Misregulation of spermatogenesis genes in Drosophila hybrids is lineage‐specific and driven by the combined effects of sterility and fast male regulatory divergence

Hybrid male sterility is a common outcome of crosses between different species. Gene expression studies have found that a number of spermatogenesis genes are differentially expressed in sterile hybrid males, compared with parental species. Late‐stage sperm development genes are particularly likely to be misexpressed, with fewer early‐stage genes affected. Thus, a link has been posited between misexpression and sterility. A more recent alternative explanation for hybrid gene misexpression has been that it is independent of sterility and driven by divergent evolution of male‐specific regulatory elements between species (faster male hypothesis). The faster male hypothesis predicts that misregulation of spermatogenesis genes should be independent of sterility and approximately the same in both hybrids, whereas sterility should only affect gene expression in sterile hybrids. To test the faster male hypothesis vs. the effect of sterility on gene misexpression, we analyse spermatogenesis gene expression in different species pairs of the Drosophila phylogeny, where hybrid male sterility occurs in only one direction of the interspecies cross (i.e. unidirectional sterility). We find significant differences among genes in misexpression with effects that are lineage‐specific and caused by sterility or fast male regulatory divergence.     

Gene regulation divergence is a major contributor to the evolution of Dobzhansky-Muller incompatibilities between species of Drosophila

The Dobzhansky-Muller model denotes incompatible gene interactions between diverging populations/species and is recognized as the basis of postzygotic reproductive isolation. Little is known about the molecular nature of such gene interactions. We have carried out comparative gene expression analyses in the testes of 3 closely related species of the Drosophila melanogaster subgroup and their hybrids (all of which are sterile). We show that in hybrids 1) a higher proportion of male-biased genes (i.e., genes with a higher level of expression in males) are underexpressed (or not expressed) compared with non-sex-biased genes, 2) the majority of the underexpressed genes appear to be under stabilizing selection by virtue of showing similar levels of expression in the parental species, and only a small proportion of genes show signs of directional selection, 3) very few of the misexpressed genes are shared between species pairs, suggesting that there may not be a "common" set of "speciation genes," and 4) expression of non-testes-specific genes is observed in the testes of interspecific hybrids, and the number of such genes is positively correlated with divergence time. These results suggest that gene regulation divergence of sex- and reproduction-related genes is a major contributor to the evolution of Dobzhansky-Muller incompatibilities between species of Drosophila.     

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.     

Rapid evolution and gene-specific patterns of selection for three genes of spermatogenesis in Drosophila

Hybrid males resulting from crosses between closely related species of Drosophila are sterile. The F1 hybrid sterility phenotype is mainly due to defects occurring during late stages of development that relate to sperm individualization, and so genes controlling sperm development may have been subjected to selective diversification between species. It is also possible that genes of spermatogenesis experience selective constraints given their role in a developmental pathway. We analyzed the molecular evolution of three genes playing a role during the sperm developmental pathway in Drosophila at an early (bam), a mid (aly), and a late (dj) stage. The complete coding region of these genes was sequenced in different strains of Drosophila melanogaster and Drosophila simulans. All three genes showed rapid divergence between species, with larger numbers of nonsynonymous to synonymous differences between species than polymorphisms. Although this could be interpreted as evidence for positive selection at all three genes, formal tests of selection do not support such a conclusion. Departures from neutrality were detected only for dj and bam but not aly. The role played by selection is unique and determined by gene-specific characteristics rather than site of expression. In dj, the departure was due to a high proportion of neutral synonymous polymorphisms in D. simulans, and there was evidence of purifying selection maintaining a high lysine amino acid protein content that is characteristic of other DNA-binding proteins. The earliest spermatogenesis gene surveyed, which plays a role in both male and female gametogenesis, was bam, and its significant departure from neutrality was due to an excess of nonsynonymous substitutions between species. Bam is degraded at the end of mitosis, and rapid evolutionary changes among species might be a characteristic shared with other degradable transient proteins. However, the large number of nonsynonymous changes between D. melanogaster and D. simulans and a phylogenetic comparative analysis among species confirms evidence of positive selection driving the evolution of Bam and suggests an yet unknown germ cell line developmental adaptive change between these two species.     

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.     

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.     

KNOCKDOWN OF ATPsyn‐b CAUSED LARVAL GROWTH DEFECT AND MALE INFERTILITY IN Drosophila

The ATPsyn‐b encoding for subunit b of ATP synthase in Drosophila melanogaster is proposed to act in ATP synthesis and phagocytosis, and has been identified as one of the sperm proteins in both Drosophila and mammals. At present, its details of functions in animal growth and spermatogenesis have not been reported. In this study, we knocked down ATPsyn‐b using Drosophila lines expressing inducible hairpin RNAi constructs and Gal4 drivers. Ubiquitous knockdown of ATPsyn‐b resulted in growth defects in larval stage as the larvae did not grow bigger than the size of normal second‐instar larvae. Knockdown in testes did not interrupt the developmental excursion to viable adult flies, however, these male adults were sterile. Analyses of testes revealed disrupted nuclear bundles during spermatogenesis and abnormal shaping in spermatid elongation. There were no mature sperm in the seminal vesicle of ATPsyn‐b knockdown male testes. These findings suggest us that ATPsyn‐b acts in growth and male fertility of Drosophila.     

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.     

Gene expression analysis of the ovary of hybrid females of <Emphasis Type="Italic">Xenopus laevis</Emphasis> and <Emphasis Type="Italic">X. muelleri</Emphasis>

Background  

Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis) and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri). In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females.     

Genetics of Hybrid Inviability and Sterility in Drosophila: Dissection of Introgression of D. simulans Genes in D. melanogaster Genome

Interspecific crosses between Drosophila melanogaster and Drosophila simulans usually produce sterile unisexual hybrids. The barrier preventing genetic analysis of hybrid inviability and sterility has been taken away by the discovery of a D. simulans strain which produces fertile female hybrids. D. simulans genes in the cytological locations of 21A1 to 22C1-23B1 and 30F3-31C5 to 36A2-7 have been introgressed into the D. melanogaster genetic background by consecutive backcrosses. Flies heterozygous for the introgression are fertile, while homozygotes are sterile both in females and males. The genes responsible for the sterility have been mapped in the introgression. The male sterility is caused by the synergistic effect of multiple genes, while the female sterility genes have been localized to a 170 kb region (32D2 to 32E4) containing 20 open reading frames. Thus, the female sterility might be attributed to a single gene with a large effect. We have also found that the Lethal hybrid rescue mutation which prevents the inviability of male hybrids from the cross of D. melanogaster females and D. simulans males cannot rescue those carrying the introgression, suggesting that D. simulans genes maybe non-functional in this hybrid genotype. The genes responsible for the inviability have not been separated from the female sterility genes by recombination.     

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]     

Drosophila bipectinata species complex: study of phylogenetic relationship among four members through the analysis of morphology of testes and seminal vesicles

Species maintain their identity through reproductive isolating mechanisms, which are broadly classified into prezygotic and postzygotic isolating mechanisms. In the Drosophila bipectinata species complex, investigations were made on the degree of crossability (a prezygotic isolating mechanism) and the causes of hybrid male sterility (a postzygotic isolating mechanism) to analyse the phylogenetic relationship. Among the four species, D. bipectinata crosses with Drosophila parabipectinata freely in one direction and both of them also cross with Drosophila malerkotliana easily but it is difficult to cross all the three species with Drosophila pseudoananassae . In the hybrids involving D. pseudoananassae , no sperm were observed indicating high degree of perturbance during spermatogenesis while in the other hybrids immotile sperm were present indicating comparatively less disturbance during spermatogenesis. Testis size, which is an indicator of degree of perturbance during spermatogenesis and used as a proxy for sterility was measured in the four species and their hybrids. It was of same size in D. bipectinata , D. parabipectinata and D. malerkotliana but larger in D. pseudoananassae . In the hybrids involving D. pseudoananassae , testes were atrophied while in other hybrids it was larger. Since, the size of testis does not exhibit uniform pattern of variation in hybrids, it cannot be used as a good indicator for sterility. Therefore, we also measured the size of seminal vesicles (storing organ of sperm) in the four species and their hybrids. Interestingly, the size of seminal vesicles was reduced uniformly in all the hybrids indicating its use as better proxy for sterility. Further, the seminal vesicle size in D. pseudoananassae was smaller than that in the other three species. These observations provide evidence for phylogenetic proximity of D. bipectinata , D. parabipectinata and D. malerkotliana and their remote relationships with D. pseudoananassae .     

A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility

Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.     

Reproductive Isolation and Morphogenetic Evolution in Drosophila Analyzed by Breakage of Ethological Barriers

This article reports the breaking of ethological barriers through the constitution of soma-germ line chimeras between species of the melanogaster subgroup of Drosophila, which are ethologically isolated. Female Drosophila yakuba and D. teissieri germ cells in a D. melanogaster ovary produced functional oocytes that, when fertilized by D. melanogaster sperm, gave rise to sterile yakuba-melanogaster and teissieri-melanogaster male and female hybrids. However, the erecta-melanogaster and orena-melanogaster hybrids were lethal, since female D. erecta and D. orena germ cells in a D. melanogaster ovary failed to form oocytes with the capacity to develop normally. This failure appears to be caused by an altered interaction between the melanogaster soma and the erecta and orena germ lines. Germ cells of D. teissieri and D. orena in a D. melanogaster testis produced motile sperm that was not stored in D. melanogaster females. This might be due to incompatibility between the teissieri and orena sperm and the melanogaster seminal fluid. A morphological analysis of the terminalia of yakuba-melanogaster and teissieri-melanogaster hybrids was performed. The effect on the terminalia of teissieri-melanogaster hybrids of a mutation in doublesex, a regulatory gene that controls the development of the terminalia, was also investigated.     

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.     

Variable modes of inheritance of morphometrical traits in hybrids between Drosophila melanogaster and Drosophila simulans.

We investigated body-size inheritance in interspecific sterile hybrids by crossing a Drosophila simulans strain with 13 strains of Drosophila melanogaster, which were of various origins and chosen for their broad range of genetic variation. A highly significant parent-offspring correlation was observed, showing that the D. melanogaster genes for size are still expressed in a hybrid background. Superimposed on to this additive inheritance, the size of hybrids was always less than the mid-parent value. This phenomenon, which at first sight might be described as dominance or overdominance, is more precisely interpreted as a consequence of a hybrid breakdown, that is, a dysfunction of the parental genes for size when put to work together. This interpretation is enforced by the fact that phenotypic variability was much more prevalent in hybrids than in parents. We also analysed body pigmentation inheritance in the same crosses and got a very different picture. There was no increase in the phenotypic variance of F(1) hybrids and only a low parent-offspring correlation. Apparent overdominance could be observed but in opposite directions, with no evidence of hybrid breakdown. Our data point to the possibility of analysing a diversity of quantitative traits in interspecific hybrids, and indicate that breakdown might be restricted to some traits only.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.