duplication of an immune-related gene. This exception immediately posed the question of how a gene outside from the classical sex differentiation cascade could acquire its function as a male SD gene. Here we show that SdY became integrated in the classical vertebrate sex differentiation cascade by interacting with the Forkhead box domain of the female-determining transcription factor, Foxl2. In the presence of Foxl2, SdY is translocated to the nucleus where the SdY:Foxl2 complex prevents activation of the aromatase (cyp19a1a) promoter in cooperation with Nr5a1 (Sf1). Hence, by blocking a positive loop of regulation needed for the synthesis of estrogens in the early differentiating gonad, SdY disrupts a preset female differentiation pathway, consequently allowing testicular differentiation to proceed. These results also suggest that the evolution of unusual vertebrate master sex determination genes recruited from outside the classical pathway like sdY is strongly constrained by their ability to interact with the canonical gonadal differentiation pathway.