First record of Stigmatomyces (Ascomycota: Laboulbeniales) on Drosophilidae from Japan.

Three Stigmatomyces species were detected on five drosophilid species from Japan. We report Stigmatomyces majewskii on Drosophila rufa and Drosophila suzukii, Stigmatomyces scaptodrosophilae on Scaptodrosophila coracina and Scaptodrosophila subtilis, and Stigmatomyces sacaptomyzae on Scaptomyza graminum. Except for Scaptomyza graminum, each of these species is a newly identified Stigmatomyces host. Our discovery that D. suzukii is a host of S. majewskii may provide new pest management approaches for this global agricultural pest insect.

Genetic Analyses of Elys Mutations in Drosophila Show Maternal-Effect Lethality and Interactions with Nucleoporin Genes.

ELYS determines the subcellular localizations of Nucleoporins (Nups) during interphase and mitosis. We made loss-of-function mutations of Elys in Drosophila melanogaster and found that ELYS is dispensable for zygotic viability and male fertility but the maternal supply is necessary for embryonic development. Subsequent to fertilization, mitotic progression of the embryos produced by the mutant females is severely disrupted at the first cleavage division, accompanied by irregular behavior of mitotic centrosomes. The Nup160 introgression from D. simulans shows close resemblance to that of the Elys mutations, suggesting a common role for those proteins in the first cleavage division. Our genetic experiments indicated critical interactions between ELYS and three Nup107-160 subcomplex components; hemizygotes of either Nup37, Nup96 or Nup160 were lethal in the genetic background of the Elys mutation. Not only Nup96 and Nup160 but also Nup37 of D. simulans behave as recessive hybrid incompatibility genes with D. melanogaster An evolutionary analysis indicated positive natural selection in the ELYS-like domain of ELYS. Here we propose that genetic incompatibility between Elys and Nups may lead to reproductive isolation between D. melanogaster and D. simulans, although direct evidence is necessary.

A Natural Population Derived from Species Hybridizationin the Drosophila ananassae Species Complexon Penang Island, Malaysia.

We surveyed natural population of the Drosophila ananassae species complex on Penang Island, Malaysia. Analyses of phenotypic traits, chromosome arrangements, molecular markers, and reproductive isolation suggest the existence of two species: D. ananassae and D. cf. parapallidosa. Molecular marker analysis indicates that D. cf. parapallidosa carries chromosome Y and 4 introgressions from D. ananassae. Thus, D. cf. parapallidosa seems to be a hybrid descendant that recently originated from a natural D. parapallidosaâ™€× D. ananassae♂ cross. Furthermore, D. cf. parapallidosa behaves differently from authentic D. parapallidosa with respect to its reproductive isolation from D. ananassae. Premating isolation is usually seen in only the D. ananassaeâ™€× D. parapallidosa♂ cross, but we observed it in crosses of both directions between D. ananassae and D. cf. parapallidosa. In addition, hybrid males from the D. ananassaeâ™€× D. parapallidosa♂ cross are usually sterile, but they were fertile when D. ananassae♀ were mated with D. cf. parapallidosa ♂. We attempted an artificial reconstruction of the hybrid species to simulate the evolutionary process(es) that produced D. cf. parapallidosa. This is a rare case of natural hybrid population in Drosophila and may be a useful system for elucidating speciation with gene flow.

A test of double interspecific introgression of nucleoporin genes in Drosophila.

In interspecific hybrids between Drosophila melanogaster and Drosophila simulans, the D. simulans nucleoporin-encoding Nup96(sim) and Nup160(sim) can cause recessive lethality if the hybrid does not also inherit the D. simulans X chromosome. In addition, Nup160(sim) leads to recessive female sterility in the D. melanogaster genetic background. Here, we conducted carefully controlled crosses to better understandthe relationship between Nup96(sim) and Nup160(sim). Nup96(sim) did not lead to female sterility in the D. melanogaster genetic background, and double introgression of Nup96(sim) and Nup160(sim) did not generally lead to lethality when one was heterozygous and the other homozygous (hemizygous). It appears that introgression of additional autosomal D. simulans genes is necessary to cause lethality and that the effect of the introgression is dominant to D. melanogaster alleles. Interestingly, the genetic background affected dominance of Nup96(sim), and double introgression carrying homozygous Nup96(sim) and hemizygous Nup160(sim) resulted in lethality. Thus, Nup96(sim) and Nup160(sim) seem to be two components of the same incompatibility.

Allelic asymmetry of the Lethal hybrid rescue (Lhr) gene expression in the hybrid between Drosophila melanogaster and D. simulans: confirmation by using genetic variations of D. melanogaster.

In the cross between Drosophila melanogaster females and D. simulans males, hybrid males die at the late larval stage, and the sibling females also die at later stages at high temperatures. Removing the D. simulans allele of the Lethal hybrid rescue gene (Lhr (sim) ) improves the hybrid incompatibility phenotypes. However, the loss-of-function mutation of Lhr (sim) (Lhr (sim0) ) does not rescue the hybrid males in crosses with several D. melanogaster strains. We first describe the genetic factor possessed by the D. melanogaster strains. It has been suggested that removing the D. melanogaster allele of Lhr (Lhr (mel) ), that is Lhr (mel0) , does not have the hybrid male rescue effect, contrasting to Lhr (sim0) . Because the expression level of the Lhr gene is known to be Lhr (sim) > Lhr (mel) in the hybrid, Lhr (mel0) may not lead to enough of a reduction in total Lhr expression. Then, there is a possibility that the D. melanogaster factor changes the expression level to Lhr (sim) < Lhr (mel) . But in fact, the expression level was Lhr (sim) > Lhr (mel) in the hybrid irrespectively of the presence of the factor. At last, we showed that Lhr (mel0) slightly improves the viability of hybrid females, which was not realized previously. All of the present results are consistent with the allelic asymmetry model of the Lhr gene expression in the hybrid.

Genetic decay of balancer chromosomes in Drosophila melanogaster.

Theoretical considerations predict that balancer chromosomes in Drosophila melanogaster should accumulate numerous deleterious mutations with time. We counted the number of recessive lethal mutations on two balancer chromosomes from the In(2LR)SM1/In(2LR)Pm strain maintained in our lab, after making the balancers heterozygous with deficiencies from second-chromosome Kyoto Deficiency kit strains. We detected 10 recessive lethal mutations in the balancer In(2LR)Pm, which is consistent with the mutation rate estimated previously. However, we detected only three mutations, a significantly smaller number, in the balancer In(2LR)SM1, although this may be an artifact. In conclusion, we observed genetic decay over an estimable timescale by using balancers with historical records. Thus, balancers of any strain may have accumulated many unidentified recessive lethal mutations.

Genetic dissection of Nucleoporin 160 (Nup160), a gene involved in multiple phenotypes of reproductive isolation in Drosophila.

Previous reports have suggested that the Nucleoporin 160 (Nup160) gene of Drosophila simulans (Nup160(sim)) causes the hybrid inviability, female sterility, and morphological anomalies that are observed in crosses with D. melanogaster. Here we have confirmed this observation by transposon excision from the P{EP}Nup160(EP372) insertion mutation of D. melanogaster. Null mutations of the Nup160 gene resulted in the three phenotypes caused by Nup160(sim), but revertants of the gene did not. Interestingly, several mutations produced by excision partially complemented hybrid inviability, female sterility, or morphological anomalies. In the future, these mutations will be useful to further our understanding of the developmental mechanisms of reproductive isolation. Based on our analyses with the Nup160(sim) introgression line, the lethal phase of hybrid inviability was determined to be during the early pupal stage. Our analysis also suggested that homozygous Nup160(sim) in D. melanogaster leads to slow development. Thus, Nup160(sim) is involved in multiple aspects of reproductive isolation between these two species.

Chromatin evolution and molecular drive in speciation.

Are there biological generalities that underlie hybrid sterility or inviability? Recently, around a dozen "speciation genes" have been identified mainly in Drosophila, and the biological functions of these genes are revealing molecular generalities. Major cases of hybrid sterility and inviability seem to result from chromatin evolution and molecular drive in speciation. Repetitive satellite DNAs within heterochromatin, especially at centromeres, evolve rapidly through molecular drive mechanisms (both meiotic and centromeric). Chromatin-binding proteins, therefore, must also evolve rapidly to maintain binding capability. As a result, chromatin binding proteins may not be able to interact with chromosomes from another species in a hybrid, causing hybrid sterility and inviability.

Copulatory courtship behavior and sine song as a mate recognition cue in Drosophila lini and its sibling species.

Most Drosophila species sing species-specific pulse songs during their "precopulatory courtship." Three sibling species of the Drosophila montium species subgroup performed "copulatory courtship": males generated courtship songs by vibrating either wing only after mounting and during copulation. In these three species, strong sexual isolation was detected between D. ohnishii and D. lini and between D. ohnishii and D. ogumai, but not between D. lini and D. ogumai. Female showed strong repelling behavior when they were mounted by a heterospecific male in the species combinations including D. ohnishii, resulting in failure of the copulation attempt of the male. Acoustic analyses of courtship songs revealed that the pulse song was irregular, without any species-specific parameters, but that the frequency of the sine song was different among the three species in accordance with the modes of sexual isolation between them; it was significantly lower in D. ohnishii (mean ± SE = 193.0 ± 1.7 Hz) but higher in D. lini (253.4 ± 2.7 Hz) and D. ogumai (246.7 ± 5.3 Hz). We suggest that this difference in the sine song frequency is a sexual signal in the Specific Mate Recognition System (SMRS) among these three Drosophila species.

Introgression of Drosophila simulans nuclear pore protein 160 in Drosophila melanogaster alone does not cause inviability but does cause female sterility.

We have been analyzing genes for reproductive isolation by replacing Drosophila melanogaster genes with homologs from Drosophila simulans by interspecific backcrossing. Among the introgressions established, we found that a segment of the left arm of chromosome 2, Int(2L)S, carried recessive genes for hybrid sterility and inviability. That nuclear pore protein 160 (Nup160) in the introgression region is involved in hybrid inviability, as suggested by others, was confirmed by the present analysis. Male hybrids carrying an X chromosome of D. melanogaster were not rescued by the Lethal hybrid rescue (Lhr) mutation when the D. simulans Nup160 allele was made homozygous or hemizygous. Furthermore, we uniquely found that Nup160 is also responsible for hybrid sterility. Females were sterile when D. simulans Nup160 was made homozygous or hemizygous in the D. melanogaster genetic background. Genetic analyses indicated that the D. simulans Nup160 introgression into D. melanogaster was sufficient to cause female sterility but that other autosomal genes of D. simulans were also necessary to cause lethality. The involvement of Nup160 in hybrid inviability and female sterility was confirmed by transgene experiment.

Evolutionary relationships in the Drosophila ananassae species cluster based on introns of multiple nuclear loci.

The Drosophila ananassae species cluster includes D. ananassae, D. pallidosa, D. parapallidosa, and the cryptic species "pallidosa-like", "pallidosa-like Wau" and "papuensis-like" Some of the taxa are sympatric in the South Pacific, Papua New Guinea, and Southeast Asia, and gene flow between different taxa has been suspected for a handful of genes. In the present analysis, we examined DNA sequences of introns in four loci: alpha actinin (Actn) on XL, white (w) on XR, CG7785 on 2L, and zinc ion transmembrane transporter 63C (ZnT63C) on 2R. Phylogenetic trees (neighbor-joining and haplotype network) were inconsistent among these loci. Some haplotypes shared between taxa were found for w, CG7785, and ZnT63C, suggesting recent gene flow. However, no haplotypes were shared, for example, between D. ananassae and D. pallidosa for CG7785, which is close to the proximal breakpoint of In(2L)D. This suggests that taxon-specific inversions prevent gene flow, as predicted by the chromosomal speciation hypothesis.

Potential gene flow in natural populations of the Drosophila ananassae species cluster inferred from a nuclear mitochondrial pseudogene.

A pseudogene with 94% similarity to mitochondrial cytochrome c oxidase subunit I (COI) was identified and localized to chromosome 4 of Drosophila ananassae. Because this chromosome is believed to have reduced recombination, its history can be traced using the pseudo-COI sequence. Pseudo-COI sequences were obtained from 27 iso-female lines of six taxa belonging to the D. ananassae species cluster in which reproductive isolation is incomplete. The phylogenetic network constructed from seven recognized haplotypes (#0-#6) indicated that different taxa inhabiting the same geographic area share the haplotypes: #1 from Papua New Guinean populations of D. ananassae and pallidosa-like-Wau; #2 from Papua New Guinean populations of D. ananassae, pallidosa-like, and papuensis-like; and #4 from South Pacific populations of D. ananassae and D. pallidosa. Taxon-K has a unique haplotype (#6), and 18 mutation steps separate it from the closest haplotype, #2. We discuss the possibility of chromosome 4 introgression beyond taxon boundaries.

Genetic analysis of female mating recognition between Drosophila ananassae and Drosophila pallidosa: application of interspecific mosaic genome lines.

Drosophila ananassae and Drosophila pallidosa are closely related species that can produce viable and fertile hybrids of both sexes, although strong sexual isolation exists between the two species. Females are thought to discriminate conspecific from heterospecific males based on their courtship songs. The genetic basis of female discrimination behavior was analyzed using isogenic females from interspecific mosaic genome lines that carry homozygous recombinant chromosomes. Multiple regression analysis indicated a highly significant effect of the left arm of chromosome 2 (2L) on the willingness of females to mate with D. ananassae males. Not only 2L but also the left arm of chromosome X (XL) and the right arm of chromosome 3 (3R) had significant effects on the females' willingness to mate with D. pallidosa males. All regions with strong effects on mate choice have chromosome arrangements characterized by species-specific inversions. Heterospecific combinations of 2L and 3R have previously been suggested to cause postzygotic reproductive isolation. Thus, genes involved in premating as well as postmating isolation are located in or near chromosomal inversions. This conclusion is consistent with the recently proposed hypothesis that "speciation genes" accumulate at a higher rate in non-recombining genome regions when species divergence occurs in the presence of gene flow.

Establishing interspecific mosaic genome lines between Drosophila ananassae and Drosophila pallidosa by means of parthenogenesis.

Strong sexual isolation exists between the closely related species Drosophila ananassae and D. pallidosa, but there is no obvious post-mating isolation; both sexes of the hybrids and their descendants appear to be completely viable and fertile. Strains exhibiting parthenogenesis have been derived from wild populations of both species. We intercrossed such strains and established iso-female lines after the second generation of parthenogenesis. These lines are clones, carrying homozygous chromosomes that are interspecific recombinants. We established 266 such isogenic lines and determined their genetic constitution by using chromosomal and molecular markers. Strong pseudo-linkage was seen between loci on the left arm of chromosome 2 and on the right arm of chromosome 3; the frequency of inheriting the two chromosome regions from the same species was significantly larger than expected. One possible cause of pseudo-linkage is female meiotic bias, so that chromosomes of the same species origin tend to be distributed to the same gamete. But this possibility is ruled out; backcross analysis indicated that the two chromosome regions segregated independently in female hybrids. The remaining possibility is elimination of low-fitness flies carrying the two chromosome regions from different species. Thus, genetic incompatibility was detected in the species pair for which no hybrid breakdown had previously been indicated. The 'interspecific mosaic genome' lines reported here will be useful for future research to identify genes involved in speciation and phenotypic evolution.

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.

Genetic complexity underlying hybrid male sterility in Drosophila.

Recent genetic analyses of closely related species of Drosophila have indicated that hybrid male sterility is the consequence of highly complex synergistic effects among multiple genes, both conspecific and heterospecific. On the contrary, much evidence suggests the presence of major genes causing hybrid female sterility and inviability in the less-related species, D. melanogaster and D. simulans. Does this contrast reflect the genetic distance between species? Or, generally, is the genetic basis of hybrid male sterility more complex than that of hybrid female sterility and inviability? To clarify this point, the D. simulans introgression of the cytological region 34D-36A to the D. melanogaster genome, which causes recessive male sterility, was dissected by recombination, deficiency, and complementation mapping. The 450-kb region between two genes, Suppressor of Hairless and snail, exhibited a strong effect on the sterility. Males are (semi-)sterile if this region of the introgression is made homozygous or hemizygous. But no genes in the region singly cause the sterility; this region has at least two genes, which in combination result in male sterility. Further, the males are less fertile when heterozygous with a larger introgression, which suggests that dominant modifiers enhance the effects of recessive genes of male sterility. Such an epistatic view, even in the less-related species, suggests that the genetic complexity is special to hybrid male sterility.

The minimal interspecific introgression resulting in male sterility in Drosophila.

Introgression of Drosophila simulans genes into the Drosophila melanogaster genome provides an ideal system for analysing genetic incompatibility between species. Females and males homozygous for the introgression Int(2L)S (cytologically, 30F3-31C5 to 36A2-7) are sterile. Genetic dissection of the proximal part of the introgression (34D1-3 to 36A2-7) has indicated that introgressions of 0.7-1.6 Mb size result in male sterility when homozygous. In the present analysis we examine the distal part of the introgression (30F3-31C to 34D1-3) and reveal that introgressions with similar DNA content (1.8-2.1 Mb) result in male sterility. Compared with introgressions between the more closely related species Drosophila mauritiana and D. simulans, the minimal introgression resulting in male sterility is smaller by several-fold.