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.     

Population genetics of Drosophila ananassae: genetic differentiation among Indian natural populations at the level of inversion polymorphism

The present study, which is one of the longest temporal (two decades) and largest spatial (different parts of India covered) investigations on inversion polymorphism in natural populations of D. ananassae, was undertaken to understand the dynamics of inversion polymorphism in a broad and comprehensive manner. Forty-five natural populations from different ecogeographic regions of the country (covering the regions from Kashmir to Kanniyakumari and Gujarat to Nagaland) were analysed for chromosomal inversions. All the populations show the presence of the three cosmopolitan inversions, frequencies of which vary among the populations analysed. Simple correlations between frequencies of different inversions and regression analysis of inversion frequencies with latitude, longitude and altitude were insignificant. This reinforces the concept of rigid polymorphism in D. ananassae. Genetic divergence (spatial and temporal) at the level of chromosomal polymorphism among natural populations was calculated. Results show spatial divergence but no temporal divergence. Rigid polymorphic systems of D. ananassae did not show long-term directional trends. On the basis of the present study, and after including comparisons with the studies conducted more than two decades ago, the most important conclusion to be drawn is that the three cosmopolitan inversions in D. ananassae segregate within populations at fairly similar frequencies, and the general geographic pattern has remained constant.     

Intra-and interchromosomal effects of heterozygous inversions on recombination in the third chromosome of Drosophila ananassae

In order to study intra-and interchromosomal effects of heterozygous inversions on recombination in the third chromosome of D.ananassae, experiments were conducted using Stw-pr marker stock and five wild stocks with known karyotypes. The stocks used were homozygous for standard or inverted gene sequence in 2L, 3L, and 3R. Recombination was investigated in both sexes. There was complete absence of crossing-over in males in all the experiments which appeared to be the characteristic of marker stock as spontaneous male crossing-over was reported earlier with the same wild stocks when the second chromosome markers were used. Based on the data of karyotypically homozygous F1 females, the map distance between stw-pr was 36.55 map units. The heterozygosity due to a lengthy inversion in 2L increased the level of crossing-over between stw-pr genes of the third chromosome indicating interchromosomal effect. There was a considerable reduction in the rate of recombination between the same markers due to inversion heterozygosity in 3R indicating intrachromosomal effect. However, 3L inversion heterozygosity had no effect on crossover rate. These results provide evidence for intra-and interchromosomal effects of inversions on crossing-over in the third chromosome of D. ananassae.     

Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster

The evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sex-linked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterized by intra-species variation in hybridization signal size, appeared on the fourth chromosome related to heterochromatic rearrangements. Nucleolar material is thought to be required for sex chromosome pairing and disjunction in a variety of organisms including Drosophila. Thus, either remnant sequences, possibly intergenic spacer repeats, are still present in the sex chromosomes which have lost their NORs (as observed in D. simulans and D. sechellia), or an alternative mechanism has evolved.     

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.     

Intra- and interspecies variations in pupation height in Drosophila.

In order to study the pupation site preference which is an important component of larval behaviour, pupation height was scored in Drosophila ananassae, D. bipectinata and D. malerkotliana by using a large number of strains of these species. The mean pupation height of different strains ranged from 1.1 to 8.7 mm in D. ananassae, from 0.41 to 0.75 mm in D. bipectinata and from 1.0 to 1.3 mm in D. malerkotliana. The analysis of variance and t-test were performed to test intre- and interspecies variations in pupation height. These tests revealed significant variation among three species. Significant variations among different strains of the same species were also found in D. ananassae and D. bipectinata. These observations provide evidence for intra- and interspecies variations in pupation height in Drosophila. Variations among different strains of the same species in pupation height are attributable to genetic heterogeneity among the strains.     

Changes in the recombinational environment affect divergence in the yellow gene of Drosophila

The complete coding region of the yellow (y) gene was sequenced in different Drosophila species. In the species of the melanogaster subgroup (D. melanogaster, D. simulans, D. mauritiana, D. yakuba, and D. erecta), this gene is located at the tip of the X chromosome in a region with a strong reduction in recombination rate. In contrast, in D. ananassae (included in the ananassae subgroup of the melanogaster group) and in the obscura group species (D. subobscura, D. madeirensis, D. guanche, and D. pseudoobscura), the y gene is located in regions with normal recombination rates. As predicted by the hitchhiking and background selection models, this change in the recombinational environment affected synonymous divergence in the y-gene-coding region. Estimates of the number of synonymous substitutions per site were much lower between the obscura group species and D. ananassae than between the species of the obscura group and the melanogaster subgroup. In fact, a highly significant increase in the rate of synonymous substitution was detected in all lineages leading to the species of the melanogaster subgroup relative to the D. ananassae lineage. This increase can be explained by a higher fixation rate of mutations from preferred to unpreferred codons (slightly deleterious mutations). The lower codon bias detected in all species of the melanogaster subgroup relative to D. ananassae (or to the obscura group species) would be consistent with this proposal. Therefore, at least in Drosophila, changes in the recombination rate in different lineages might cause deviations of the molecular-clock hypothesis and contribute to the overdispersion of the rate of synonymous substitution. In contrast, the change in the recombinational environment of the y gene has no detectable effect on the rate of amino acid replacement in the Yellow protein.     

Behavioral genetics of Drosophila ananassae

Drosophila ananassae has a unique status among Drosophila species because of certain peculiarities in its genetic behavior. The most unusual feature of this species is its relatively high frequency of spontaneous male recombination. The results of studies on non-sexual behavior, such as phototactic responses, eclosion rhythm, and preferences for oviposition and pupation sites, lead us to suggest that this behavior is under polygenic control, with a substantial amount of additive genetic variation. Sexual isolation has been reported in D. ananassae with the degree of such isolation being stronger in isofemale lines than in natural populations. The significant variations seen in the mating propensity of several isofemale strains, inversion karyotypes and wild type strains, the diminishing effects of certain mutations on the sexual activity of males, and the positive responses to selection for high and low mating propensity point to a genetic control of sexual behavior in D. ananassae. Males contribute more to variation and thus are more subject to intrasexual selection than females. There is a positive correlation between sternopleural bristle number, mating propensity and fertility in D. ananassae. This correlation between morphometric traits and mating success suggests that larger flies are more successful in mating than smaller ones. There is also evidence for adaptive plasticity and a trade-off between longevity and productivity in D. ananassae. Rare, specific courtship song parameters that provide males with a mating advantage have also been reported in different geographic strains of D. ananassae. The remating behavior of males and females, sperm displacement, and the bi-directional selection for female remating speed indicate that post-mating behavior in this species may also be under genetic control. The occurrence of size assortative mating further indicates that there is size-dependent sexual selection in D. ananassae.     

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.     

Investigations on gene copy number, introns and chromosomal arrangement of genes encoding the fucoxanthin chlorophyll a/c-binding proteins of the centric diatom Cyclotella cryptica

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.     

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.     

Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera

Transposable elements of the mariner family are widespread among insects and other invertebrates, and initial analyses of their relationships indicated frequent occurrence of horizontal transfers between hosts. A specific PCR assay was used to screen for additional members of the irritans subfamily of mariners in more than 400 arthropod species. Phylogenetic analysis of cloned PCR fragments indicated that relatively recent horizontal transfers had occurred into the lineages of a fruit fly Drosophila ananassae, the horn fly Haematobia irritans, the African malaria vector mosquito Anopheles gambiae, and a green lacewing Chrysoperla plorabunda. Genomic dot-blot analysis revealed that the copy number in these species varies widely, from about 17,000 copies in the horn fly to three copies in D. ananassae. Multiple copies were sequenced from genomic clones from each of these species and four others with related elements. These sequences confirmed the PCR results, revealing extremely similar elements in each of these four species (greater than 88% DNA and 95% amino acid identity). In particular, the consensus sequence of the transposase gene of the horn fly elements differs by just two base pairs out of 1,044 from that of the lacewing elements. The mosquito lineage has diverged from the other Diptera for over 200 Myr, and the neuropteran last shared a common ancestor with them more than 265 Myr ago, so this high similarity implies that these transposons recently transferred horizontally into each lineage. Their presence in only the closest relatives in at least the lacewing lineage supports this hypothesis. Such horizontal transfers provide an explanation for the evolutionary persistence and widespread distribution of mariner transposons. We propose that the ability to transfer horizontally to new hosts before extinction by mutation in the current host constitutes the primary selective constraint maintaining the sequence conservation of mariners and perhaps other DNA-mediated elements.      

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.     

Patterns of inversion polymorphism in three species of the Drosophila melanogaster species group

In Drosophila, chromosomal polymorphism due to paracentric inversions is very common and constitutes an adaptive character. The degree of chromosomal variability varies in different species and also in different populations of the same species. Chromosomal polymorphism in Indian natural populations of three species, D. melaonogaster, D. ananassae and D. bipectinata which belong to the melanogaster species group has been studied and the quantitative data on frequency of inversions have been reported. Behaviour of chromosome inversions has also been studied in laboratory conditions. The present review summarises the work done on inversion polymorphism in Indian populations of three species which clearly demonstrates that these three species vary in their patterns of inversion polymorphism and have evolved different mechanisms for adjustment to their environments although they belong to the same species group.     

Imprinted genes: identification by chromosome rearrangements and post-genomic strategies

Imprinted genes are differentially expressed from the maternally and paternally inherited alleles. Accordingly, inheritance of both copies of an imprinted chromosome or region from a single parent leads to the mis-expression of the imprinted genes present in the selected region. Strains of mice with reciprocal and Robertsonian chromosomal translocations or mice with engineered chromosomal rearrangements can be used to produce progeny where both copies of a chromosomal region are inherited from one parent. In combination with systematic differential expression and methylation-based approaches, these mice can be used to identify novel imprinted genes. Advances in genome sequencing and computer-based technologies have facilitated this approach to finding imprinted genes.     

Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.     

Investigations on Gene Copy Number, Introns and Chromosomal Arrangement of Genes Encoding the Fucoxanthin Chlorophyll a/c-Binding Proteins of the Centric Diatom Cyclotella cryptica

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.     

Genomewide comparative analysis of the highly abundant transposable element DINE-1 suggests a recent transpositional burst in Drosophila yakuba

DINE-1 (Drosophila interspersed element) is the most abundant repetitive sequence in the Drosophila genome derived from transposable elements. It comprises >1% of the Drosophila melanogaster genome (DMG) and is believed to be a relic from an ancient transpositional burst that occurred approximately 5-10 MYA. We performed a genomewide comparison of the abundance, sequence variation, and chromosomal distribution of DINE-1 in D. melanogaster and D. yakuba. Unlike the highly diverged copies in the DMG (pairwise distance approximately 15%), DINE-1's in the Drosophila yakuba genome (DYG) have diverged by only 3.4%. Moreover, the chromosomal distribution of DINE-1 in the two species is very different, with a significant number of euchromatic insertions found only in D. yakuba. We propose that these different patterns are caused by a second transpositional burst of DINE-1's in the D. yakuba genome approximately 1.5 MYA. On the basis of the sequence of these recently transposed copies, we conclude that DINE-1 is likely to be a family of nonautomomous DNA transposons. Analysis of the chromosomal distribution of two age groups of DINE-1's in D. yakuba indicates that (1) there is a negative correlation between recombination rates and the density of DINE-1's and (2) younger copies are more evenly distributed in the chromosome arms, while older copies are mostly located near the centromere regions. Our results fit the predictions of a selection-transposition balance model. Our data on whole-genome comparison of a highly abundant TE among Drosophila sibling species demonstrate the unexpectedly dynamic nature of TE activity in different host genomes.     

Tracing the evolution of H-2 D region genes using sequences associated with a repetitive element

The class I genes in the murine MHC are genetically divided into the K, D, Qa, and T1a region subfamilies. These genes presumably arose by duplication from a common class I ancestor. Oligonucleotide probes specific for sequences associated with a moderately repetitive B2 SINE element, which is inserted into the 3' untranslated region of the H-2D and H-2L genes, were used to examine the evolutionary relationship between these classically defined D region genes (H-2D and H-2L) and the other members of the class I gene family. Hybridization analyses of recombinant cosmid and genomic DNA indicated that the D region genes separated genetically from the other members of the class I gene family 12 to 14 million years ago. The evidence suggests that during this time frame the chromosomal segment harboring the characteristic insertion became fixed in the ancestral population which gave rise to Mus domesticus. Previous studies have shown that the number of genes present in the Qa and T1a regions varies among inbred strains and among laboratory stocks of wild mice derived from more distant species on the genus Mus. No evidence was found in this study to support the hypothesis that variation in class I gene number is the result of recent duplications of the functionally defined class I genes of the D region, H-2D and H-2L.