Heterogametic Research Articles

Haldane’s duel: intragenomic conflict, selfish Y chromosomes and speciation

Haldane's rule, which states that the heterogametic sex (XY or ZW females) fares more poorly in interspecific hybrids, is generally attributed to absence of one of the two species' X/Z chromosomes. However, Haldane's rule is also observed in mouse placentas despite paternal X silencing. This pattern could reflect Y chromosomes having evolved to promote growth due to maternal-paternal conflict. If so, balanced sex investment arises from a complex intra- and intergenomic duel.

Asymmetric Phenotypes of Sterile Hybrid Males From Reciprocal Crosses Between Species of the Anopheles gambiae Complex

Haldane’s rule of speciation states that sterility or inviability affects the heterogametic sex of inter-species hybrids. Darwin’s corollary to Haldane’s rule implies that there are asymmetric phenotypes in inter-species hybrids from reciprocal crosses. Studying the phenotypes of F1 hybrids among closely related species of malaria mosquitoes can assist researchers in identifying the genetic factors and molecular mechanisms of speciation. To characterize phenotypes of sterile hybrid males in the Anopheles gambiae complex, we performed crosses between laboratory strains of An. merus and either An. gambiae or An. coluzzii. The reproductive tracts had normal external morphology in hybrid males from crosses between female An. merus and male An. gambiae or An. coluzzii. Despite being sterile, these males could copulate with females for a normal period of time and could transfer a mating plug to induce female oviposition and monogamy. In contrast, the entire reproductive tracts in hybrid males from crosses between female An. gambiae or An. coluzzii and male An. merus were severely underdeveloped. These males had atrophic testes and reduced somatic organs of the reproductive system including male accessary glands and ejaculatory duct. In addition, hybrid males with underdeveloped reproductive tracts displayed a shorter copulation time with females and failed to induce female oviposition and monogamy due to their inability to form and transfer a plug to females during mating. The asymmetry of the phenotypes associated with hybrid male sterility suggests that different genetic factors and molecular mechanisms are responsible for reproductive isolation in reciprocal crosses among species of the An. gambiae complex.

Toxic Y chromosome: Increased repeat expression and age-associated heterochromatin loss in male Drosophila with a young Y chromosome.

Sex-specific differences in lifespan are prevalent across the tree of life and influenced by heteromorphic sex chromosomes. In species with XY sex chromosomes, females often outlive males. Males and females can differ in their overall repeat content due to the repetitive Y chromosome, and repeats on the Y might lower survival of the heterogametic sex (toxic Y effect). Here, we take advantage of the well-assembled young Y chromosome of Drosophila miranda to study the sex-specific dynamics of chromatin structure and repeat expression during aging in male and female flies. Male D. miranda have about twice as much repetitive DNA compared to females, and live shorter than females. Heterochromatin is crucial for silencing of repetitive elements, yet old D. miranda flies lose H3K9me3 modifications in their pericentromere, with heterochromatin loss being more severe during aging in males than females. Satellite DNA becomes de-repressed more rapidly in old vs. young male flies relative to females. In contrast to what is observed in D. melanogaster, we find that transposable elements (TEs) are expressed at higher levels in male D. miranda throughout their life. We show that epigenetic silencing via heterochromatin formation is ineffective on the TE-rich neo-Y chromosome, presumably due to active transcription of a large number of neo-Y linked genes, resulting in up-regulation of Y-linked TEs already in young males. This is consistent with an interaction between the evolutionary age of the Y chromosome and the genomic effects of aging. Our data support growing evidence that "toxic Y chromosomes" can diminish male fitness and a reduction in heterochromatin can contribute to sex-specific aging.

The Diversity and Evolution of Sex Chromosomes in Frogs.

Frogs are ideal organisms for studying sex chromosome evolution because of their diversity in sex chromosome differentiation and sex-determination systems. We review 222 anuran frogs, spanning ~220 Myr of divergence, with characterized sex chromosomes, and discuss their evolution, phylogenetic distribution and transitions between homomorphic and heteromorphic states, as well as between sex-determination systems. Most (~75%) anurans have homomorphic sex chromosomes, with XY systems being three times more common than ZW systems. Most remaining anurans (~25%) have heteromorphic sex chromosomes, with XY and ZW systems almost equally represented. There are Y-autosome fusions in 11 species, and no W-/Z-/X-autosome fusions are known. The phylogeny represents at least 19 transitions between sex-determination systems and at least 16 cases of independent evolution of heteromorphic sex chromosomes from homomorphy, the likely ancestral state. Five lineages mostly have heteromorphic sex chromosomes, which might have evolved due to demographic and sexual selection attributes of those lineages. Males do not recombine over most of their genome, regardless of which is the heterogametic sex. Nevertheless, telomere-restricted recombination between ZW chromosomes has evolved at least once. More comparative genomic studies are needed to understand the evolutionary trajectories of sex chromosomes among frog lineages, especially in the ZW systems.

Identification of a novel sex determining chromosome in cichlid fishes that acts as XY or ZW in different lineages

Sex determination systems are highly conserved among most vertebrates with genetic sex determination, but can be variable and evolve rapidly in some. Here, we study sex determination in a clade with exceptionally high sex chromosome turnover rates. We identify the sex determining chromosomes in three interspecific crosses of haplochromine cichlid fishes from Lakes Victoria and Malawi. We find evidence for different sex determiners in each cross. In the Malawi cross and one Victoria cross the same chromosome is sex-linked but while females are the heterogametic sex in the Malawi species, males are the heterogametic sex in the Victoria species. This chromosome has not previously been reported to be sex determining in cichlids, increasing the number of different chromosomes shown to be sex determining in cichlids to 12. All Lake Victoria species of our crosses are less than 15,000 years divergent, and we identified different sex determiners among them. Our study provides further evidence for the diversity and evolutionary flexibility of sex determination in cichlids, factors which might contribute to their rapid adaptive radiations.

Transcriptional regulation of dosage compensation in Carica papaya

Sex chromosome evolution results in the disparity in gene content between heterogametic sex chromosomes and creates the need for dosage compensation to counteract the effects of gene dose imbalance of sex chromosomes in males and females. It is not known at which stage of sex chromosome evolution dosage compensation would evolve. We used global gene expression profiling in male and female papayas to assess gene expression patterns of sex-linked genes on the papaya sex chromosomes. By analyzing expression ratios of sex-linked genes to autosomal genes and sex-linked genes in males relative to females, our results showed that dosage compensation was regulated on a gene-by-gene level rather than whole sex-linked region in papaya. Seven genes on the papaya X chromosome exhibited dosage compensation. We further compared gene expression ratios in the two evolutionary strata. Y alleles in the older evolutionary stratum showed reduced expression compared to X alleles, while Y alleles in the younger evolutionary stratum showed elevated expression compared to X alleles. Reduced expression of Y alleles in the older evolutionary stratum might be caused by accumulation of deleterious mutations in regulatory regions or transposable element-mediated methylation spreading. Most X-hemizygous genes exhibited either no or very low expression, suggesting that gene silencing might play a role in maintaining transcriptional balance between females and males.

Sex assignment in a non-model organism in the absence of field records using Diversity Arrays Technology (DArT) data

Conservation genomics research often relies on accurate sex information to make inferences about species demography, dispersal, and population structure. However, field determined sex data are not always available and can be subject to human error, while laboratory sex assignment methods such as PCR assays can often be costly and challenging for non-model species. Conservation genomics programs increasingly use reduced-representation genome sequencing to assess neutral and functional genetic diversity, population structure, gene flow and pedigrees in threatened species. Here we demonstrate that sex can be determined from reduced-representation sequencing data produced by the increasingly popular Diversity Arrays Technology sequencing workflow (DArT-seq) using a program originally designed for application to shotgun data. This program—sexassign—compares the “dosage” of sequencing reads mapping to autosomes versus the X chromosome. In the present study, sexassign was used to identify the sex of 60 field-collected Greater Stick-Nest Rat (Leporillus conditor) samples, despite the absence of an annotated reference genome for the species. This “read-dosage” approach is not only more accurate and affordable than traditional sex assignment methods, but can be applied to any diploid organism with a heterogametic sex determination system—including non-model and understudied species of conservation importance—by using FASTQs generated by DArT.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Gonadal histology and concentration of 11-ketotesterone of meiotic gynogens confirm female heterogametic sex determination in sterlet (Acipenser ruthenus)

Gynogenetic progeny of sterlet Acipenser ruthenus were obtained by inseminating eggs with UV-inactivated sperm followed by meiotic restoration of ploidy by heat shock. Gynogenetic origin of progeny was assessed by parentage assignment using 15 microsatellite markers. Sex ratio was investigated by gonad histology in 15 3-year-old fish, eight of which were confirmed gynogenotes, with five of those containing primary oocytes (62.5%). Blood plasma concentration of 11-ketotestosterone (KT-11) was analyzed in 63 4-year-old juveniles, 46 of which were genetically confirmed gynogenotes. Measurement of KT-11 showed the proportion of female sterlet to be 65.2%. This sex ratio conformed to the current hypothesis of female heterogametic sex determination but was at the low end of the reported female ratio in sturgeon gynogenetic progeny.

Chromosomal evolution in Raphicerus antelope suggests divergent X chromosomes may drive speciation through females, rather than males, contrary to Haldane's rule

Chromosome structural change has long been considered important in the evolution of post-zygotic reproductive isolation. The premise that karyotypic variation can serve as a possible barrier to gene flow is founded on the expectation that heterozygotes for structurally distinct chromosomal forms would be partially sterile (negatively heterotic) or show reduced recombination. We report the outcome of a detailed comparative molecular cytogenetic study of three antelope species, genus Raphicerus, that have undergone a rapid radiation. The species are largely conserved with respect to their euchromatic regions but the X chromosomes, in marked contrast, show distinct patterns of heterochromatic amplification and localization of repeats that have occurred independently in each lineage. We argue a novel hypothesis that postulates that the expansion of heterochromatic blocks in the homogametic sex can, with certain conditions, contribute to post-zygotic isolation. i.e., female hybrid incompatibility, the converse of Haldane’s rule. This is based on the expectation that hybrids incur a selective disadvantage due to impaired meiosis resulting from the meiotic checkpoint network’s surveillance of the asymmetric expansions of heterochromatic blocks in the homogametic sex. Asynapsis of these heterochromatic regions would result in meiotic silencing of unsynapsed chromatin and, if this persists, germline apoptosis and female infertility.

Transcriptome profiling of differentially expressed genes of male and female inflorescences in spinach (Spinacia oleracea L.).

Spinach (Spinacia oleracea L.) is commonly considered a dioecious plant with heterogametic (XY) and homogametic (XX) sex chromosomes. The characteristic is also utilized for the production of spinach hybrid seeds. However, the molecular mechanisms of sex determination in spinach are still unclear because of a lack of genomic and transcriptomic information. In this study, RNA sequencing (RNA-seq) was performed in male and female inflorescences to provide insight into the molecular basis of sex determination in spinach. Comparative transcriptome analyses showed that 2278 differentially expressed genes (DEGs) were identified between male and female inflorescences. A high correlation between the RNA-Seq and qRT-PCR validation for DEGs was observed. Among these, 182 DEGs were annotated to transcription factors including the MYB family protein, bHLH family, and MADS family, suggesting these factors might play a vital role in sex determination. Moreover, 26 DEGs related to flower development, including nine ABCE class genes, were detected. Expression analyses of hormone pathways showed that brassinosteroids may be key hormones related to sex determination in spinach. Overall, this study provides a large amount of DEGs related to sexual expression and lays a foundation for unraveling the regulatory mechanism of sex determination in spinach.

Dosage-sensitive functions in embryonic development drove the survival of genes on sex-specific chromosomes in snakes, birds, and mammals.

Different ancestral autosomes independently evolved into sex chromosomes in snakes, birds, and mammals. In snakes and birds, females are ZW and males are ZZ; in mammals, females are XX and males are XY. Although X and Z Chromosomes retain nearly all ancestral genes, sex-specific W and Y Chromosomes suffered extensive genetic decay. In both birds and mammals, the genes that survived on sex-specific chromosomes are enriched for broadly expressed, dosage-sensitive regulators of gene expression, subject to strong purifying selection. To gain deeper insight into the processes that govern survival on sex-specific chromosomes, we carried out a meta-analysis of survival across 41 species—three snakes, 24 birds, and 14 mammals—doubling the number of ancestral genes under investigation and increasing our power to detect enrichments among survivors relative to nonsurvivors. Of 2564 ancestral genes, representing an eighth of the ancestral amniote genome, only 324 survive on present-day sex-specific chromosomes. Survivors are enriched for dosage-sensitive developmental processes, particularly development of neural crest–derived structures, such as the face. However, there was no enrichment for expression in sex-specific tissues, involvement in sex determination or gonadogenesis pathways, or conserved sex-biased expression. Broad expression and dosage sensitivity contributed independently to gene survival, suggesting that pleiotropy imposes additional constraints on the evolution of dosage compensation. We propose that maintaining the viability of the heterogametic sex drove gene survival on amniote sex-specific chromosomes, and that subtle modulation of the expression of survivor genes and their autosomal orthologs has disproportionately large effects on development and disease.

Classical and molecular cytogenetics of Belontia hasselti (Perciformes: Osphronemidae): Insights into the ZZ/ZW sex chromosome system

Abstract. Chaiyasan P, Mingkwan B, Jantarat S, Suwannapoom C, Cioffi MDB, Liehr T, Talumphai S, Tanomtong A, Supiwong W. 2021. Classical and molecular cytogenetics of Belontia hasselti (Perciformes: Osphronemidae): Insights into the ZZ/ZW sex chromosome system. Biodiversitas 22: 546-554. Karyotype of Java combtail fish, Belontia hasselti, from To Daeng peat swamp forest, Narathiwat Province, southern Thailand, was studied for the first time. Mitotic chromosome preparations were prepared directly from kidney cells from ten male and ten female fish. Conventional staining, NOR banding, and molecular cytogenetics with fluorescence in situ hybridization (FISH) using 5S and 18S rDNAs, as well as microsatellites d(CA)15 and d(CAC)10 as probes were applied. The diploid chromosome number (2n) was 48 and a female heterogametic sex chromosome system (ZZ/ZW) is suggested. The fundamental numbers (NF) were 48 and 49 in males and females, respectively. The karyotype of males comprised 48 telocentric chromosomes while the female ones were composed of one metacentric and 47 telocentric chromosomes. A single Ag-NOR-bearing chromosomal pair was identified. The NOR positions were characterized at the interstitial sub-centromeric region of pair 13, which coincided with signals of 18S rDNA and d(CAC)10 probes. The 5S rDNA signals were located at interstitial sites of the largest telocentric pair. Microsatellite d(CA)15 repeats were highly distributed throughout almost all entire chromosomes except for centromeric regions on some chromosome pairs, including sex chromosomes. The present study is a novel report for a ZZ/ZW sex chromosome system of this fish family in Thailand.

The rates of introgression and barriers to genetic exchange between hybridizing species: sex chromosomes vs autosomes.

Interspecific crossing experiments have shown that sex chromosomes play a major role in reproductive isolation between many pairs of species. However, their ability to act as reproductive barriers, which hamper interspecific genetic exchange, has rarely been evaluated quantitatively compared to Autosomes. This genome-wide limitation of gene flow is essential for understanding the complete separation of species, and thus speciation. Here, we develop a mainland-island model of secondary contact between hybridizing species of an XY (or ZW) sexual system. We obtain theoretical predictions for the frequency of introgressed alleles, and the strength of the barrier to neutral gene flow for the two types of chromosomes carrying multiple interspecific barrier loci. Theoretical predictions are obtained for scenarios where introgressed alleles are rare. We show that the same analytical expressions apply for sex chromosomes and autosomes, but with different sex-averaged effective parameters. The specific features of sex chromosomes (hemizygosity and absence of recombination in the heterogametic sex) lead to reduced levels of introgression on the X (or Z) compared to autosomes. This effect can be enhanced by certain types of sex-biased forces, but it remains overall small (except when alleles causing incompatibilities are recessive). We discuss these predictions in the light of empirical data comprising model-based tests of introgression and cline surveys in various biological systems.

Karyotype Characterisation of Two Australian Dragon Lizards (Squamata: Agamidae: Amphibolurinae) Reveals Subtle Chromosomal Rearrangements Between Related Species with Similar Karyotypes

Agamid lizards (Squamata: Agamidae) are karyotypically heterogeneous. Among the 101 species currently described from Australia, all are from the subfamily Amphibolurinae. This group is, with some exceptions, karyotypically conserved, and all species involving heterogametic sex show female heterogamety. Here, we describe the chromosomes of 2 additional Australian agamid lizards, Tympanocryptis lineata and Rankinia diemensis. These species are phylogenetically and cytogenetically sisters to the well-characterised Pogona vitticeps, but their sex chromosomes and other chromosomal characteristics are unknown. In this study, we applied advanced molecular cytogenetic techniques, such as fluorescence in situ hybridisation (FISH) and cross-species gene mapping, to characterise chromosomes and to identify sex chromosomes in these species. Our data suggest that both species have a conserved karyotype with P. vitticeps but with subtle rearrangements in the chromosomal landscapes. We could identify that T. lineata possesses a female heterogametic system (ZZ/ZW) with a pair of sex microchromosomes, while R. diemensis may have heterogametic sex chromosomes, but this requires further investigations. Our study shows the pattern of chromosomal rearrangements between closely related species, explaining the speciation within Australian agamid lizards of similar karyotypes.

Male hybrid sterility in the cattle-yak and other bovines: a review.

Hybridization is important for both animal breeders attempting to fix new phenotypic traits and researchers trying to unravel the mechanism of reproductive barriers in hybrid species and the process of speciation. In interspecies animal hybrids, gains made in terms of adaptation to environmental conditions and hybrid vigor may be offset by reduced fertility or sterility. Bovine hybrids exhibit remarkable hybrid vigor compared to their parents. However, the F1 male hybrid exhibits sterility, whereas the female is fertile. This male-biased sterility is consistent with the Haldane rule where heterogametic sex is preferentially rare, absent, or sterile in the progeny of two different species. The obstacle of fixing favorable traits and passing them to subsequent generations due to the male sterility is a major setback in improving the reproductive potential of bovines through hybridization. Multiperspective approaches such as molecular genetics, proteomics, transcriptomics, physiology, and endocrinology have been used by several researchers over the past decade in an attempt to unravel the potential mechanisms underlying male hybrid sterility. However, the mechanism of sterility in the hybrid male is still not completely unravelled. This review seeks to provide an update of the mechanisms of the sterility in the cattle-yak and other bovines.

Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males.

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males.

The evolution of sex chromosome dosage compensation in animals

The evolution of heteromorphic sex chromosomes shall lead to gene expression dosage problems, as in at least one of the sexes, the sex-linked gene dose has been reduced by half. It has been proposed that the transcriptional output of the whole X or Z chromosome should be doubled for complete dosage compensation in heterogametic sex. However, owing to the variability of the existing methods to determine the transcriptional differences between sex chromosomes and autosomes (S:A ratios) in different studies, we collected more than 500 public RNA-Seq data set from multiple tissues and species in major clades and proposed a unified computational framework for unbiased and comparable measurement of the S:A ratios of multiple species. We also tested the evolution of dosage compensation more directly by assessing changes in the expression levels of the current sex-linked genes relative to those of the ancestral sex-linked genes. We found that in mammals and birds, the S:A ratio is approximately 0.5, whereas in insects, fishes, and flatworms, the S:A ratio is approximately 1.0. Further analysis showed that the fraction of dosage-sensitive housekeeping genes on the X/Z chromosome is significantly correlated with the S:A ratio. In addition, the degree of degeneration of the Y chromosome may be responsible for the change in the S:A ratio in mammals without a dosage compensation mechanism. Our observations offer unequivocal support for the sex chromosome insensitivity hypothesis in animals and suggest that dosage sensitivity states of sex chromosomes are a major factor underlying different evolutionary strategies of dosage compensation.

The probability of fusions joining sex chromosomes and autosomes.

Chromosome fusion and fission are primary mechanisms of karyotype evolution. In particular, the fusion of a sex chromosome and an autosome has been proposed as a mechanism to resolve intralocus sexual antagonism. If sexual antagonism is common throughout the genome, we should expect to see an excess of fusions that join sex chromosomes and autosomes. Here, we present a null model that provides the probability of a sex chromosome autosome fusion, assuming all chromosomes have an equal probability of being involved in a fusion. This closed-form expression is applicable to both male and female heterogametic sex chromosome systems and can accommodate unequal proportions of fusions originating in males and females. We find that over 25% of all chromosomal fusions are expected to join a sex chromosome and an autosome whenever the diploid autosome count is fewer than 16, regardless of the sex chromosome system. We also demonstrate the utility of our model by analysing two contrasting empirical datasets: one from Drosophila and one from the jumping spider genus Habronattus. We find that in the case of Habronattus, there is a significant excess of sex chromosome autosome fusions but that in Drosophila there are far fewer sex chromosome autosome fusions than would be expected under our null model.

No sex differences in adult telomere length across vertebrates: a meta-analysis.

In many mammalian species, females live on average longer than males. In humans, women have consistently longer telomeres than men, and this has led to speculation that sex differences in telomere length (TL) could play a role in sex differences in longevity. To address the generality and drivers of patterns of sex differences in TL across vertebrates, we performed meta-analyses across 51 species. We tested two main evolutionary hypotheses proposed to explain sex differences in TL, namely the heterogametic sex disadvantage and the sexual selection hypotheses. We found no support for consistent sex differences in TL between males and females among mammal, bird, fish and reptile species. This absence of sex differences in TL across different classes of vertebrates does not support the heterogametic sex disadvantage hypothesis. Likewise, the absence of any negative effect of sexual size dimorphism on male TL suggests that sexual selection is not likely to mediate the magnitude of sex differences in TL across vertebrates. Finally, the comparative analyses we conducted did not detect any association between sex differences in TL and sex differences in longevity, which does not support the idea that sex differences in TL could explain the observed sex differences in longevity.