Mitotic progression and dual spindle formation caused by spindle association of de novo-formed microtubule-organizing centers in parthenogenetic embryos of Drosophila ananassae.

Facultative parthenogenesis occurs in many animal species that typically undergo sexual reproduction. In Drosophila, such development from unfertilized eggs involves diploidization after completion of meiosis, but the exact mechanism remains unclear. Here we used a laboratory stock of Drosophila ananassae that has been maintained parthenogenetically to cytologically examine the initial events of parthenogenesis. Specifically, we determined whether the requirements for centrosomes and diploidization that are essential for developmental success can be overcome. As a primal deviation from sexually reproducing (i.e. sexual) strains of the same species, free asters emerged from the de novo formation of centrosome-like structures in the cytosol of unfertilized eggs. Those microtubule-organizing centers had distinct roles in the earliest cycles of parthenogenetic embryos with respect to mitotic progression and arrangement of mitotic spindles. In the first cycle, an anastral bipolar spindle self-assembled around a haploid set of replicated chromosomes. Participation of at least one microtubule-organizing center in the spindle was necessary for mitotic progression into anaphase. In particular, the first mitosis involving a monastral bipolar spindle resulted in haploid daughter nuclei, one of which was associated with a microtubule-organizing center whereas the other was not. Remarkably, in the following cycle, biastral and anastral bipolar spindles formed that were frequently arranged in tandem by sharing an aster with bidirectional connections at their central poles. We propose that, for diploidization of haploid nuclei, unfertilized parthenogenetic embryos utilize dual spindles during the second mitosis, as occurs for the first mitosis in normal fertilized eggs.

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.

Male recombination in Brazilian populations of Drosophila ananassae.

With few exceptions, spontaneous crossing over does not normally occur in male Drosophila. Drosophila ananassae males show considerable amounts of crossing over. In wild males of D. ananassae from Asian (2008) and Brazilian populations (1986 and 2007) variable frequencies of meiotic crossing over, estimated from chiasmata counts, suggested the existence of factors controlling male crossing over in these populations. To corroborate for such prediction, we present data on spontaneous recombination in F1 males of D. ananassae heterozygous for chromosomes of the same Brazilian populations (1986) and marker chromosomes using three testers stocks. Mean recombination value was low, although high variability existed between individual frequencies. Recombination frequencies between lines in each tester stock were not significantly different, excepting when the 3ple-px and 3ple-cy testers were compared (p < 0.05). These two testers differ in respect to the regional distribution of crossovers. The occurrence of recombination in chromosomes 2 and 3 in F1 males tested with e(65) se; bri ru was not related, suggesting they are under independent genetic control. Our data are consistent with proposed genetic factors controlling male crossing over in the tester stocks and to the presence of enhancers and suppressors of male crossing over segregating in the Brazilian populations (1986).

Crossing over does occur in males of Drosophila ananassae from natural populations.

Spontaneous crossing over in males of Drosophila ananassae has been well demonstrated using F(1) individuals from crosses between marker stocks and wild type strains. However, the question of its occurrence in males from natural populations remained open. Here we present the cytological evidence that crossing over does occur in males of D. ananassae from two Brazilian populations, sampled nearly 21 years apart, and in two recently sampled populations, one from Indonesia and one from Okinawa, Japan. Cytological analysis of meiosis in males collected from nature and in sons of females from the same population inseminated in nature revealed the presence of chiasmata, inversion chiasmata, and isosite chromosome breakages in the diplotene cells in all sampled populations. These data demonstrate that reciprocal and nonreciprocal exchanges and chromosome breakages, previously reported as related events of male crossing over, do occur at variable frequencies among males from natural populations.

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.

Evolution in the Drosophila ananassae species subgroup.

Drosophila ananassae and its relatives have many advantages as a model of genetic differentiation and speciation. In this report, we examine evolutionary relationships in the ananassae species subgroup using a multi-locus molecular data set, karyotypes, meiotic chromosome configuration, chromosomal inversions, morphological traits, and patterns of reproductive isolation. We describe several new taxa that are the closest known relatives of D. ananassae. Analysis of Y-chromosomal and mitochondrial haplotypes, shared chromosome arrangements, pre-mating isolation and hybrid male sterility suggests that these taxa represent a recent evolutionary radiation and may experience substantial gene flow. We discuss possible evolutionary histories of these species and give a formal description of one of them as D. parapallidosa Tobari sp. n. The comparative framework established by this study, combined with the recent sequencing of the D. ananassae genome, will facilitate future studies of reproductive isolation, phenotypic variation and genome evolution in this lineage.

BAC library construction and BAC end sequencing of five Drosophila species: the comparative map with the D. melanogaster genome.

We constructed and characterized arrayed bacterial artificial chromosome (BAC) libraries of five Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. auraria, and D. ananassae), which are genetically well characterized in the studies of meiosis, evolution, population genetics, and developmental biology. The BAC libraries comprise 8,000 to 12,500 clones for each species, estimated to cover the most of the genomes. We sequenced both ends of most of these BAC clones with a success rate of 91%. Of these, 53,701 clones consisting of non-repetitive BAC end sequences (BESs) were mapped with reference of the public D. melanogaster genome sequences. The BES mapping estimated that the BAC libraries of D. auraria and D. ananassae covered 47% and 57% of the D. melanogaster genome, respectively, and those of D. melanogaster, D. sechellia, and D. simulans covered 94-97%. The low coverage by BESs of D. auraria and D. ananassae may be due to the high sequence divergence with D. melanogaster. From the comparative BES mapping, 111 possible breakpoints of chromosomal rearrangements were identified in these four species. The breakpoints of the major chromosome rearrangement between D. simulans and D. melanogaster on the third chromosome were determined within 20 kb in 84E and 30 kb in 93E/F. Corresponding breakpoints were also identified in D. sechellia. The BAC clones described here will be an important addition to the Drosophila genomic resources.

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.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

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.

Chiasmata and chromosome breakages are related to crossing over in Drosophila ananassae males.

A cytogenetic analysis of male crossing over in Drosophila ananassae revealed that cytological exchanges resulted in genetic crossing over, and that chiasma frequency and the genetic recombination correlated positively in chromosomes 2 and 3. Furthermore, the frequency of chromosome breakages correlated positively with chiasma frequency. Paracentric inversion heterozygosity had no detectable influence on the chromosome pairing or exchange events within the inversion loop at meiosis. Scoring of the chiasma demonstrated that males homozygous for the previously mapped enhancers of male crossing over had low frequencies of chiasmata, whereas higher frequencies of chiasmata were observed in males heterozygous for enhancers. The results presented here indicate that the genetic factors controlling male crossing over are involved in the origin of chromosome breakages and in exchange events.

Reproductive isolation among geographical populations of Drosophila bipectinata Duda (Diptera, Drosophilidae) with recognition of three subspecies.

Among D. bipectinata Duda, 1923, three subspecies, bipectinata from Southeast Asia (SEA) and Okinawa (OKN), szentivanii stat. nov. from Papua New Guinea (PNG) and pacificiae ssp. nov. from South Pacific Ocean (SPO), are recognized. The external morphology of the reproductive organs and the numbers of teeth per row in the sex combs are different between the three subspecies. Furthermore, the sterility of hybrid males between strains from the different regions confirms the subspecies status of each population from SEA, PNG and SPO, together with different gene arrangements in the geographical populations. Although males of the strains from OKN (Okinawa), the northernmost population, show significant differences in the number of teeth of sex combs from males of SEA (Southeast Asia) strains, hybrid males between them are fertile.

Chromosomal phylogeny and geographical divergence in the Drosophila bipectinata complex.

We have prepared reference polytene photographic maps as a standard sequence for the Drosophila bipectinata complex using structurally homozygous flies derived from a stock of Drosophila parabipectinata from Brunei, Borneo, in 1971. We found 87 inversions in the D. bipectinata complex and described their breakpoints on the reference maps. Only 2 arrangements were shared interspecifically: 2R-AB was shared with 3 species, D. parabipectinata, D. bipectinata, and Drosophila malerkotliana, and 3L-A was found in 2 species, D. parabipectinata and D. malerkotliana. The 2 subspecies of D. malerkotliana and the 2 subspecies of Drosophila pseudoananassae shared half of the total gene arrangements detected in each species. The number of different inversions found between species in the complex ranges from 7 (between D. parabipectinata and D. malerkotliana) to at least 24 (between D. bipectinata and D. pseudoananassae). On the basis of the characteristic differences of their gene arrangements, we propose a reliable chromosomal phylogeny of the D. bipectinata complex.

Genetic analyses of several Drosophila ananassae-complex species show a low-frequency major gene for parthenogenesis that maps to chromosome 2.

Parthenogenetic strains of several species have been found in the genus Drosophila. The mode of diploidization in the eggs of females has been found to be post-meiotic nuclear fusion. The genetic basis for this parthenogenesis is not understood but is believed to be under the control of a complex polygenic system. We found parthenogenetic females in an isofemale strain (LAE345) of D. pallidosa-like collected in 1981 at Lae, Papua New Guinea, and established a parthenogenetically reproducing strain. Parthenogenetic strains of D. ananassae and D. pallidosa collected at Taputimu, American Samoa had also been established by Futch (1972). D. ananassae, D. pallidosa and D. pallidosa-like are very closely related species belonging to the ananassae complex of the ananassae species subgroup of the melanogaster species group. Using these three species, we found that more than 80% of females from parthenogenetic strains produced progeny parthenogenetically and that inter-specific hybrid females also produced impaternate progeny. In the present report, we demonstrate that the mode of parthenogenesis of D. ananassae appears to be the post-meiotic nuclear doubling of a single meiotic product, and that a major gene responsible for the parthenogenesis maps to the left arm of the second chromosome of D. ananassae. We also suggest that the genetic basis for parthenogenesis capacity may be identical among the three closely related species. We discuss the function of the gene required for parthenogenesis and its significance for the evolutionary process.

Search for species-specific mating signal in courtship songs of sympatric sibling species, Drosophila ananassae and D. pallidosa.

Sexual isolation is one of the most important mechanisms that may lead to speciation. Drosophila ananassae and D. pallidosa are useful for the study of sexual isolation, because of their sympatric distribution and no postmating isolation between them. Courtship songs are considered to play a crucial role in sexual isolation between D. ananassae and D. pallidosa. We recorded and analyzed male courtship songs of D. ananassae and D. pallidosa for eight and four geographical strains, respectively. Courtship behaviors of the two species were consistent with those previously described, however, male's middle leg shaking, which had not before been described, was observed in both species. Males sing by wing vibration only during courtship. Their song oscillograms were distinct between species, but those of conspecific strains were very similar, in spite of their different geographical derivation. We found species-specificity in burst length, pulse length, cycle number in a pulse, and frequency spectra of bursts; these results suggest that these song parameters may play a role in mate recognition that enforces their sexual isolation. The specific values of interpulse interval, cycle number in a pulse and intrapulse frequency were involved with the determination of specificity in frequency spectra of bursts. We discussed the possibility that the specific frequency spectra of bursts are recognized by females as the species-specific signal rather than each parameter individually.

Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa.

Sexual isolation has been considered one of the primary causes of speciation and its genetic study has the potential to reveal the genetics of speciation. In Drosophila, the importance of courtship songs in sexual isolation between closely related species has been well investigated, but studies analysing the genetic basis of the difference in the courtship songs associated with sexual isolation are less well documented. Drosophila ananassae and Drosophila pallidosa are useful for studies of sexual isolation, because of their sympatric distribution and absence of postmating isolation. Courtship songs are known to play a crucial role in sexual isolation between these two species, and the female discrimination behaviour against the courting male has been revealed to be controlled by a very narrow region on the second chromosome. In this study we investigated the genetic basis controlling the song differences associated with their sexual isolation, using intact and wingless males with chromosomes substituted between species. The results obtained from F1 hybrid males between these species indicate the dominance of the song characters favoured by D. pallidosa females. In addition, the results obtained from backcross F2 males indicate that chromosome 2 had a major effect on the control of the song characters associated with sexual isolation.