The phylogeny of nine species of the Drosophila obscura group inferred by the banding homologies of chromosomal regions. I. Element B

The phylogenetic relationships among nine Drosophila species belonging to the obscura group were investigated by establishing the segments displaying banding homologies in their element B (equivalent to the U element of D. subobscura). The phylogenetic ordering of the species was accomplished using overlapping inversions. Two African species, D. kitumensis and D. microlabis, were investigated. These species are homosequential for their element B gene arrangement but differ from that of D. obscura by several rearrangements. Drosophila obscura seems to be most closely related to D. subsilvestris, from which the respective element B gene arrangements differ at least by six inversions. Three species, D. obscura, D. ambigua, and D. tristis, are closely related and form a cluster. Drosophila obscura displays an element B polymorphism for a pericentric inversion for which D. ambigua is fixed for one gene arrangement and D. tristis for the other. Both D. ambigua and D. tristis share a short distal inversion in the small arm of the chromosome, and differ in this respect from D. obscura. Drosophila madeirensis, D. guanche, and D. subobscura all share the same element B gene arrangement, which is acrocentric, but metacentric in all the other species mentioned. It was found that the gene arrangements of the species from the obscura cluster seem to occupy an intermediate position between those of the species of the D. subobscura cluster and those of the African one. The data reported generally are in good agreement with information provided in the literature.     

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.     

Differentiation of Muller''s Chromosomal Elements D and E in the Obscura Group of Drosophila

Twenty-two markers located on Muller's elements D or E have been mapped by in situ hybridization in six species of the obscura group of Drosophila and in D. melanogaster. The obscura species can be grouped into a Palearctic cluster (D. subobscura, D. madeirensis and D. guanche) and a Nearctic one (D. pseudoobscura, D. persimilis and D. miranda). Eleven of the probes contain known genes: E74, Acp70A, Est5, hsp28/23, hsp83, emc, hsp70, Xdh, Acph-1, Cec and rp49. The remaining probes are recombinant phages isolated from a D. subobscura genomic library. All these markers hybridize to the putative homologous chromosome or chromosomal arm of elements D and E. Thus, these elements have conserved their genic content during species divergence. Chromosomal homologies proposed previously for each element among the species of the same cluster have been compared with the present results. The distribution of markers within each element has changed considerably as inferred from pairwise comparisons of obscura species included in the two different clusters. Only chromosomal segments defined by closely linked markers have been conserved: one such segment has been detected in element D and three in element E between D. subobscura and D. pseudoobscura.     

The phylogeny of nine species of the Drosophila obscura group inferred by the banding homologies of chromosomal regions. III. Element D.

The phylogenetic relationships among nine species belonging to the obscura group of the genus Drosophila were deduced, based on similarities of the banding pattern of their polytene chromosomal element D. These similarities were inferred by the comparison of chromosomal photomaps. The phylogenetic reconstruction was the most parsimonious based on seriation by overlapping inversions and on the principle of conservation/disassociation of nearby located segments. The gene sequences of element D for all species studied were relatively easy to recognize in terms of the map of D. obscura, already found to occupy a relative central position in this group. Thus, three clusters of closely related species could be identified: obscura (D. obscura, D. ambigua, and D. tristis), African (D. kitumensis and D. microlabis), and subobscura (D. subobscura, D. madeirensis and D. guanche), with D. subsilvestris standing apart. The results are in agreement with those from the previously studied elements B and E, but element D was found to be much more conclusive concerning the links among the different clusters. Thus, it is inferred that D. guanche occupies an intermediate position between the other two species of its own cluster and all the others. The gene arrangement of D. obscura, directly related to those of the other species, has been identified. In the phylogenetic tree proposed, both the African cluster and D. subsilvestris derive from a hypothetical gene arrangement, intermediate in the pathway between the subobscura and obscura clusters.     

Insertion polymorphism of retrotransposable elements in populations of the insular, endemic species Drosophila madeirensis

The insertion site numbers of the retrotransposable elements (TE) 412, gypsy and bilbo were determined in individuals of five distinct natural populations of the endemic species Drosophila madeirensis from the island of Madeira. The TE distributions were compared to those of the paleartic, widespread and phylogenetically closely related species, D. subobscura. In situ hybridization and Southern blots showed that in D. madeirensis the number of insertion sites ranged between 10 and 15, three and six, and 35 and 42 for elements 412, gypsy and bilbo, respectively. The corresponding values for D. subobscura were similar. Two of these elements, 412 and gypsy, had very few insertions in the heterochromatin, unlike bilbo, which displayed a high heterochromatic insertion number. The Southern band polymorphism was very high, leading to within-population variation of 97.2%, whatever the population and the TE concerned. Using the polymorphic TE insertion sites as markers to analyse population structure by AMOVA, adapted for RAPD (Randomly Amplified Polymorphic DNA) data, we found small but significant genetic differences between the populations on Madeira. This slight differentiation, coupled with similar copy numbers for each TE between populations, suggests that the D. madeirensis species consists of a single, only slightly subdivided population. These data also show that insular populations and endemic species of Drosophila can have as many copies of TEs as more widespread species.     

Adaptive significance of amylase polymorphism in Drosophila XIII. Old World obscura species subgroup divergence according to biochemical properties of alpha-amylase

Biochemical properties of enzyme alpha-amylase were surveyed in Drosophila obscura Old world group of species (D. subobscura, D. ambigua, D. obscura and D. tristis) sampled in the same habitat, with the aim to reveal some ecological and evolutionary aspects of amylase polymorphism, which has been studied extensively in D. subobscura, but not compared with other species in the group. The data obtained show that D. subobscura is distinct from the other three species regarding all biochemical amylase properties. Such a divergence also correlates with the niche breadth and relative abundance of these species in the same habitat.     

Chromosomal evolution of elements B and C in the Sophophora subgenus of Drosophila: evolutionary rate and polymorphism

The locations of 77 markers along the chromosomal elements B (41 markers) and C (36 markers) of Drosophila subobscura, D. pseudoobscura, and D. melanogaster were obtained by in situ hybridization on polytene chromosomes. In comparisons between D. subobscura and D. pseudoobscura, 10 conserved segments (accounting for 32% of the chromosomal length) were detected on element B and eight (17% of the chromosomal length) on element C. The fixation rate of paracentric inversions inferred by a maximum likelihood approach differs significantly between elements. Muller's element C (0.17 breakpoints/Mb/million years) is evolving two times faster than element B (0.08 breakpoints/Mb/million years). This difference in the evolutionary rate is paralleled by differences in the extent of chromosomal polymorphism in the corresponding lineages. Element C is highly polymorphic in D. subobscura, D. pseudoobscura, and in other obscura group species such as D. obscura and D. athabasca. In contrast, the level of polymorphism in element B is much lower in these species. The fixation rates of paracentric inversions estimated in the present study between species of the Sophophora subgenus are the highest estimates so far reported in the genus for the autosomes. At the subgenus level, there is also a parallelism between the high fixation rate and the classical observation that the species of the Sophophora subgenus tend to be more polymorphic than the species of the Drosophila subgenus. Therefore, the detected relationship between level of polymorphism and evolutionary rate might be a general characteristic of chromosomal evolution in the genus Drosophila.     

Molecular Organization of the X Chromosome in Different Species of the Obscura Group of Drosophila

Nine single copy regions located on the X chromosome have been mapped by in situ hybridization in six species of the obscura group of Drosophila. Three Palearctic species, D. subobscura, D. madeirensis and D. guanche, and three Nearctic species, D. pseudoobscura, D. persimilis and D. miranda, have been studied. Eight of the regions include known genes from D. melanogaster (Pgd, zeste, white, cut, vermilion, RNA polymerase II 215, forked and suppressor of forked) and the ninth region (lambda DsubF6) has not yet been characterized. In all six species, as in D. melanogaster, all probes hybridize to a single site. Established chromosomal arm homologies of Muller's element A are only partly supported by present results since two of the probes (Pgd and zeste) hybridize at the proximal end of the XR chromosomal arm in the three Nearctic species. In addition to the centric fusion of Muller's A (= XL) and D (= XR) elements, the metacentric X chromosome of the Nearctic species requires a pericentric inversion to account for this result. Previously proposed homologies of particular chromosomal regions of the A (= X) chromosome in the three species of the D. subobscura cluster and of the XL chromosomal arm in the three species of the D. pseudoobscura cluster are discussed in light of the present results. Location of the studied markers has changed drastically not only since the divergence between the melanogaster and obscura groups but also since the Palearctic and Nearctic species of the obscura group diverged.     

Reevaluation of phylogeny in the Drosophila obscura species group based on combined analysis of nucleotide sequences.

The Drosophila obscura species group has served as an important model system in many evolutionary and population genetic studies. Despite the amount of study this group has received, some phylogenetic relationships remain unclear. While individual analysis of different nuclear, mitochondrial, allozyme, restriction fragment, and morphological data partitions are able to discern relationships among closely related species, they are unable to resolve relationships among the five obscura species subgroups. A combined analysis of several nucleotide data sets is able to provide resolution and support for some nodes not seen or well supported in analyses of individual loci. A phylogeny of the obscura species group based on combined analysis of nucleotide sequences from six mitochondrial and five nuclear loci is presented here. The results of several different combined analyses indicate that the Old World obscura and subobscura subgroups form a monophyletic clade, although they are unable to resolve the relationships among the major lineages within the obscura species group.     

Population densities in Drosophila obscura Fallén and D. subobscura Collin

Abstract. 1. The population densities of Drosophila obscura and D. subobscura in mixed deciduous woodland were investigated by multiple recapture techniques, marking flies with micronized fluorescent dusts.
2. Approximate densities per 100 m² were as follows: September 1974: D.subobscura males 5, D.subobscura females 31. April 1975: D.obscura males 1, D.obscura females 2; D.subobscura males 0.3, D.subobscura females 1. June 1975: D.subobscura males 6, D.subobscura females 9.
3. Actual estimates have been compared using relative estimates derived from a reanalysis of Shorrocks's (1975) data.
4. Throughout most of the year D.subobscura was numerically dominant, rising to a peak of 750/100 m² in the winter; but in late spring D.obscura was dominant, having reduced winter mortality by diapausing.
5. D.obscura 's density was comparable with those of its Nearctic relatives; D.subobscura 's was considerably higher.
6. The effects of previous workers' approximations on the estimation of population parameters have been examined, and possible theoretical consequences have been indicated.     

Phylogeny of the Drosophila obscura group as inferred from one- and two-dimensional protein electrophoresis

The phylogenetic relationships of 15 species of the obscura group of Drosophila were analysed by use of one- and two-dimensional electrophoresis. Genetic distances based on two-dimensional data are five times smaller than those based on native proteins. From the data, it is proposed that the species radiation of the obscura group happened in two evolutionary bursts, the first one giving rise to at least four palearctic proto-lineages ( bifasciata, obscura (including D. subsilvestris ), subobscura , and microlabis ) and one or two proto-nearctic lineages ( affinis, pseudoobscura ), and the second, more recent burst giving rise to the current speciation within lineages.

Zusammenfassung

Phylogenie der Arten der Drosophila obscura-Gruppe abgeleitet von ein- und zweidimensionaler Protein-Elektrophorese
Die phylogenetischen Verwandtschaftsbeziehungen von 15 Arten der obscura -Gruppe der Gattung Drosophila wurden mit Hilfe von ein-und zweidimensionaler Elektrophorese von Proteinen untersucht. Die genetische Distanzen, die aus den Ergebnissen der zweidimensionalen Elektrophoresen ermittelt wurden, waren fünfmal kleiner als solche, die von nativen Proteinen kommen. Aufgrund der Untersuchungsergebnisse wird angenommen, daß die Radiation der Arten der obscura-Gruppe in zwei evolutiven Schüben erfolgt sei; der erste Schub hätte zu zumindest vier palaerktischen ( bifasciata, obscura mit D. subsilvestris, subobscura und microlabis ) und zwei proto-ne arktischen Linien ( affinis, pseudoobscura ) geführt. In einem zweiten Schub wären dann die endgültigen rezenten Arten entstanden.     

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.     

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.     

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.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

The Drosophila subobscura Adh genomic region contains valuable evolutionary markers.

We have sequenced 4 kb of the genomic region comprising the Adh (Alcohol dehydrogenase) gene of Drosophila subobscura. In agreement with other species which belong to the same subgenus, two structural genes, Adh and Adh-dup, are contained in this region. The main features of these two genes of D. subobscura have been inferred from the sequence data and compared with the homologous region of D. ambigua and D. pseudoobscura. Drosophila subobscura Adh and Adh-dup differ from those of D. ambigua at a corrected estimation of 10.1% and 12.5%, respectively, while from those of D. pseudoobscura they differ by 9.5% and 8.1%, respectively. Our data suggest that Adh and Adh-dup are evolving independently, showing a species-specific pattern. Moreover, particular features of some regions of these genes make them valuable evolutionary hallmarks. For instance, replacement substitutions in the third exon of Adh may indicate the branching of the melanogaster-obscura groups, whereas replacement substitutions in the third exon of the Adh-dup could be used to assess speciation within the obscura group.     

Chromosome homologies within the Drosophila obscura group probed by in situ hybridization

Probes specific to chromosome elements were used to investigate chromosome homologies between seven species of the Drosophila obscura group by in situ hybridization. Our results were in perfect agreement with the already established chromosome element homologies between D. subobscura, D. pseudoobscura, D. persimilis, and D. miranda. Furthermore, we were able to identify the chromosomal elements of D. obscura, D. ambigua, and D. subsilvestris. Of special interest was the localization of the two D. melanogaster-derived representatives of the tandemly repetitive genes, cDm500 and 12D8. In contrast to the findings with the element-specific probes, the localizations of the repetitive genes varied in the various species. Whereas D. melanogaster, D. subobscura, D. pseudoobscura, D. persimilis, and D. miranda showed only one strong block of label in the cross in situ hybridizations with cDm500, three labeling blocks were found on two elements for both D. ambigua and D. obscura. The two labeling blocks on one element occur in very close proximity, but are clearly separated in both species by cytologically detectable chromosomal material. We used the distribution of the cDm500 labeling sites to postulate a series of chromosomal rearrangements involved in the karyotype evolution of the analyzed species. Our results support the conclusion that the chromosomal elements retain their essential identity and that the observed gross structural rearrangements are due to fusions and paracentric or pericentric inversions. Cytologically obvious translocations were not recorded and are considered by us to be rare. The frequently occurring translocations of the tandemly repeated gene clusters observed in this study are probably due to a different mechanism, which may be an intrinsic property of this category of genes.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday with our best wishes     

The utilization of naturally occurring yeasts by Drosophila species, using chemically defined substrates

The ability of three Drosophila species; Drosophila obscura, D. subobscura and D. phalerata, to utilize a range of naturally occurring yeasts as food sources was determined. The yeasts used were the dominant species of two commonly used breeding sites, Sorbus aucuparia fruits and the fruiting bodies of the fungus Phallus impudicus. The range of yeast species used was found to reflect the degree ot substrate specialization exhibited by these three species, with D. subobscura being broader niched than the other two species. There was an indication that D. phalerata was adapted to those yeasts normally found on its natural breeding site, Phallus impudicus. Many factors were found to affect the nutritional adequacy of the yeasts, including their growth period, the larval density, the presence of competitors and particularly the chemical composition of the basal growth medium.     

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.