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.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin’s importance for illuminating vertebrate biology and diversity in the genomic era.

Foxl2 is a member of the large family of Forkhead Box (Fox) domain transcription factors. It emerged during the last 15 years as a key player in ovarian differentiation and oogenesis in vertebrates and especially mammals. This review focuses on Foxl2 genes in light of recent findings on their evolution, expression, and implication in sex differentiation in animals in general. Homologs of Foxl2 and its paralog Foxl3 are found in all metazoans, but their gene evolution is complex, with multiple gains and losses following successive whole genome duplication events in vertebrates. This review aims to decipher the evolutionary forces that drove Foxl2/3 gene specialization through sub- and neo-functionalization during evolution. Expression data in metazoans suggests that Foxl2/3 progressively acquired a role in both somatic and germ cell gonad differentiation and that a certain degree of sub-functionalization occurred after its duplication in vertebrates. This generated a scenario where Foxl2 is predominantly expressed in ovarian somatic cells and Foxl3 in male germ cells. To support this hypothesis, we provide original results showing that in the pea aphid (insects) foxl2/3 is predominantly expressed in sexual females and showing that in bovine ovaries FOXL2 is specifically expressed in granulosa cells. Overall, current results suggest that Foxl2 and Foxl3 are evolutionarily conserved players involved in somatic and germinal differentiation of gonadal sex.

To date, more than 20 different vertebrate master sex-determining genes have been identified on different sex chromosomes of mammals, birds, frogs and fish. Interestingly, six of these genes are transcription factors ( Dmrt1 - or Sox3 - related) and 13 others belong to the TGF-β signalling pathway ( Amh , Amhr2 , Bmpr1b , Gsdf and Gdf6 ). This pattern suggests that only a limited group of factors/signalling pathways are prone to become top regulators again and again. Although being clearly a subordinate member of the sex-regulatory network in mammals, the TGF-β signalling pathway made it to the top recurrently and independently. Facing this rolling wave of TGF-β signalling pathways, this review will decipher how the TGF-β signalling pathways cope with the canonical sex gene regulatory network and challenge the current evolutionary concepts accounting for the diversity of sex-determining mechanisms. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

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 evidences 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 depicts an unexpected level of limited differentiation within a pair of sex chromosomes harboring an old MSD gene in a wild population of teleost fish, highlights 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.Author SummaryIn stark contrast to mammals and birds, teleosts have predominantly homomorphic sex chromosomes and display a high diversity of sex determining genes. Yet, population level knowledge of both the sex chromosome and the master sex determining gene is only available for the Japanese medaka, a model species. Here we identified and provided functional proofs of an old duplicate of anti-Müllerian hormone (Amh), a member of the Tgf-β family, as the male master sex determining gene in the Northern pike, Esox lucius. We found that this duplicate, named amhby (Y-chromosome-specific anti-Müllerian hormone paralog b), was translocated to the sub-telomeric region of the new sex chromosome, and now amhby shows strong sequence divergence as well as substantial expression pattern differences from its autosomal paralog, amha. We assembled a male genome sequence using Nanopore long reads and identified a restricted region of differentiation within the sex chromosome pair in a wild population. Our results provide insight on the conserved players in sex determination pathways, the mechanisms of sex chromosome turnover, and the diversity of levels of differentiation between homomorphic sex chromosomes in teleosts.

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex-linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y-chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

The study of sex determination and sex chromosome organisation in non-model species has long been technically challenging, but new sequencing methodologies are now enabling precise and high-throughput identification of sex-specific genomic sequences. In particular, Restriction Site-Associated DNA Sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software designed to specifically search for sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analyzed a published dataset of Japanese medaka, Oryzias latipes , where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyze new RAD-Seq datasets from 15 fish species spanning multiple systematic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in non-model species and outperforms the commonly used RAD-Seq analysis software STACKS.RADSex in speed, resource usage, ease of application, and visualization options. Furthermore, our analysis of new datasets from 15 species provides new insights on sex determination in fish.