Evolution of overwintering strategies in Eurasian species of the Drosophila obscura species group

The phylogenetic relationship of Eurasian species of the Drosophila obscura species group remains ambiguous in spite of intensive analyses based on morphology, allozymes and DNA sequences. The present analysis based on sequence data for cytochrome oxidase subunit I (COI) and a-glycerophosphate dehydrogenase (Gpdh) suggests that the phylogenetic position of D. alpina is also ambiguous. These ambiguities have been considered to be attributable to rapid phyletic radiation in this group at an early stage of its evolution. Overwintering strategies are diversified among these species: D. alpina and D. subsihestris pass the winter in pupal diapause, D. bifasciata and D. obscura in reproductive diapause, and D. subobscura and D. guanche without entering diapause. This diversity may also suggest rapid radiation at an early phase of adaptations to temperate climates. On the other hand, adult tolerance of cold was closely related to overwintering strategy and distribution: D. obscura and D. bifasciata with reproductive diapause were very tolerant; D. alpina and D. subsilvestris which pass the winter in pupal diapause were less tolerant; D. subobscura having no diapause was moderately tolerant and D. guanche occurring in the Canary Islands was rather susceptible. Tolerance of high temperature at the preimaginal stages seemed to be also associated with overwintering strategy; i.e. lower in the species with pupal diapause than in those with reproductive diapause or without diapause mechanism.     

Comparative and functional studies of Drosophila species invasion by the gypsy endogenous retrovirus.

Gypsy is an endogenous retrovirus of Drosophila melanogaster. Phylogenetic studies suggest that occasional horizontal transfer events of gypsy occur between Drosophila species. gypsy possesses infective properties associated with the products of the envelope gene that might be at the origin of these interspecies transfers. We report here the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster, suggesting that potentially infective gypsy copies able to spread between sexually isolated species can occur. The ability of gypsy to invade the genome of a new species is conditioned by its capacity to be expressed in the naive genome. The genetic basis for the regulation of gypsy activity in D. melanogaster is now well known, and it has been assigned to an X-linked gene called flamenco. We established an experimental simulation of the invasion of the D. melanogaster genome by gypsy elements derived from other Drosophila species, which demonstrates that these non- D. melanogaster gypsy elements escape the repression exerted by the D. melanogaster flamenco gene.     

Evolutionary patterns of the gypsy and bilbo retrotransposon families in the Drosophila species of the obscura group

We analyse in this paper the evolutionary patterns of two types of Drosophila retrotransposons, gypsy (a virus-like element), and bilbo (a LINE-like element), in host species from the Drosophila and Scaptomyza genus. Phylogenetic analysis of the retrotransposon sequences amplified by PCR, revealed concordance with the phylogeny of the Drosophila host species from the obscura group, which is consistent with vertical transmission during differentiation of the species. However, in the species outside of the obscura group, horizontal transmission can be considered. The amplified sequences that presented intact open reading frames were used in an analysis of the evolutionary constraints on the amino acid sequences. The analysed sequences seem to be functional, and the selective constraints are evidenced, especially when sequences from distant species are compared. Comparison of the evolutionary rates of both retrotransposons in the same species, suggests that bilbo seems to evolve more rapidly than gypsy.     

The endogenous Drosophila melanogaster retrovirus gypsy can propagate in Drosophila hydei cells

The endogenous Drosophila melanogaster retrovirus gypsy (mdg4) forms virus-like particles (VLPs) which are found as extracellular particles in the medium used to culture D. melanogaster cells. The D. hydei somatic cell line DH14, which does not harbour gypsy sequences, was exposed to D. melanogaster VLPs. Subsequent PCR and Southern analysis revealed that gypsy elements had penetrated into the D. hydei cells, suggesting interspecific transmission of the retrovirus. A D. hydei cell line containing gypsy sequences was established and grown in a mixed culture together with the G418-resistant D. hydei cell line DH33, and gypsy was shown to be transmitted from cell to cell. The proportion of cells carrying gypsy increased with time. The rate of gypsy invasion of the lines DH14 and DH33 was 10(-3) and 10(-2) per cell per generation, respectively. The results demonstrate the possibility of interspecific horizontal transfer of gypsy in the form of its VLPs.     

Evolution of the Gypsy endogenous retrovirus in the Drosophila melanogaster subgroup

We conducted a phylogenetic survey of the endogenous retrovirus Gypsy in the eight species of the Drosophila melanogaster subgroup. A 362-bp fragment from the integrase gene (int) was amplified, cloned, and sequenced. Phylogenetic relationships of the elements isolated from independent clones were compared with the host phylogeny. Our results indicate that two main lineages of Gypsy exist in the melanogaster subgroup and that vertical and horizontal transmission have played a crucial role in the evolution of this insect endogenous retrovirus.     

Differentiation of the vitellogenin proteins in species of the Drosophila obscura group

The three female specific vitellogenin proteins of eight Drosophila species of the obscura group were detected in discontinuous SDS-polyacrylamide gels. The molecular weights of these proteins were estimated. These three genetic markers represent an useful addition to the electrophoretic variation that has been used to construct phylogenies in the genus Drosophila.     

Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene

The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.     

Gene activity of polytene chromosomes in Drosophila species of the obscura group

The polytene chromosome puffing patterns of Drosophila guanche were established and compared with those of Drosophila subobscura. A total of 150 loci, active in some of the 17 developmental stages studied, were described and 23 of them were found to form the characteristic puffing pattern of D. guanche. Taking into account the number of puffs as well as the gene activity of each chromosome and the total gene activity, D. guanche seems to be less active than D. subobscura. Although both species show a degree of homology in their puffing patterns lower than that found for sibling species, the degree of homology is stronger than that between species belonging to the same group but to different subgroups. Thus, D. guanche and D. subobscura must be considered as phylogenetically closely related species, belonging to the same subgroup.     

Evolutionary dynamics of the SGM transposon family in the Drosophila obscura species group

SGM (Drosophila subobscura, Drosophila guanche, and Drosophila madeirensis) transposons are a family of transposable elements (TEs) in Drosophila with some functional and structural similarities to miniature inverted-repeat transposable elements (MITEs). These elements were recently active in D. subobscura and D. madeirensis (1-2 MYA), but in D. guanche (3-4 MYA), they gave rise to a species-specifically amplified satellite DNA making up approximately 10% of its genome. SGM elements were already active in the common ancestor of all three species, giving rise to the A-type specific promoter section of the P:-related neogene cluster. SGM sequences are similar to elements found in other obscura group species, such as the ISY elements in D. miranda and the ISamb elements in Drosophila ambigua. SGM elements are composed of different sequence modules, and some of them, i.e., LS and LS-core, are found throughout the Drosophila and Sophophora radiation with similarity to more distantly related TEs. The LS-core module is highly enriched in the noncoding sections of the Drosophila melanogaster genome, suggesting potential regulatory host gene functions. The SGM elements can be considered as a model system elucidating the evolutionary dynamics of mobile elements in their arms race with host-directed silencing mechanisms and their evolutionary impact on the structure and composition of their respective host genomes.     

Phylogeny of the Drosophila obscura species group deduced from mitochondrial DNA sequences

Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., afftnis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two complexes (i.e., subobscura and obscura).     

Distribution of Drosophila melanogaster transposable element sequences in species of the obscura group

Fifteen species belonging to the obscura group of the genus Drosophila were screened for sequences homologous to Drosophila melanogaster transposable elements (TEs) as an initial step in the examination of the possible occurrence of TEs at chromosomal inversion breakpoints. Blots of genomic DNAs from species of the obscura group were hybridized at three different stringencies with 14 probes representing the major families of TEs described in D. melanogaster. The probe DNAs included copia, gypsy, 412, 297, mdg1, mdg3, 3S18, F, G, I, jockey, P, hobo, and FB3. D. melanogaster TEs were not well represented in the species of the obscura group analyzed. The TEs that were observed generally exhibited heterogeneous distributions, with the exception of F, gypsy and 412 which were ubiquitous, and 297, G, Sancho 2, hobo and FB which were not detected.by A. Bird     

Morphometrical evolution in a Drosophila clade: the Drosophila obscura group

Five morphometrical traits (wing and thorax length, ovariole number, and thoracic and female abdomen pigmentation) were investigated in laboratory stocks of 20 species belonging to the Drosophila obscura group (subgenus Sophophora). These species originated from four biogeographical regions and represent all five of the presently recognized, taxonomic subgroups. Size‐related traits (wing and thorax length) were highly variable across species, and interspecific variation explained more than 90% of total variability. In both traditional and phylogenetic analyses, wing size was positively correlated with latitude of origin. These interspecific correlations were however notably weaker than those for intraspecific correlations. Wing/thorax ratio, which may be related to flight capacity, showed little variation. Ovariole number was highly variable (range 27–53) both within and between species, and was positively correlated with the wing/thorax ratio, suggesting that species with relatively large ovaries have relatively low wing loading. Although many species are completely dark, 11 had some regions of light coloration. A light thorax with a median darkening was observed in six species. A variable pigmentation of abdominal tergites, in females only, was found in nine species, belonging to three subgroups only. With respect to both molecular phylogeny and morphometrical evolution, the D. obscura subgroup is probably now the best investigated clade in Drosophila.     

A new species of Drosophila obscura species group (Diptera:Drosophilidae) from China

A new species of the Drosophila obscura species group is described here, namely Drosophila glabra sp. nov. It was recently found from the Maoershan National Nature Reserve, Guangxi, China. The characteristics of the new species are based not only on morphological characters but also on DNA sequences of the mitochondrial COII(cytochrome c oxidase subunit II) gene.     

Protein determinants of insertional specificity for the Drosophila gypsy retrovirus.

The gypsy retrovirus invades the germ line of Drosophila females, inserting with a high frequency into the ovo locus. Gypsy insertion sites in ovo are clustered within a region in the promoter of the ovo gene that contains multiple binding sites for the OvoA and OvoB proteins. We found that a 1.3-kb DNA fragment containing this region is able to confer gypsy insertional specificity independent of its genomic location. The frequency of gypsy insertions into the ovo gene is significantly lower in wild-type females than in ovoD1 females. In addition, gypsy insertions in ovoD1 females occur during most stages of germ-line development whereas insertions in wild-type females occur only in late stages. This pattern of temporally specific insertions, as well as the higher frequency of insertion in ovoD1 females, correlates with the presence of the OvoA or OvoD1 proteins. The results suggest that gypsy insertional specificity might be determined by the binding of the OvoA repressor isoform to the promoter region of the gene.     

Relationships in the Drosophila obscura species group, inferred from mitochondrial cytochrome oxidase II sequences

We compare the sequences for the mitochondrial cytochrome oxidase II gene of 13 species of the Drosophila obscura group. The survey includes six members of the D. affinis subgroup, four of the D. pseudoobscura subgroup, and three of the D. obscura subgroup. In all species, the gene is 688 nucleotides in length, encoding a protein of 229 amino acids plus the first position T of the stop codon. The sequences show the typical high-transition bias for closely related species, but that bias is essentially eliminated for species pairs of > 5% sequence divergence. The phylogenetic relationships in the species group are inferred using both neighbor-joining and maximum parsimony. The two procedures give comparable results, showing that the D. affinis and D. pseudoobscura subgroups are monophyletic groupings that appear to have closer affinities to one another than either has to the D. obscura subgroup. We use transversion distances to estimate times of divergence, on the basis of three different estimates of the time of separation of the D. obscura species group from the D. melanogaster group. If that event occurred 35 Mya, then we can estimate the origin of the nearctic forms at approximately 22 Mya and the separation of the D. affinis and D. pseudoobscura subgroups at approximately 17 Mya.      

Evolutionary history of microsatellites in the obscura group of Drosophila

The evolutionary origins of microsatellites are not well understood. Some investigators have suggested that point mutations that expand repeat arrays beyond a threshold size trigger microsatellites to become variable. However, little empirical data has been brought forth on this and related issues. In this study, we examine the evolutionary history of microsatellites in six species within the obscura group of Drosophila, tracing changes in microsatellite alleles using both PCR product size and sequence data. We found little evidence supporting a general role of point mutations triggering initial microsatellite expansion, and no consistent threshold size for expansion was observed. Flanking region length variation was extensive when alleles were sequenced in distantly related species, and some species possessed altogether different repeat arrays between the same primer binding sites. Our results suggest extreme caution in using microsatellite allele sizes for phylogenetic analyses or to infer divergences between populations.     

Functional and biochemical features of alcohol dehydrogenase in four species of the obscura group of Drosophila

The biochemical features of ADH of four Drosophila species of the obscura group have been studied. The relationship between ethanol tolerance and ADH activity has been investigated. Propan-2-ol and acetone concentrations have been determined in propan-2-ol treated flies and ADH activity has been followed during 96 h of propan-2-ol treatment. Data on the ADH system confirm constructed phylogenies based on electrophoretic variation and chromosome homologies.     

Characterization of the length polymorphism in the A+T-rich region of the Drosophila obscura group species

Summary In the twelve Drosophila obscura group species studied, belonging to the affinis, obscura, and pseudoobscura subgroups, the mitochondrial DNA length ranges from 15.8 to 17.2 kb. This length polymorphism is mainly due to insertions/deletions in the variable region of the A+T-rich region. In addition, one species (D. tristis) possess a tandem duplication of a 470-bp fragment that contains the replication origin.The same duplication has occurred at least twice in the Drosophila evolutionary history due to the fact that the repetition is analogous to repetitions found in the four species of the D. melanogaster complex.By comparing the nucleotide sequence of the conserved region in D. ambigua, D. obscura, D. yakuba, D. teissieri, and D. virilis, we show the presence of a secondary structure, likely implied in the replication origin, which could favor the generation of this kind of duplications.Finally, we propose that the high A and T content in the variable region of the A + T-rich region favors the formation of less-stable secondary structures, which could explain the generation of minor insertion/deletions found in this region.Offprint requests to: A. Latorre