Heterogametic Females Research Articles

New primers for sex identification in the Chukar partridges (Alectoris chukar)

ABSTRACT 1. Male and female Chukar partridges are difficult to differentiate based on their morphology or by the Chromobox-Helicase-DNA binding (CHD) during early growth. 2. The current study developed a novel, simple, low-cost and rapid sexing protocol for Chukar partridges based on the newly defined sexing gene ubiquitin-associated protein 2 (UBAP2). 3. The length of polymorphism between UBAP2-W and UBAP2-Z homologous genes allows for easy sex discrimination in this species. Molecular sexing analysis was based on the simultaneous amplification of both genes, resulting in two distinct amplicons (947 bp and 535 bp) in heterogametic females and only a single band (535 bp) in homogametic males, which is easy to detect with agarose gel electrophoresis. 4. This technique is simple and convenient for genetic sex determination in Chukar partridges.

Genomic analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria moupinensis).

The strawberry genus, Fragaria, exhibits a wide range of sexual systems and natural ploidy variation. Nearly, all polyploid strawberry species exhibit separate sexes (dioecy). Research has identified the sex-determining sequences as roughly conserved but with repeatedly changed genomic locations across octoploid strawberries. However, it remains unclear whether tetraploid wild strawberries evolved dioecy independently or shared a common origin with octoploid strawberries. In this study, we investigated the sex determinants of F. moupinensis, a dioecious plant with heterogametic females (ZW). Utilizing a combination of haplotype-resolved genome sequencing of the female F. moupinensis, k-mer-based and coverage-based genome-wide association studies (GWAS), and transcriptomic analysis, we discovered a non-recombining, approximately 33.6 kb W-specific region on chromosome 2a. Within this region, only one candidate sex-determining gene (FmoAFT) was identified. Furthermore, an extensive resequencing of the entire Fragaria genus indicated that the W-specific region displays conservative female specificity across all tetraploid species. This observation suggests that dioecy evolved independently in tetraploid and octoploid strawberries. Moreover, employing virus-induced gene silencing (VIGS), we knocked down the expression of the FmoAFT homologue transcript in cultivated strawberries, revealing its potential role in promoting female functions during early carpel development. We also applied DNA affinity purification sequencing (DAP-seq) and yeast one-hybrid assays to identify potential direct targets of FmoAFT. These insights shed new light on the genetic basis and evolutionary history of sex determination in strawberries, thereby facilitating the formulation of strategies to manipulate sex determination in breeding programs.

Understanding the genetic sex-determining mechanism in Hyla eximia treefrog inferred from H-Y antigen.

Genetic sex-determining mechanisms have been extensively elucidated in mammals; however, the sex chromosomes, sex-determining genes, and gene regulatory networks involved in sex differentiation remain poorly understood in amphibians. In this study, we investigated the sex-determining mechanism in the Hyla eximia treefrog based on karyotypic analysis and identification of H-Y antigen, a sex-linked peptide that is present in the gonads of the heterogametic sex (XY or ZW) in all vertebrates. Results show a diploid chromosome number 2n = 24 with homomorphic sex chromosomes. The heterogametic sex, ZW-female, were hypothesized based on H-Y antigen mRNA expression in female gonads (24,ZZ/24,ZW). The treefrog H-Y peptide exhibited a high percentage of identity with other vertebrate sequences uploaded to GenBank database. To obtain gene expression profiles, we also obtained the coding sequence of the housekeeping Actb gene. High H-Y antigen expression levels were further confirmed in ovaries using real-time polymerase chain reaction (RT-PCR) during non-breeding season, we noted a decrease in the expression of the H-Y antigen during breeding season. This study provides evidence that sex hormones might suppress H-Y antigen expression in the gonads of heterogametic females 24,ZW during the breeding season. These findings suggest that H-Y gene expression is a well-suited model for studying heterogametic sex by comparing the male and female gonads.

A polygenic explanation for Haldane's rule in butterflies.

Two robust rules have been discovered about animal hybrids: Heterogametic hybrids are more unfit (Haldane's rule), and sex chromosomes are disproportionately involved in hybrid incompatibility (the large-X/Z effect). The exact mechanisms causing these rules in female heterogametic taxa such as butterflies are unknown but are suggested by theory to involve dominance on the sex chromosome. We investigate hybrid incompatibilities adhering to both rules in Papilio and Heliconius butterflies and show that dominance theory cannot explain our data. Instead, many defects coincide with unbalanced multilocus introgression between the Z chromosome and all autosomes. Our polygenic explanation predicts both rules because the imbalance is likely greater in heterogametic females, and the proportion of introgressed ancestry is more variable on the Z chromosome. We also show that mapping traits polygenic on a single chromosome in backcrosses can generate spurious large-effect QTLs. This mirage is caused by statistical linkage among polygenes that inflates estimated effect sizes. By controlling for statistical linkage, most incompatibility QTLs in our hybrid crosses are consistent with a polygenic basis. Since the two genera are very distantly related, polygenic hybrid incompatibilities are likely common in butterflies.

The evolutionary maintenance of ancient recombining sex chromosomes in the ostrich.

Sex chromosomes have evolved repeatedly across the tree of life and often exhibit extreme size dimorphism due to genetic degeneration of the sex-limited chromosome (e.g. the W chromosome of some birds and Y chromosome of mammals). However, in some lineages, ancient sex-limited chromosomes have escaped degeneration. Here, we study the evolutionary maintenance of sex chromosomes in the ostrich (Struthio camelus), where the W remains 65% the size of the Z chromosome, despite being more than 100 million years old. Using genome-wide resequencing data, we show that the population scaled recombination rate of the pseudoautosomal region (PAR) is higher than similar sized autosomes and is correlated with pedigree-based recombination rate in the heterogametic females, but not homogametic males. Genetic variation within the sex-linked region (SLR) (π = 0.001) was significantly lower than in the PAR, consistent with recombination cessation. Conversely, genetic variation across the PAR (π = 0.0016) was similar to that of autosomes and dependent on local recombination rates, GC content and to a lesser extent, gene density. In particular, the region close to the SLR was as genetically diverse as autosomes, likely due to high recombination rates around the PAR boundary restricting genetic linkage with the SLR to only ~50Kb. The potential for alleles with antagonistic fitness effects in males and females to drive chromosome degeneration is therefore limited. While some regions of the PAR had divergent male-female allele frequencies, suggestive of sexually antagonistic alleles, coalescent simulations showed this was broadly consistent with neutral genetic processes. Our results indicate that the degeneration of the large and ancient sex chromosomes of the ostrich may have been slowed by high recombination in the female PAR, reducing the scope for the accumulation of sexually antagonistic variation to generate selection for recombination cessation.

Sex-related differences in aging rate are associated with sex chromosome system in amphibians.

Sex‐related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females) systems. Taking advantage of a unique collection of capture–recapture datasets in amphibians, a vertebrate group where XY and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex‐specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the “unguarded X/Z effect”) or repeat‐rich Y/W chromosome (the “toxic Y/W effect”) could accelerate aging in the heterogametic sex in some vertebrate clades.

Absence of W Chromosome in Psychidae Moths and Implications for the Theory of Sex Chromosome Evolution in Lepidoptera.

Moths and butterflies (Lepidoptera) are the largest group with heterogametic females. Although the ancestral sex chromosome system is probably Z0/ZZ, most lepidopteran species have the W chromosome. When and how the W chromosome arose remains elusive. Existing hypotheses place the W origin either at the common ancestor of Ditrysia and Tischeriidae, or prefer independent origins of W chromosomes in these two groups. Due to their phylogenetic position at the base of Ditrysia, bagworms (Psychidae) play an important role in investigating the W chromosome origin. Therefore, we examined the W chromosome status in three Psychidae species, namely Proutia betulina, Taleporia tubulosa, and Diplodoma laichartingella, using both classical and molecular cytogenetic methods such as sex chromatin assay, comparative genomic hybridization (CGH), and male vs. female genome size comparison by flow cytometry. In females of all three species, no sex chromatin was found, no female-specific chromosome regions were revealed by CGH, and a Z-chromosome univalent was observed in pachytene oocytes. In addition, the genome size of females was significantly smaller than males. Overall, our study provides strong evidence for the absence of the W chromosome in Psychidae, thus supporting the hypothesis of two independent W chromosome origins in Tischeriidae and in advanced Ditrysia.

Genomic islands of differentiation in two songbird species reveal candidate genes for hybrid female sterility.

Hybrid sterility is a common first step in the evolution of postzygotic reproductive isolation. According to Haldane's Rule, it affects predominantly the heterogametic sex. While the genetic basis of hybrid male sterility in organisms with heterogametic males has been studied for decades, the genetic basis of hybrid female sterility in organisms with heterogametic females has received much less attention. We investigated the genetic basis of reproductive isolation in two closely related avian species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (L.luscinia), that hybridize in a secondary contact zone and produce viable hybrid progeny. In accordance with Haldane's Rule, hybrid females are sterile, while hybrid males are fertile, allowing gene flow to occur between the species. Using transcriptomic data from multiple individuals of both nightingale species, we identified genomic islands of high differentiation (FST ) and of high divergence (Dxy ), and we analysed gene content and patterns of molecular evolution within these islands. Interestingly, we found that these islands were enriched for genes related to female meiosis and metabolism. The islands of high differentiation and divergence were also characterized by higher levels of linkage disequilibrium than the rest of the genome in both species indicating that they might be situated in genomic regions of low recombination. This study provides one of the first insights into genetic basis of hybrid female sterility in organisms with heterogametic females.

New Insights into the Evolution of the W Chromosome in Lepidoptera

Moths and butterflies (Lepidoptera) represent the most diverse group of animals with heterogametic females. Although the vast majority of species has a WZ/ZZ (female/male) sex chromosome system, it is generally accepted that the ancestral system was Z/ZZ and the W chromosome has evolved in a common ancestor of Tischeriidae and Ditrysia. However, the lack of data on sex chromosomes in lower Lepidoptera has prevented a formal test of this hypothesis. Here, we performed a detailed analysis of sex chromosomes in Tischeria ekebladella (Tischeriidae) and 3 species representing lower Ditrysia, Cameraria ohridella (Gracillariidae), Plutella xylostella (Plutellidae), and Tineola bisselliella (Tineidae). Using comparative genomic hybridization we show that the first 3 species have well-differentiated W chromosomes, which vary considerably in their molecular composition, whereas T. bisselliella has no W chromosome. Furthermore, our results suggest the presence of neo-sex chromosomes in C. ohridella. For Z chromosomes, we selected 5 genes evenly distributed along the Z chromosome in ditrysian model species and tested their Z-linkage using qPCR. The tested genes (Henna, laminin A, Paramyosin, Tyrosine hydroxylase, and 6-Phosphogluconate dehydrogenase) proved to be Z-linked in all species examined. The conserved synteny of the Z chromosome across Tischeriidae and Ditrysia, along with the W chromosome absence in the lower ditrysian families Psychidae and Tineidae, suggests a possible independent origin of the W chromosomes in these 2 lineages.

Sexual Dimorphism in Body Size and the Origin of Sex-Determination Systems

Diverse sex-determining systems occur in vertebrates, including environmental sex determination (ESD), genetic sex determination (GSD) of type XX/XY (heterogametic males), and GSD of type ZZ/ZW (heterogametic females). The origins of the two genetic types are poorly understood. We use protected invasion theory to derive a model that generates testable predictions about the origins of the two genetic types from ESD. Protected invasion theory predicts biases in the evolutionary origins of new traits by focusing on the probability that a selectively favored trait will avoid loss by genetic drift when rare. We show that the theory makes predictions about the conditions under which XY or ZW systems are more likely to arise from an ancestral state of ESD. In particular, assuming that there is an average trend toward increasing body size in lineages, the origins of XY systems are predicted to be accompanied by increases in male∶female body size ratio. In contrast, ZW systems are predicted to be accompanied by decreases in male∶female body size ratio. We find support for these predictions in the form of a marked association among vertebrates between sex-determining system and body size dimorphism in paired comparisons independent of shared phylogeny.

Sexing and genetic diversity in the genus Charadrius based on CHD gene

Charadrius spp. are migratory shorebirds in Thailand during the non-breeding season inwhich adult plumages are not clearly sexually dimorphic. In this work, a molecular approach was used to determine the gender and genetic diversity. Amplification of intron length polymorphisms in chromo-helicase-DNA-binding ( CHD ) genes on both Z and W chromosomes of three Charadrius spp. included 87 lesser sand plover ( Charadrius mongolus ), 27 greater sand plover ( C. leschenaultii ) and 3 kentish plover ( C. alexandrinus ). The resulting PCR products from 2550F/2718R primer showed fragments on a conventional agarose gel electrophoresis with size differences ranging from 200 bp between the two respective ZW allele. Males were identified by the presence of a single band about 650 bp ( CHD-Z ). Heterogametic females were identified by the presence of a second additional fragment length of approximately 450 bp ( CHD-W ). The CHD-Z fragments were purified and analysed by sequencing. The multiple sequence comparison was accomplished using ClustalX and PHYLIP software. These data demonstrate that the CHD-Z sequence related with morphology and can be used for genetic diversity study. The results of molecular sex determination and genetic diversity analyses will also have implications for shorebird conservation.

THE VARIABILITY IS IN THE SEX CHROMOSOMES

Sex differences in the mean trait expression are well documented, not only for traits that are directly associated with reproduction. Less is known about how the variability of traits differs between males and females. In species with sex chromosomes and dosage compensation, the heterogametic sex is expected to show larger trait variability ("sex-chromosome hypothesis"), yet this central prediction, based on fundamental genetic principles, has never been evaluated in detail. Here we show that in species with heterogametic males, male variability in body size is significantly larger than in females, whereas the opposite can be shown for species with heterogametic females. These results support the prediction of the sex-chromosome hypothesis that individuals of the heterogametic sex should be more variable. We argue that the pattern demonstrated here for sex-specific body size variability is likely to apply to any trait and needs to be considered when testing predictions about sex-specific variability and sexual selection.

Sex-biased gene expression on the avian Z chromosome: highly expressed genes show higher male-biased expression.

Dosage compensation, the process whereby expression of sex-linked genes remains similar between sexes (despite heterogamety) and balanced with autosomal expression, was long believed to be essential. However, recent research has shown that several lineages, including birds, butterflies, monotremes and sticklebacks, lack chromosome-wide dosage compensation mechanisms and do not completely balance the expression of sex-linked and autosomal genes. To obtain further understanding of avian sex-biased gene expression, we studied Z-linked gene expression in the brain of two songbirds of different genera (zebra finch, Taeniopygia guttata, and common whitethroat, Sylvia communis) using microarray technology. In both species, the male-bias in gene expression was significantly higher for Z than for autosomes, although the ratio of Z-linked to autosomal expression (Z:A) was relatively close to one in both sexes (range: 0.89–1.01). Interestingly, the Z-linked male-bias in gene expression increased with expression level, and genes with low expression showed the lowest degree of sex-bias. These results support the view that the heterogametic females have up-regulated their single Z-linked homologues to a high extent when the W-chromosome degraded and thereby managed to largely balance their Z:A expression with the exception of highly expressed genes. The male-bias in highly expressed genes points towards male-driven selection on Z-linked loci, and this and other possible hypotheses are discussed.

Sex-linkage Identification of Microsatellite TUT1*

The microsatellite TUT1 was used in several grouse species as an autosomal locus with "heterozygote deficit",but recently found with null alleles.By amplifying in 45 adult Chinese grouse(Tetrastes sewerzowi) and five broods with known parents,it was found that TUT1 was homozygous in heterogametic females and heterozygous in 84% males,and transmitted from fathers to both sons and daughters but from mothers to sons only.By BLAST query,a nucleotide sequence(460 bp) that contains TUT1 site was most similar to a portion of the sequence of Z chromosome of chicken(Gallus gallus),with the max score of 329 and expect value of 8e-87.Both methods showed that TUT1 was based on Z chromosome.These findings indicated that microsatellite "null alleles" might be originated from sex-linkage and highlighted the importance of gender-specific analysis when publishing and using microsatellites.Tab 1,Ref 17

Faster evolution of Z-linked duplicate genes in chicken

It has been shown that duplicate genes on the X chromosome evolve much faster than duplicate genes on autosomes in Drosophila melanogaster. However, whether this phenomenon is general and can be applied to other species is not known. Here we examined this issue in chicken that have heterogametic females (females have ZW sex chromosome). We compared sequence divergence of duplicate genes on the Z chromosome with those on autosomes. We found that duplications on the Z chromosome indeed evolved faster than those on autosomes and show distinct patterns of molecular evolution from autosomal duplications. Examination of the expression of duplicate genes revealed an enrichment of duplications on the Z chromosome having male-biased expression and an enrichment of duplications on the autosomes having female-biased expression. These results suggest an evolutionary trend of the recruitment of duplicate genes towards reproduction-specific function. The faster evolution of duplications on Z than on the autosomes is most likely contributed by the selective forces driving the fixation of adaptive mutations on Z. Therefore, the common phenomena observed in both flies and chicken suggest that duplicate genes on sex chromosomes have distinct dynamics and are more influenced by natural selection than autosomal duplications, regardless of the kind of sex determination systems.

Female heterogamety and speciation: reduced introgression of the Z chromosome between two species of nightingales.

Several lines of evidence suggest that the X chromosome plays a large role in intrinsic postzygotic isolation. The role of the Z chromosome in speciation is much less understood. To explore the role of the Z chromosome in reproductive isolation, we studied nucleotide variation in two closely related bird species, the Thrush Nightingale (Luscinia luscinia) and the Common Nightingale (L. megarhynchos). These species are isolated by incomplete prezygotic isolation and female hybrid sterility. We sequenced introns of four Z-linked and eight autosomal loci and analyzed patterns of polymorphism and divergence using a divergence-with-gene flow framework. Our results suggest that the nightingale species diverged approximately 1.8 Mya. We found strong evidence of gene flow after divergence in both directions, although more introgression occurred from L. megarhynchos into L. luscinia. Gene flow was significantly higher on the autosomes than on the Z chromosome. Our results support the idea that the Z chromosome plays an important role in intrinsic postzygotic isolation in birds, although it may also contribute to the evolution of prezygotic isolation through sexual selection. This highlights the similarities in the genetic basis of reproductive isolation between organisms with heterogametic males and organisms with heterogametic females during the early stages of speciation.

Can you feminise a crustacean?

The ability of anthropogenic chemicals to cause reproductive disorders has been the focus of toxicologists for many years. Whilst the focus of endocrine disrupting chemicals has mainly been associated with vertebrate groups, there have been continued calls for more research on the invertebrates. Surprisingly, within the Crustacea, many studies have focussed on female or growth/moulting related endpoints despite many of the vertebrate studies highlighting male related effects such as abnormal male reproductive development. Furthermore, a large number of the invertebrate studies have focussed on vertebrate estrogens or their mimics. Considering the biology of the crustacean endocrine systems, this paper shall argue that unlike the vertebrates, it is a lot more difficult to feminise a crustacean than it is to de-masculinise one. Consequently, crustacean toxicologists, by following the tact of vertebrate biologists, may have been trying to address the right questions, but in the wrong way. Studies have shown that intersexuality in crustaceans may arise through the masculinisation of heterogametic (WZ) females or the de-masculinisation of males through aberrations in male androgenic gland activity. It is recommended that the focus be put on understanding the mechanisms of sex determination in Crustacea, and the expression of male secondary sexual characteristics at the molecular, biochemical and physiological level are fully explored so that appropriate assessments can be made as to whether sexual endocrine disruption is occurring in this ecologically important group.

Unusual sex chromosome inheritance in six species of small ermine moths (Yponomeuta, Yponomeutidae, Lepidoptera)

First meiotic division in females and males of Yponomeuta evonyrnellus (L.), Y. cagnagellus (Hiibner). Y. rnalinellus Zeller, Y. padellus (L), Y. rorellus (Hiibner) and Y. gigas Rebel was studied in the light microscope. In the heterogametic females the sex chromosomes were found to be associated in a sex chromosome trivalent. The sex chromosome trivalent, written AAWZ, consisted of a W-chromosome translocated to an autosome to form an Aw-chromosome, which was paired with the homologous auto-some and the 2-chromosome. In the homogametic males the two Z-chromosomes were paired in a usual bivalent. The sex chromosome inheritance can be balanced in this way. The haploid chromosome number was n = 29 A + AAWZ in females and n = 30 A + ZZ in males, for all species examined. It is of interest to notice that the chromosome number of Y. rorellus earlier has been differently determined to be n = 29 for males. Especially in females the bivalents were observed to be nonhomologously associated at the telomeres during meiotic prophase.

Polytene chromosomes of Simulium (Psaroniocompsa) daltanhani (Diptera: Simuliidae) from Central Amazonia, Brazil

Last-instar larvae of Simulium (Psaroniocompsa) daltanhani Hamada and Adler from a stream in Central Amazonia were analyzed cytologically by mapping their polytene chromosomes. Simulium daltanhani has the nucleolar organizer in the short arm of chromosome I, heterogametic females, and an absence of autosomal polymorphisms. The chromosomes carry multiple rearrangements relative to other analyzed members of the S. quadrifidum species group in the subgenus Psaroniocompsa. One-third of the chromosomal complement is rearranged relative to the sequence of S. ulyssesi, the species with the most similar banding pattern among studied members of the S. quadrifidum group.

Characterization of a secondary contact zone of the Great Tit Parus major and the Japanese Tit P. minor (Aves: Passeriformes) in Far Eastern Siberia with DNA markers

The Great Tit Parus major and the Japanese Tit P. minor meet and hybridize in a secondary contact zone in Far Eastern Amur River Valley. The genetic characteristics of this hybrid zone were studied by nuclear microsatellites and mitochondrial DNA sequences. Patterns of introgression of nuclear markers, mitochondrial markers and phenotypic characters were found to be different. The possible reasons, such as stronger selection against heterogametic females according to Haldane’s rule, lowered hybrid fitness, assortative mating and factors enforcing introgression, like density-dependent pair-formation, are discussed.