Sex chromosome turnover and biodiversity in fishes.

The impact of sex chromosomes and their turnover in speciation remains a subject of ongoing debate in the field of evolutionary biology. Fishes are the largest group of vertebrates, and they exhibit unparalleled sexual plasticity, as well as diverse sex-determining (SD) genes, sex chromosomes, and sex determination mechanisms. This diversity is hypothesized to be associated with the frequent turnover of sex chromosomes in fishes. Although it is evident that amh and amhr2 are repeatedly and independently recruited as SD genes, their relationship with the rapid turnover of sex chromosomes and the biodiversity of fishes remains unknown. We summarize the canonical models of sex chromosome turnover and highlight the vital roles of gene mutation and hybridization with empirical evidence. We revisit Haldane's rule and the large X-effect and propose the hypothesis that sex chromosomes accelerate speciation by multiplying genotypes via hybridization. By integrating recent findings on the turnover of SD genes, sex chromosomes, and sex determination systems in fish species, this review provides insights into the relationship between sex chromosome evolution and biodiversity in fishes.

Genome assemblies for Chromidotilapia guntheri (Teleostei: Cichlidae) identify a novel candidate gene for vertebrate sex determination, RIN3.

Advances in genome sequencing have greatly accelerated the identification of sex chromosomes in a variety of species. Many of these species have experienced structural rearrangements that reduce recombination between the sex chromosomes, allowing the accumulation of sequence differences over many megabases. Identification of the genes that are responsible for sex determination within these sometimes large regions has proved difficult. Here, we identify an XY sex chromosome system on LG19 in the West African cichlid fish Chromidotilapia guntheri in which the region of differentiation extends over less than 400 kb. We develop high-quality male and female genome assemblies for this species, which confirm the absence of structural variants, and which facilitate the annotation of genes in the region. The peak of differentiation lies within rin3, which has experienced several debilitating mutations on the Y chromosome. We suggest two hypotheses about how these mutations might disrupt endocytosis, leading to Mendelian effects on sexual development.

Diversity of Sex Chromosomes in Vertebrates: Six Novel Sex Chromosomes in Basal Haplochromines (Teleostei: Cichlidae).

African cichlid fishes are known for their high rates of phenotypic evolution. A rapid rate of diversification is apparent also in the diversity of their sex chromosomes. To date, sex determiners have been identified on 18 of 22 chromosomes in the standard karyotype. Here, we use whole-genome sequencing to characterize the sex chromosomes of seven populations of basal haplochromines, focusing on the genus Pseudocrenilabrus. We identify six new sex chromosome systems, including the first report of a cichlid sex-determining system on linkage group 12. We then quantify the rates and patterns of sex chromosome turnover in this clade. Finally, we test whether some autosomes become sex chromosomes in East African cichlids more often than expected by chance.

Mutation of mpv17 results in loss of iridophores due to mitochondrial dysfunction in tilapia.

Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Turnover of sex chromosomes in the Lake Tanganyika cichlid tribe Tropheini (Teleostei: Cichlidae).

Sex chromosome replacement is frequent in many vertebrate clades, including fish, frogs, and lizards. In order to understand the mechanisms responsible for sex chromosome turnover and the early stages of sex chromosome divergence, it is necessary to study lineages with recently evolved sex chromosomes. Here we examine sex chromosome evolution in a group of African cichlid fishes (tribe Tropheini) which began to diverge from one another less than 4 MYA. We have evidence for a previously unknown sex chromosome system, and preliminary indications of several additional systems not previously reported in this group. We find a high frequency of sex chromosome turnover and estimate a minimum of 14 turnovers in this tribe. We date the origin of the most common sex determining system in this tribe (XY-LG5/19) near the base of one of two major sub-clades of this tribe, about 3.4 MY ago. Finally, we observe variation in the size of one sex-determining region that suggests independent evolution of evolutionary strata in species with a shared sex-determination system. Our results illuminate the rapid rate of sex chromosome turnover in the tribe Tropheini and set the stage for further studies of the dynamics of sex chromosome evolution in this group.

Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

Sex chromosomes in the tribe Cyprichromini (Teleostei: Cichlidae) of Lake Tanganyika.

Sex determining loci have been described on at least 12 of 22 chromosomes in East African cichlid fishes, indicating a high rate of sex chromosome turnover. To better understand the rates and patterns of sex chromosome replacement, we used new methods to characterize the sex chromosomes of the cichlid tribe Cyprichromini from Lake Tanganyika. Our k-mer based methods successfully identified sex-linked polymorphisms without the need for a reference genome. We confirm the three previously reported sex chromosomes in this group. We determined the polarity of the sex chromosome turnover on LG05 in Cyprichromis as ZW to XY. We identified a new ZW locus on LG04 in Paracyprichromis brieni. The LG15 XY locus in Paracyprichromis nigripinnis was not found in other Paracyprichromis species, and the sample of Paracyprichromis sp. "tembwe" is likely to be of hybrid origin. Although highly divergent sex chromosomes are thought to develop in a stepwise manner, we show two cases (LG05-ZW and LG05-XY) in which the region of differentiation encompasses most of the chromosome, but appears to have arisen in a single step. This study expands our understanding of sex chromosome evolution in the Cyprichromini, and indicates an even higher level of sex chromosome turnover than previously thought.

Polymorphism of Sex Determination Amongst Wild Populations Suggests its Rapid Turnover Within the Nile Tilapia Species.

Sex-determining regions have been identified in the Nile tilapia on linkage groups (LG) 1, 20 and 23, depending on the domesticated strains used. Sex determining studies on wild populations of this species are scarce. Previous work on two wild populations, from Lake Volta (Ghana) and from Lake Koka (Ethiopia), found the sex-determining region on LG23. These populations have a Y-specific tandem duplication containing two copies of the Anti-Müllerian Hormone amh gene (named amhY and amhΔY). Here, we performed a whole-genome short-reads analysis using male and female pools on a third wild population from Lake Hora (Ethiopia). We found no association of sex with LG23, and no duplication of the amh gene. Furthermore, we found no evidence of sex linkage on LG1 or on any other LGs. Long read whole genome sequencing of a male from each population confirmed the absence of a duplicated region on LG23 in the Lake Hora male. In contrast, long reads established the structure of the Y haplotype in Koka and Kpandu males and the order of the genes in the duplicated region. Phylogenies constructed on the nuclear and mitochondrial genomes, showed a closer relationship between the two Ethiopian populations compared to the Ghanaian population, implying an absence of the LG23Y sex-determination region in Lake Hora males. Our study supports the hypothesis that the amh region is not the sex-determining region in Hora males. The absence of the Y amh duplication in the Lake Hora population reflects a rapid change in sex determination within Nile tilapia populations. The genetic basis of sex determination in the Lake Hora population remains unknown.

New Sex Chromosomes in Lake Victoria Cichlid Fishes (Cichlidae: Haplochromini).

African cichlid fishes harbor an extraordinary diversity of sex-chromosome systems. Within just one lineage, the tribe Haplochromini, at least 6 unique sex-chromosome systems have been identified. Here we focus on characterizing sex chromosomes in cichlids from the Lake Victoria basin. In Haplochromis chilotes, we identified a new ZW system associated with the white blotch color pattern, which shows substantial sequence differentiation over most of LG16, and is likely to be present in related species. In Haplochromis sauvagei, we found a coding polymorphism in amh that may be responsible for an XY system on LG23. In Pundamilia nyererei, we identified a feminizing effect of B chromosomes together with XY- and ZW-patterned differentiation on LG23. In Haplochromis latifasciatus, we identified a duplication of amh that may be present in other species of the Lake Victoria superflock. We further characterized the LG5-14 XY system in Astatotilapia burtoni and identified the oldest stratum on LG14. This species also showed ZW differentiation on LG2. Finally, we characterized an XY system on LG7 in Astatoreochromis alluaudi. This report brings the number of distinct sex-chromosome systems in haplochromine cichlids to at least 13, and highlights the dynamic evolution of sex determination and sex chromosomes in this young lineage.

CRISPR Knockouts of pmela and pmelb Engineered a Golden Tilapia by Regulating Relative Pigment Cell Abundance.

Premelanosome protein (pmel) is a key gene for melanogenesis. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study, we found that most fishes have 2 pmel genes arising from the teleost-specific whole-genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypopigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a golden body color. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, although both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.

Identification of sex chromosome and sex-determining gene of southern catfish (Silurus meridionalis) based on XX, XY and YY genome sequencing.

Teleosts are important models to study sex chromosomes and sex-determining (SD) genes because they present a variety of sex determination systems. Here, we used Nanopore and Hi-C technologies to generate a high-contiguity chromosome-level genome assembly of a YY southern catfish (Silurus meridionalis). The assembly is 750.0 Mb long, with contig N50 of 15.96 Mb and scaffold N50 of 27.22 Mb. We also sequenced and assembled an XY male genome with a size of 727.2 Mb and contig N50 of 13.69 Mb. We identified a candidate SD gene through comparisons to our previous assembly of an XX individual. By resequencing male and female pools, we characterized a 2.38 Mb sex-determining region (SDR) on Chr24. Analysis of read coverage and comparison of the X and Y chromosome sequences showed a Y specific insertion (approx. 500 kb) in the SDR which contained a male-specific duplicate of amhr2 (named amhr2y). amhr2y and amhr2 shared high-nucleotide identity (81.0%) in the coding region but extremely low identity in the promotor and intron regions. The exclusive expression in the male gonadal primordium and loss-of-function inducing male to female sex reversal confirmed the role of amhr2y in male sex determination. Our study provides a new example of amhr2 as the SD gene in fish and sheds light on the convergent evolution of the duplication of AMH/AMHR2 pathway members underlying the evolution of sex determination in different fish lineages.

A Chromosome-Level Genome Assembly of Mozambique Tilapia (Oreochromis mossambicus) Reveals the Structure of Sex Determining Regions.

The Mozambique tilapia (Oreochromis mossambicus) is a fascinating taxon for evolutionary and ecological research. It is an important food fish and one of the most widely distributed tilapias. Because males grow faster than females, genetically male tilapia are preferred in aquaculture. However, studies of sex determination and sex control in O. mossambicus have been hindered by the limited characterization of the genome. To address this gap, we assembled a high-quality genome of O. mossambicus, using a combination of high coverage of Illumina and Nanopore reads, coupled with Hi-C and RNA-Seq data. Our genome assembly spans 1,007 Mb with a scaffold N50 of 11.38 Mb. We successfully anchored and oriented 98.6% of the genome on 22 linkage groups (LGs). Based on re-sequencing data for male and female fishes from three families, O. mossambicus segregates both an XY system on LG14 and a ZW system on LG3. The sex-patterned SNPs shared by two XY families narrowed the sex determining regions to ∼3 Mb on LG14. The shared sex-patterned SNPs included two deleterious missense mutations in ahnak and rhbdd1, indicating the possible roles of these two genes in sex determination. This annotated chromosome-level genome assembly and identification of sex determining regions represents a valuable resource to help understand the evolution of genetic sex determination in tilapias.

Nile Tilapia: A Model for Studying Teleost Color Patterns.

The diverse color patterns of cichlid fishes play an important role in mate choice and speciation. Here we develop the Nile tilapia (Oreochromis niloticus) as a model system for studying the developmental genetics of cichlid color patterns. We identified 4 types of pigment cells: melanophores, xanthophores, iridophores and erythrophores, and characterized their first appearance in wild-type fish. We mutated 25 genes involved in melanogenesis, pteridine metabolism, and the carotenoid absorption and cleavage pathways. Among the 25 mutated genes, 13 genes had a phenotype in both the F0 and F2 generations. None of F1 heterozygotes had phenotype. By comparing the color pattern of our mutants with that of red tilapia (Oreochromis spp), a natural mutant produced during hybridization of tilapia species, we found that the pigmentation of the body and eye is controlled by different genes. Previously studied genes like mitf, kita/kitlga, pmel, tyrb, hps4, gch2, csf1ra, pax7b, and bco2b were proved to be of great significance for color patterning in tilapia. Our results suggested that tilapia, a fish with 4 types of pigment cells and a vertically barred wild-type color pattern, together with various natural and artificially induced color gene mutants, can serve as an excellent model system for study color patterning in vertebrates.

Identification, Expression and Evolution of Short-Chain Dehydrogenases/Reductases in Nile Tilapia (Oreochromis niloticus).

The short-chain dehydrogenases/reductases (SDR) superfamily is involved in multiple physiological processes. In this study, genome-wide identification and comprehensive analysis of SDR superfamily were carried out in 29 animal species based on the latest genome databases. Overall, the number of SDR genes in animals increased with whole genome duplication (WGD), suggesting the expansion of SDRs during evolution, especially in 3R-WGD and polyploidization of teleosts. Phylogenetic analysis indicated that vertebrates SDRs were clustered into five categories: classical, extended, undefined, atypical, and complex. Moreover, tandem duplication of hpgd-a, rdh8b and dhrs13 was observed in teleosts analyzed. Additionally, tandem duplications of dhrs11-a, dhrs7a, hsd11b1b, and cbr1-a were observed in all cichlids analyzed, and tandem duplication of rdh10-b was observed in tilapiines. Transcriptome analysis of adult fish revealed that 93 SDRs were expressed in more than one tissue and 5 in one tissue only. Transcriptome analysis of gonads from different developmental stages showed that expression of 17 SDRs were sexually dimorphic with 11 higher in ovary and 6 higher in testis. The sexually dimorphic expressions of these SDRs were confirmed by in situ hybridization (ISH) and qPCR, indicating their possible roles in steroidogenesis and gonadal differentiation. Taken together, the identification and the expression data obtained in this study contribute to a better understanding of SDR superfamily evolution and functions in teleosts.

Chromosome-level assembly of southern catfish (silurus meridionalis) provides insights into visual adaptation to nocturnal and benthic lifestyles.

The Southern catfish (Silurus meridionalis) is a nocturnal and benthic freshwater fish endemic to the Yangtze River and its tributaries. In this study, we constructed a chromosome-level draft genome of S. meridionalis using 69.7-Gb Nanopore long reads and 49.5-Gb Illumina short reads. The genome assembly was 741.2 Mb in size with a contig N50 of 13.19 Mb. An additional 116.4 Gb of Bionano and 77.4 Gb of Hi-C data were applied to assemble contigs into scaffolds and further into 29 chromosomes, resulting in a 738.9-Mb genome with a scaffold N50 of 28.04 Mb. A total of 22,965 protein-coding genes were predicted from the genome with 22,519 (98.06%) genes functionally annotated. Comparative genomic and transcriptomic analyses revealed a rod-dominated visual system which was responsible for scotopic vision. The absence of cone opsins SWS1 and SWS2 resulted in the lack of ultraviolet and blue violet sensitivity. Mutations at key amino acid sites of RH1.1, RH1.2 and RH2 resulted in spectral tuning good for dim light vision and narrow colour vision. A higher expression level of rod phototransduction genes than that of cone genes and higher rod-to-cone ratio led to higher optical sensitivity under dim light conditions. In addition, analysis of the genes involved in eye morphogenesis and development revealed the loss of some conserved noncoding elements, which might be associated with the small eyes in catfish. Together, our study provides important clues for the adaptation of the catfish visual system to the nocturnal and benthic lifestyles. The draft genome of S. meridionalis represents a valuable resource for studies of the molecular mechanisms of ecological adaptation.

Origin of a Giant Sex Chromosome.

Chromosome size and morphology vary within and among species, but little is known about the proximate or ultimate causes of these differences. Cichlid fish species in the tribe Oreochromini share an unusual giant chromosome that is ∼3 times longer than the other chromosomes. This giant chromosome functions as a sex chromosome in some of these species. We test two hypotheses of how this giant sex chromosome may have evolved. The first hypothesis proposes that it evolved by accumulating repetitive elements as recombination was reduced around a dominant sex determination locus, as suggested by canonical models of sex chromosome evolution. An alternative hypothesis is that the giant sex chromosome originated via the fusion of an autosome with a highly repetitive B chromosome, one of which carried a sex determination locus. We test these hypotheses using comparative analysis of chromosome-scale cichlid and teleost genomes. We find that the giant sex chromosome consists of three distinct regions based on patterns of recombination, gene and transposable element content, and synteny to the ancestral autosome. The WZ sex determination locus encompasses the last ∼105 Mb of the 134-Mb giant chromosome. The last 47 Mb of the giant chromosome shares no obvious homology to any ancestral chromosome. Comparisons across 69 teleost genomes reveal that the giant sex chromosome contains unparalleled amounts of endogenous retroviral elements, immunoglobulin genes, and long noncoding RNAs. The results favor the B chromosome fusion hypothesis for the origin of the giant chromosome.

Network architecture and sex chromosome turnovers: Do epistatic interactions shape patterns of sex chromosome replacement?

Recent studies have revealed an astonishing diversity of sex chromosomes in many vertebrate lineages, prompting questions about the mechanisms of sex chromosome turnover. While there is considerable population genetic theory about the evolutionary forces promoting sex chromosome replacement, this theory has not yet been integrated with our understanding of the molecular and developmental genetics of sex determination. Here, we review recent data to examine four questions about how the structure of gene networks influences the evolution of sex determination. We argue that patterns of epistasis, arising from the structure of genetic networks, may play an important role in regulating the rates and patterns of sex chromosome replacement.

Movement of transposable elements contributes to cichlid diversity.

African cichlid fishes are a prime model for studying speciation mechanisms. Despite the development of extensive genomic resources, it has been difficult to determine which sources of genetic variation are responsible for cichlid phenotypic variation. One of their most variable phenotypes is visual sensitivity, with some of the largest spectral shifts among vertebrates. These shifts arise primarily from differential expression of seven cone opsin genes. By mapping expression quantitative trait loci (eQTL) in intergeneric crosses of Lake Malawi cichlids, we previously identified four causative genetic variants that correspond to indels in the promoters of either key transcription factors or an opsin gene. In this comprehensive study, we show that these indels are the result of the movement of transposable elements (TEs) that correlate with opsin expression variation across the Malawi flock. In tracking the evolutionary history of these particular indels, we found they are endemic to Lake Malawi, suggesting that these TEs are recently active and are segregating within the Malawi cichlid lineage. However, an independent indel has arisen at a similar genomic location in one locus outside of the Malawi flock. The convergence in TE movement suggests these loci are primed for TE insertion and subsequent deletions. Increased TE mobility may be associated with interspecific hybridization, which disrupts mechanisms of TE suppression. This might provide a link between cichlid hybridization and accelerated regulatory variation. Overall, our study suggests that TEs may be an important driver of key regulatory changes, facilitating rapid phenotypic change and possibly speciation in African cichlids.