Rapid Male-Specific Regulatory Divergence and Down Regulation of Spermatogenesis Genes in Drosophila Species Hybrids

In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.     

Male Sex Interspecies Divergence and Down Regulation of Expression of Spermatogenesis Genes in Drosophila Sterile Hybrids

Male sex genes have shown a pattern of rapid interspecies divergence at both the coding and gene expression level. A common outcome from crosses between closely-related species is hybrid male sterility. Phenotypic and genetic studies in Drosophila sterile hybrid males have shown that spermatogenesis arrest is postmeiotic with few exceptions, and that most misregulated genes are involved in late stages of spermatogenesis. Comparative studies of gene regulation in sterile hybrids and parental species have mainly used microarrays providing a whole genome representation of regulatory problems in sterile hybrids. Real-time PCR studies can reject or reveal differences not observed in microarray assays. Moreover, differences in gene expression between samples can be dependant on the source of RNA (e.g., whole body vs. tissue). Here we survey expression in D. simulans, D. mauritiana and both intra and interspecies hybrids using a real-time PCR approach for eight genes expressed at the four main stages of sperm development. We find that all genes show a trend toward under expression in the testes of sterile hybrids relative to parental species with only the two proliferation genes (bam and bgcn) and the two meiotic class genes (can and sa) showing significant down regulation. The observed pattern of down regulation for the genes tested can not fully explain hybrid male sterility. We discuss the down regulation of spermatogenesis genes in hybrids between closely-related species within the contest of rapid divergence experienced by the male genome, hybrid sterility and possible allometric changes due to subtle testes-specific developmental abnormalities.     

Gene expression divergence and the origin of hybrid dysfunctions

Hybrids between closely related species are often sterile or inviable as a consequence of failed interactions between alleles from the different species. Most genetic studies have focused on localizing the alleles associated with these failed interactions, but the mechanistic/biochemical nature of the failed interactions is poorly understood. This review discusses recent studies that may contribute to our understanding of these failed interactions. We focus on the possible contribution of failures in gene expression as an important contributor to hybrid dysfunctions. Although regulatory pathways that share elements in highly divergent taxa may contribute to hybrid dysfunction, various studies suggest that misexpression may be disproportionately great in regulatory pathways containing rapidly evolving, particularly male-biased, genes. We describe three systems that have been analyzed recently with respect to global patterns of gene expression in hybrids versus pure species, each in Drosophila. These studies reveal that quantitative misexpression of genes is associated with hybrid dysfunction. Misexpression of genes has been documented in sterile hybrids relative to pure species, and variation in upstream factors may sometimes cause the over- or under-expression of genes resulting in hybrid sterility or inviability. Studying patterns of evolution between species in regulatory pathways, such as spermatogenesis, should help in identifying which genes are more likely to be contributors to hybrid dysfunction. Ultimately, we hope more functional genetic studies will complement our understanding of the genetic disruptions leading to hybrid dysfunctions and their role in the origin of species.     

Patterns of evolution of genes disrupted in expression in Drosophila species hybrids

Divergence between species in regulatory pathways may contribute to hybrid incompatibilities such as sterility. Consistent with this idea, genes involved in male fertility often evolve faster than most other genes both in amino acid sequence and in expression. Previously, we identified a panel of male-specific genes under-expressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to pure species, and we showed that this under-expression is associated with infertility. In a preliminary effort to assess the generalities in the patterns of evolution of these genes, I examined patterns of mRNA expression in three of these genes in sterile F 1 hybrid males of D. pseudoobscura and D. persimilis . F 1 hybrid males bearing D. persimilis X chromosomes under-expressed all these genes relative to the parental species, while hybrids bearing D. pseudoobscura X chromosomes under-expressed two of these three genes. Interestingly, the third gene, CG5762 , has undergone extensive amino acid evolution within the D. pseudoobscura species group, possibly driven by positive natural selection. We conclude that some of the same genes exhibit disruptions in expression within each of the two species groups, which could suggest commonalities in the regulatory architecture of sterility in these groups. Alternative explanations are also considered.     

Genome-wide patterns of expression in Drosophila pure species and hybrid males

One of the most fundamental questions for understanding the origin of species is why genes that function to cause fertility in a pure-species genetic background fail to produce fertility in a hybrid genetic background. A related question is why the sex that is most often sterile or inviable in hybrids is the heterogametic (usually male) sex. In this survey, we have examined the extent and nature of differences in gene expression between fertile adult males of two Drosophila species and sterile hybrid males produced from crosses between these species. Using oligonucleotide microarrays and real-time quantitative polymerase chain reaction, we have identified and confirmed that differences in gene expression exist between pure species and hybrid males, and many of these differences are quantitative rather than qualitative. Furthermore, genes that are expressed primarily or exclusively in males, including several involved in spermatogenesis, are disproportionately misexpressed in hybrids, suggesting a possible genetic cause for their sterility.     

Genomic imprinting,disrupted placental expression,and speciation

The importance of regulatory incompatibilities to the early stages of speciation remains unclear. Hybrid mammals often show extreme parent‐of‐origin growth effects that are thought to be a consequence of disrupted genetic imprinting (parent‐specific epigenetic gene silencing) during early development. Here, we test the long‐standing hypothesis that abnormal hybrid growth reflects disrupted gene expression due to loss of imprinting (LOI) in hybrid placentas, resulting in dosage imbalances between paternal growth factors and maternal growth repressors. We analyzed placental gene expression in reciprocal dwarf hamster hybrids that show extreme parent‐of‐origin growth effects relative to their parental species. In massively enlarged hybrid placentas, we observed both extensive transgressive expression of growth‐related genes and biallelic expression of many genes that were paternally silenced in normal sized hybrids. However, the apparent widespread disruption of paternal silencing was coupled with reduced gene expression levels overall. These patterns are contrary to the predictions of the LOI model and indicate that hybrid misexpression of dosage‐sensitive genes is caused by other regulatory mechanisms in this system. Collectively, our results support a central role for disrupted gene expression and imprinting in the evolution of mammalian hybrid inviability, but call into question the generality of the widely invoked LOI model.     

Compensatory cis-trans evolution and the dysregulation of gene expression in interspecific hybrids of Drosophila

Hybrids between species are often characterized by novel gene-expression patterns. A recent study on allele-specific gene expression in hybrids between species of Drosophila revealed cases in which cis- and trans-regulatory elements within species had coevolved in such a way that changes in cis-regulatory elements are compensated by changes in trans-regulatory elements. We hypothesized that such coevolution should often lead to gene misexpression in the hybrid. To test this hypothesis, we estimated allele-specific expression and overall expression levels for 31 genes in D. melanogaster, D. simulans, and their F1 hybrid. We found that 13 genes with cis-trans compensatory evolution are in fact misexpressed in the hybrid. These represent candidate genes whose dysregulation might be the consequence of coevolution of cis- and trans-regulatory elements within species. Using a mathematical model for the regulation of gene expression, we explored the conditions under which cis-trans compensatory evolution can lead to misexpression in interspecific hybrids.     

A biotechnology‐based male‐sterility system for hybrid seed production in tomato

Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male‐sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology‐based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male‐sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated mutagenesis of a stamen‐specific gene SlSTR1 and devised a transgenic maintainer by transforming male‐sterile plants with a fertility‐restoration gene linked to a seedling‐colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male‐sterile plant segregated into 50% non‐transgenic male‐sterile plants and 50% male‐fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male‐sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.     

Dominance effects strengthen premating hybridization barriers between sympatric species of grasshoppers (Acrididae,Orthoptera)

Sexual selection can lead to the rapid evolution of premating hybridization barriers and allows accelerated diversification and speciation within an evolutionary lineage. Especially during early stages of divergence, hybridization may impede further divergence, which strongly depends on the reproductive success of hybrids. Behavioural sterility of hybrids can limit or even prevent homogenizing gene flow. In this study, we investigated the attractiveness of male courtship songs for females of the grasshopper species Chorthippus biguttulus and C. brunneus and their interspecific F1 and F2 hybrids. Song preferences of females of both species are highly species specific and differ in three parameters: shape of the preference function, preference for syllable pattern and phrase duration. F1 hybrid females of both reciprocal crosses as well as F2 hybrid females resembled closely pure C. biguttulus females in respect of shape of the preference function and preference for syllable pattern, while preference for phrase duration showed an intermediate expression. This resulted in song preferences of hybrid females that closely resembled those of one parental species, that is C. biguttulus females. Such strong dominance effects were rarely reported so far. They represent an effective barrier limiting gene flow between the two species, since hybrid females will backcross to only one parental species and discriminate against hybrid males, which are behaviourally sterile. Such taxon‐specific modes of inheritance may have facilitated the rapid divergence of acoustically communicating grasshoppers of the species group of Chorthippus biguttulus. Our findings have novel implications on the expression of neuronal filters and the evolution of complex courtship signals.     

Association of Misexpression with Sterility in Hybrids of <Emphasis Type="Italic">Drosophila simulans</Emphasis>and <Emphasis Type="Italic">D. mauritiana</Emphasis>

Recent studies have identified genes associated with hybrid sterility and other hybrid dysfunctions, but the consequences of introgressions of these speciation genes are often poorly understood. Previously, we identified a panel of genes that are underexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to pure species. Here, we build on this reverse-genetics approach to demonstrate that the underexpression of at least five of these genes in hybrids is associated with hybrid sterility and that these five genes are coordinately regulated. We map one upstream regulator of these genes to a region previously shown to harbor one or more factors causing hybrid sterility. Finally, we show that the genes underexpressed in hybrids are often highly conserved, as might be predicted for downstream targets of the genetic changes that cause hybrid sterility. This approach integrates forward genetics with reverse genetics to show a proximate consequence of the introgression of particular hybrid sterility-conferring regions between species: underexpression of genes necessary for normal spermatogenesis.[Reviewing Editor: Martin Kreitman]     

Hybrid sterility,Haldane's rule and speciation in Heliconius cydno and H. melpomene

Most genetic studies of Haldane's rule, in which hybrid sterility or inviability affects the heterogametic sex preferentially, have focused on Drosophila. It therefore remains unclear to what extent the conclusions of that work apply more generally, particularly in female-heterogametic taxa such as birds and Lepidoptera. Here we present a genetic analysis of Haldane's rule in Heliconius butterflies. Female F(1) hybrids between Heliconius melpomene and H. cydno are completely sterile, while males have normal to mildly reduced fertility. In backcrosses of male F(1) hybrids, female offspring range from completely sterile to fully fertile. Linkage analysis using the Z-linked triose-phosphate isomerase locus demonstrates a "large X" (Z) effect on sterility. Expression of female sterility varies among crosses in this and a previous study of Heliconius. Sterility may result from the production of normal but infertile eggs, production of small infertile eggs, or from a complete failure to develop ovarioles, which suggests multiple routes to the evolution of hybrid sterility in these Heliconius species. These results conform to the expectations of the "dominance" rather than "faster male" theories of Haldane's rule and suggest that relatively few loci are responsible. The two species are broadly sympatric and hybridize in the wild, so that female hybrid sterility forms one of several strong but incomplete barriers to gene flow in nature. The effect of female sterility is comparable to that of selection against non-mimetic hybrids, while mate choice forms a much stronger barrier to gene transfer.     

GENETIC ARCHITECTURE OF ISOLATION BETWEEN TWO SPECIES OF SILENE WITH SEX CHROMOSOMES AND HALDANE'S RULE

Examination of the genetic architecture of hybrid breakdown can provide insight into the genetic mechanisms of commonly observed isolating phenomena such as Haldane's rule. We used line‐cross analysis to dissect the genetic architecture of divergence between two plant species that exhibit Haldane's rule for male sterility and rarity, Silene latifolia and Silene diclinis. We made 15 types of crosses, including reciprocal F₁, F₂, backcrosses, and later‐generation crosses, grew the seeds to flowering, and measured the number of viable ovules, proportion of viable pollen, and sex ratio. Typically, Haldane's rule for male rarity in XY animal hybrids is explained by interactions involving recessive X‐linked alleles that are deleterious when hemizygous (dominance theory), whereas sterility is explained by rapid evolution of spermatogenesis genes (faster‐male evolution). In contrast, we found that the genetic mechanisms underlying Haldane's rule between the two Silene species did not follow these conventions. Dominance theory was sufficient to explain male sterility, but male rarity likely involved faster‐male evolution. We also found an effect of the neo‐sex chromosomes of S. diclinis on the extreme rarity of some hybrid males. Our findings suggest that the genetic architecture of Haldane's rule in dioecious plants may differ from those commonly found in animals.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.