Characterization of the complete mitochondrial genome of flower-breeding <Emphasis Type="Italic">Drosophila incompta</Emphasis> (Diptera,Drosophilidae)

Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A?T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis–repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.     

Transfer of mitochondrial DNA to nuclear genome of cells of passaged cell line of Drosophila virilis

The variability of the chromosomal fragments of the atp6 mitochondrial gene, which is integrated into chromosomal DNA in the lines of flies of different geographic origins and in the passaged cell lines of D. virilis has been analyzed. We did not reveal any nucleotide variability in this DNA marker among the studied fly lines. This result is consistent with the proposition that the D. virilis species is monomorphic. The new fragments of the atp6 gene that are associated with the insertions of the Tv1 retrotransposon and are absent in the fly genome are revealed in the genome of the passaged cell line of D. virilis. This fact is evidence of the activation of the mitochondrial DNA transfer into the nuclear genome of the cells of passaged cell culture.     

Variation of wing shape in the Drosophila virilis species group (Diptera: Drosophilidae)

Wing shape variation was analysed with geometric morphometric methods in 17 laboratory strains, representing 11 closely related species (including two subspecies) of the Drosophila virilis group: D. virilis, D. lummei, D. novamexicana, D. americana americana, D. americana texana, D. montana, D. lacicola, D. flavomontana, D. borealis, D. littoralis, D. ezoana and D. kanekoi. Overall shape estimated using Procrustes coordinates of 14 landmarks was highly variable among strains and very similar in females and males. The landmarks in the distal part of the wing showed higher variation across strains than those in the proximal part. Procrustes distances between species were not consistent with phylogenetic distances previously suggested for the virilis group. Moreover, Procrustes distances between strains within species and within two major phylads (virilis and montana) were comparable with those between species and between phylads, respectively. The most different from other members of the group was the endemic D. kanekoi species, currently viewed as separate subphylad within the montana phylad. Allometric effects were found to be partly responsible for shape differences between the strains. Three most significant shape transformations were considered using the relative warp analysis and the strains were ordinated in accordance with transformation values. The pattern of relative warp scores could be easily interpreted only for the third warp explaining about 13% of shape variation. It separated the largest species, D. montana, D. ezoana and D. kanekoi, from other ones and was mainly associated with shape changes in the proximal region of the wing. The results of the present work suggest that wing shape in the virilis species group is not related to the speciation process. The observed proximal‐distal contrasts and allometric effects are in agreement with data of other studies, in which wing shape variation was analysed within Drosophila species.     

Nucleotide and repeat length variation at the nonA gene of the Drosophila virilis group species and its effects on male courtship song

Genes found to affect male courtship song characters in Drosophila melanogaster are good candidates when tracing genes responsible for species-specific songs in other Drosophila species. It has previously been shown that Thr–Gly repeat length variation at the period gene affects song traits in D. melanogaster, which gives the repetitive regions a special interest. In this work, we have characterised the patterns of nucleotide variation for gene regions containing two Gly and one Gln–Ala repeat in another D. melanogaster song gene, no-on-transient A, in D. virilis group species. The levels of nucleotide variability in D. virilis nonA were similar to those found for other genes of the species, and the gene sequences showed no signs of deviation from neutrality. The Gly 2 repeat preceding the central domain of the gene exhibited length variation, which did not, however, correlate with song variation either within D. virilis or between the species of D. virilis group. The Gly 3 repeat located on the other side of the central domain showed amino acid divergence parallel to the consensus phylogeny of the D. virilis group species. The species of the virilis subgroup having Asn after the first three glycines in this repeat have simple songs with no species-specificity, while the species of the montana subgroup having two Gly or Asn–Ser in this site have unique courtship songs. Amino acid differences between the species in this repeat may, however, reflect species phylogeny rather than have an effect on song divergence per se.     

Isolation and characterization of microsatellites in Drosophila montana and their cross‐species amplification in D. virilis

We report the isolation of 20 microsatellite loci from Drosophila montana and their cross amplification in the relative D. virilis. All microsatellite loci were polymorphic in the focal species D. montana, with gene diversities ranging from 0.23 to 0.93. In D. virilis only eight loci (40%) amplified and two loci were polymorphic (10%). These markers represent the first report of microsatellites isolated in D. montana. They could be applied for studying population structure and phylogeography. The largest benefit, however, will be their use in studies of quantitative trait loci, such as the mapping of behavioural quantitative trait loci.     

Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

A RAPD fingerprinting of sibling species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

A comparative analysis of the sibling species of Drosophila virilis was performed by RAPD-PCR technique using a set of random primers. The degree of relatedness was studied by cluster analysis (UPGMA) and multidimensional scaling. The resulting pattern of species relationships contradicts the classical taxonomy. The main result of the cluster analysis is that D. virilis does not cluster with the remaining three species of its phylad, while according to multidimensional scaling, D. virilis is equidistant from all the species of its group, from both the species of its phylad and the species of the montana phylad. The montana phylad is extremely heterogeneous; moreover, the species D. littoralis, D. ezoana, and D. kanekoi appear to be closer to the virilis phylad than to the other species of the montana phylad, wherein these species are traditionally included. The phylogenetic relationships between the studied species discovered using RAPD fingerprinting comply with the results obtained using protein markers and quantitative traits.     

Mitochondrial DNA Polymorphism in Natural Populations of the Drosophila virilis Species Group

Restriction fragment length polymorphism (RFLP) analysis has been used to evaluate mitochondrial DNA (mtDNA) variation in 12 sibling species forming the Drosophila virilis species group. The variation thresholds corresponding to the interspecific and interstrain levels have been determined. The results indicate that interspecific hybridization has significantly contributed to the evolutionary history of the virilis species group.     

THE GENETIC BASIS OF EVOLUTION OF THE MALE COURTSHIP SOUNDS IN THE DROSOPHILA VIRILIS GROUP

When courting, males of the Drosophila virilis group vibrate their wings and emit species-specific courtship sounds consisting of trains of polycyclic sound pulses. To analyze the genetic basis of evolutionary changes in the sounds we made an F₁ diallel set of reciprocal crosses between the members of the virilis phylad of the group (two stocks of D. virilis and one of D. americana americana, D. a. texana, D. novamexicana, and D. lummei). We also crossed the D. virilis stocks with the members of the montana phylad of the same group (D. kanekoi, D. littoralis, D. borealis, D. flavomontana, D. lacicola, and D. montana) and made a backcross (D. virilis x D. littoralis) x D. virilis using a D. virilis marker stock (b; sv t tb gp; cd; pe). The sounds of the hybrids were analyzed using the following parameters: the length of a pulse train (PTL), the number of pulses in a train (PN), the interpulse interval (IPI), the length of a pulse (PL), the number of cycles in a pulse (CN), and the length of a cycle (CL). In the virilis phylad, the differences between species appeared to be determined mainly by autosomal genes in each sound trait. The heritabilities (narrow-/broad-sense) obtained from the diallel tables were the following: PTL 0.662/0.817, PN 0.651/0.841, IPI 0.193/0.546, PL 0.408/0.552, CN 0.425/0.719, and CL 0.361/0.764. The direction of dominance is for longer PTL, higher PN and CN, and shorter IPI and CL. PL shows ambidirectional dominance. In the sounds of the virilis phylad species, PTL and PL seem to be phenotypically the most important parameters, since their components (PN and IPI for PTL, CN and CL for PL) are negatively correlated. In crosses between D. virilis and D. littoralis or D. flavomontana reciprocal hybrids differed from each other in PTL, IPI, PL, and CN indicating X-chromosomal or cytoplasmic inheritance. In the backcrosses between D. virilis and D. littoralis the role of the X chromosome was ascertained to be decisive. We conclude that an X-chromosomal major change allowing variation in IPI has occurred during the separation of the two D. virilis group phylads, the long IPI allowing variation also in PL (and CN). The evolution of the sounds in the virilis phylad has probably gone towards longer and denser pulse trains, while in the montana phylad the sounds have evolved in different directions.     

In Situ Hybridisation Analysis of the X-Linked Genes in the Species of the Virilis Group of Drosophila

When estimating the level of DNA sequence variation within and between populations or when planning QTL analysis, it is essential to know the location of the genes under study. In the present work, five X chromosomal genes, earlier localised in Drosophila virilis and D. littoralis, were mapped by in situ hybridisation on the larval polytene chromosomes of four other virilis group species, D. a. americana, D. flavomontana, D. lacicola and D. montana. Conjugation of X chromosomes of the most interesting species pairs was studied in interspecific hybrids. Three of the marker genes were used as RFLP markers to examine the occurrence of recombination in D. flavomontana and D. montana hybrid females. The gene arrangement of all species studied, appeared to be different at the proximal end of the X chromosome, which prevented normal conjugation along the most part of the X chromosome. The data illustrating the locations of five X chromosomal marker genes are presented for D. a. americana, D. flavomontana, D. lacicola and D. montana.     

Estimation of Genetic Variability in Natural Populations of DROSOPHILA SIMULANS by Two-Dimensional and Starch Gel Electrophoresis

Genic variation in natural populations of Drosophila simulans was surveyed using allozymic and two-dimensional electrophoretic techniques. Consistent with some previous reports, allozymic heterozygosity appeared lower than in the sibling species D. melanogaster (0.07 vs. 0.16). No variation was detected by two-dimensional electrophoresis of 19 lines scored for 70 abundant proteins. This is consistent with reported reductions in estimates of genic heterozygosity by two-dimensional electrophoresis in D. melanogaster, Mus musculus, and man. Although the amount of intraspecific variation detected in abundant proteins was lower than that detected for allozymes in D. simulans and D. melanogaster, the genetic distances between the sibling species calculated from the two data sets are not significantly different (0.35 and 0.20). The allozyme and two-dimensional electrophoresis data confirmed the impression from other measures of genetic variation (mitochondrial DNA restriction maps and inversion polymorphisms) that D. simulans is substantially less variable than D. melanogaster.     

Sites of the 5s Ribosomal Genes in Drosophila. I. the Multiple Clusters in the VIRILIS Group

Drosophila melanogaster ¹²⁵I-5S RNA was annealed to salivary gland preparations of 6 species in the virilis group of Drosophila. Two patterns of annealing were found. D. virilis, D. montana and D. borealis showed three 5S gene clusters on chromosome 5; S_d–f and W_c–j were strongly labeled, but X_a–e was weakly labeled. D. montana and D. borealis have a greater percentage of their total 5S cistrons at S _d–f than does D. virilis. D. americana americana, D. americana texana and D. novamexicana showed 2 sites labeled; no label was seen at S_d–f while W_c–j was weakly labeled and X_a–e was strongly labeled, the reciprocal of the previous pattern in the W-X region. Hybrids between D. a. americana and D. virilis showed no difference in chromosome banding at the sites of the 5S clusters despite their pattern differences. D. a texana x D. virilis, on the other hand, did show a difference in staining the X_a–e region. These patterns fall squarely into the biosystematic groupings deduced by many previous workers.     

Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Morphological analysis of male mating organ in the Drosophila virilis species group: a multivariate approach

The shape of the male mating organ differs among 11 closely related species of the Drosophila virilis species group. Multivariate analyses of variation of a suite of 35 morphological traits (indices) describing the phallus shape were carried out in order to characterize interspecies variability of the traits. An overwhelming majority of the traits displayed species‐specific variability. The main result of the investigation was the revelation of the differences involved in the traits studied in the evolution of the D. virilis species group. The structure of species‐specific variability of some traits was discovered to be in accordance with the generally accepted taxonomy of the species group, while that of other traits required isolation of D. virilisper se from lummei phylad (former virilis phylad), and confirmed separation of montana phylad into three subphylads: montana proper, littoralis and kanekoi. Several subsets of traits having separate variability were determined in different parts of the male mating organ which correspond to spots of evolutionarily significant variability.     

Evolutionary divergence of the cytochrome b5 gene of Drosophila

Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b₅ (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b₅ locus encodes an essential product of the Drosophila system.Correspondence to: C.E. Rozek     

Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.