Search

Background RNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss—most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila. Results Here we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression. Conclusions These results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible., BMC Evolutionary Biology, 19, ISSN:1471-2148

TheDrosophila obscuraspecies group shows dramatic variation in karyotype, including transitions among sex chromosomes. Members of theaffinisandpseudoobscurasubgroups contain a neo-X chromosome (a fusion of the X with an autosome), and it was shown that ancestral Y genes of Drosophila have become autosomal in species that contain the neo-X. Detailed analysis in species of thepseudoobscurasubgroup revealed a translocation of ancestral Y genes to the small dot chromosome of that group. Here, we show that the Y-dot translocation is restricted to thepseudoobscurasubgroup, and translocation of Y genes in theaffinissubgroup followed a different route. We find that most ancestral Y genes moved independently to autosomal or X-linked locations in different taxa of theaffinissubgroup, and we propose a dynamic model of sex chromosome formation and turnover in theobscuraspecies group. Our results show that Y genes can find unique paths to escape an unfavorable genomic environment.

The Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromere, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

Theory predicts that selfish genetic elements that increase their transmission are prone to originate on sex chromosomes but create strong selective pressure to evolve suppressors due to reduced fertility and distorted population sex ratios. Here we show that recurrent genetic conflict over sex chromosome transmission appears to be an important evolutionary force that has shaped gene content evolution of sex chromosomes in Drosophila. We demonstrate that convergent acquisition and amplification of spermatid expressed gene families are common on Drosophila sex chromosomes, and especially on recently formed ones, and harbor characteristics typical of meiotic drivers. We carefully characterize one putative novel cryptic sex chromosome distortion system that arose independently several times in members of the Drosophila obscura group. Co-amplification of the S-Lap1/GAPsec gene pair on both the X and the Y chromosome occurred independently several times in members of the D. obscura group, where this normally autosomal gene pair is sex-linked due to a sex chromosome - autosome fusion. Investigation of gene expression and short RNA profiles at the S-Lap1/GAPsec system suggest that meiotic drive and suppression likely involves RNAi mechanisms. Our finding suggests that recurrent conflict over sex chromosome transmission has shaped widespread genomic and evolutionary patterns, including the epigenetic regulation of sex chromosomes, the distribution of sex-biased genes, and the evolution of hybrid sterility.

Baits – fermented food products – are generally attractive to many types of insects, which makes it difficult to sort through non-target insects to monitor a pest species of interest. We test the hypothesis that a chemically simpler and more defined attractant developed for a target insect is more specific and attracts fewer non-target insects than a chemically more complex food-type bait. A four-component chemical lure isolated from a food bait and optimized for the spotted wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), was compared to the original wine/vinegar bait to assess the relative responses of non-target insects. In several field experiments in Washington State, USA, it was shown that numbers of pest muscid flies, cutworm and armyworm moths, and pest yellowjackets were reduced in traps baited with the chemical lure compared to the wine/vinegar bait. In other field experiments in the states of Washington, Oregon, and New York, numbers of non-target drosophilid flies were also reduced in traps baited with the chemical lure relative to wine/vinegar bait. In Washington, numbers of Drosophila melanogaster Meigen and Drosophila obscura Fallen species groups and Drosophila immigrans Sturtevant were reduced in the chemical lure traps, whereas in New York, D. melanogaster and D. obscura species groups, D. immigrans, Drosophila putrida Sturtevant, Drosophila simulans Sturtevant, Drosophila tripunctata Loew, and Chymomyza spp. numbers were reduced. In Oregon, this same effect was observed with the D. melanogaster species group. Taken together, these results indicate that the four-component SWD chemical lure will be more selective for SWD compared to fermentation baits, which should reduce time and cost involved in trapping in order to monitor SWD.

The vinegar flies Drosophila subobscura and D. obscura frequently serve as study organisms for evolutionary biology. Their high morphological similarity renders traditional species determination difficult, especially when living specimens for setting up laboratory populations need to be identified. Here we test the usefulness of cuticular chemical profiles collected via the non-invasive method near-infrared spectroscopy for discriminating live individuals of the two species. We find a classification success for wild-caught specimens of 85%. The species specificity of the chemical profiles persists in laboratory offspring (87-92% success). Thus, we conclude that the cuticular chemistry is genetically determined, despite changes in the cuticular fingerprints, which we interpret as due to laboratory adaptation, genetic drift and/or diet changes. However, because of these changes, laboratory-reared specimens should not be used to predict the species-membership of wild-caught individuals, and vice versa. Finally, we demonstrate that by applying an appropriate cut-off value for interpreting the prediction values, the classification success can be immensely improved (to up to 99%), albeit at the cost of excluding a considerable portion of specimens from identification.

Drosophila obscura group species produce two distinct sizes of nucleated sperm that differ only in head and tail lenghts. Between both sperm there is no differences in location of the acrosome and flagellum during spermiogenesis where each sperm type develops in its own bundle. Fertile sperm accumulate in the seminal vesicles. Fertilization is ex- clusively monospermic and in a previous study we suggested that both types of sperm are fertilization-competent on the basis of similar DNA content and storage in females also if morph variations are consistent with a fertilization-related se- lection for optimal sperm size. This assumption is in agreement with previous studies that demonstrated that only long sperm fertilize eggs. In this study fertilization of Drosophila subobscura is examined using anti-sperm surface � -N- acetylhexosaminidases and � -L-fucosidase antibodies. Beta hexosaminidases are intrinsic proteins of the sperm plasma membrane in spermomomorphic species of the melanogaster group closely related to Drosophila melanogaster. These enzymes had been previously identified as putative receptors for glycoconjugates of the egg surface, structurally and func- tionally conserved. Here their localization has been investigated in Drosophila subobscura. Consistent with our previ- ous study, short and long sperm are functionally equivalent. More data are needed to clarify the consequences and adapta- tive significance of morph variations.

The chromosomal location of the histone genes was determined in seven species of the Drosophila obscura group by in situ hybridization. Histone genes occur on more than one site per genome and on non-homologous chromosome elements. In addition, the metaphase karyotypes and the banding pattern of the polytene chromosomes were compared. Based on chromosomal characters, the cladogenesis of the D. obscura group was established. From the distribution of histone sites in different species, analysed in this paper and in previous studies, the phylogenetic history of histone gene transposition was derived. The molecular mechanisms responsible for the generation of new histone sites are discussed.

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 funfmal kleiner als solche, die von nativen Proteinen kommen. Aufgrund der Untersuchungsergebnisse wird angenommen, das die Radiation der Arten der obscura-Gruppe in zwei evolutiven Schuben erfolgt sei; der erste Schub hatte zu zumindest vier palaerktischen (bifasciata, obscura mit D. subsilvestris, subobscura und microlabis) und zwei proto-ne arktischen Linien (affinis, pseudoobscura) gefuhrt. In einem zweiten Schub waren dann die endgultigen rezenten Arten entstanden.

The heterochromatin of the chromosomes of Drosophila gunche consists mainly of a satellite DNA composed of multiple, tandemly arranged copies of a 290 b p basic sequence. Five clones containing one or two copies of the basic unit were sequenced. As expected from CsCl density centrifugation and AT specific staining of mitotic chromosomes the sequence is AT rich. The average nucleotid variability between the cloned sequences is 11.6%. In situ hybridization on the mitotic chromosomes revealed, that this satellite DNA is present in the centromeric regions of all chromosomes but the Y. The nucleotide variability between copies of different tandem clusters seems to be higher than between members of the same cluster. The copy number of the sequence in the haploid genome was estimated to be approximately 80000. The sequence is species specific and is not present in the genome of sibling species D. subobscura and D. madeiren-sis. The evolutionary origin of the satellite DNA and its possible role in species formation is discussed.

The phylogenetic relationships among nine species of Drosophila belonging to the obscura group were investigated by establishing (according to their banding similarities) the homologous chromosome segments of element E (equivalent to chromosome O of D. subobscura). The phylogenetic relationships were based on the existence of segments in different triads of species, which could only be produced by overlapping inversions. This permitted the ordering of the species belonging to each triad. Drosophila obscura, D. ambigua and D. tristis were found to be very closely related and thus forming a cluster in which D. ambigua occupies an intermediate position between the other two species. Drosophila obscura seems to be the species more directly linked to three other separate lineages, that of D. subsilvestris, the two African species (D. microlabis and D. kitumensis), and the subobscura cluster. The species from this last cluster may be ordered as follows: D. subobscura-D. madeirensis-D. guanche. It is not clear which species of this triad is the direct link to D. obscura. These results completely agree with those produced in an independent study, where element B was considered for the same nine species. Furthermore, the present study clarifies some ambiguities concerning the phylogenetic relationships which remained obscure due to the conservative nature of chromosome B.

Argonaute2 (Ago2) is a rapidly evolving nuclease in theDrosophila melanogasterRNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. Here we reconstruct the history of Ago2 duplications across theDrosophila obscuragroup, and use patterns of gene expression to infer new functional specialization. We show that some duplications are old, shared by the entire species group, and that losses may be common, including previously undetected losses in the lineage leading toD. pseudoobscura. We find that while the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogues have independently specialized to testis-specific expression. Using population genetic analyses, we show that most testis-specific paralogues have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogues. We speculate that the repeated evolution of testis-specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. Our study highlights the lability of RNAi pathways, even within well-studied groups such asDrosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in non-model species.Supporting DataAll new sequences produced in this study have been submitted to Genbank asKX016642-KX016771.

The Drosophila obscura clade consists of about 41 species, of which 20 were used for analyses of wing and thorax length. Our primary goal was to investigate the magnitude of sexual size dimorphism (SSD) of these traits within this clade and to test Rensch's Rule [when females are larger than males, SSD (e.g., female/male ratio) should decrease with body size]. Our secondary goal was methodological and involved evaluating for these flies alternative measures of SSD (female/male ratio, female/male absolute difference, female/male relative difference), developing a bootstrap method to estimate the magnitude of intraspecific variation in SSD, and applying a new method of estimating allometric relationships that is phylogenetically based and incorporates error variance in both traits. All indices of SSD were strongly correlated for both size traits. Nevertheless, female/male ratio is the best index here: it is easily interpretable and essentially independent of size. For both traits, SSD (F/M) varied interspecifically, showed a strong phylogenetic signal, but did not differ for the main phylogenetic subgroups or correlate with latitude. Factors underlying variation in SSD in this clade are elusive and might include genetic drift. SSD (wing) tended to decrease with increasing size, as predicted by Rensch's Rule, though not consistently so. SSD (thorax) was unrelated to size. However, analysis of published data for thorax length of Drosophila spp. (N=42) with a larger size range showed that SSD decreased significantly with increasing size (consistent with Rensch's Rule), suggesting our ability to detect SSD-size relations in the D. obscura data may be limited by low statistical power.

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.

The actin genes of five nearctic species of the Drosophila obscura group were mapped by in situ hybridization, using the 5C actin gene of D. melanogaster as a probe. In all species but D. azteca eight actin loci were observed variously dispersed over all five (A- E) chromosomal elements. In D. azteca ten actin hybridization sites were found; four of which most probably originated by duplications or by transposition events. Although the five nearctic species differ from all other Drosophila species of the D. obscura group so far studied in the number of loci as well as in the chromosomal distribution and location of the actin loci, the uniformity of the main pattern with six actin loci throughout the genus Drosophila reinforces the hypothesis that the chromosomal elements have maintained their essential identities during the course of evolution. Our findings are in accordance with the conclusion that the nearctic D. obscura species have differentiated from a common ancestor of the palearctic species and that they belong to two distinct subgroups, the pseudoobscura and the affinis subgroups.

The bilbo element is a non-LTR retrotransposon isolated from Drosophila subobscura. We conducted a distribution survey by Southern blot for 52 species of the family Drosophilidae, mainly from the obscura and melanogaster groups. Most of the analyzed species bear sequences homologous to bilbo from D. subobscura. In the obscura group, species from the same species subgroup also share similar Southern blot patterns. To investigate the phylogenetic relationship among these elements, we analyzed eight copies of a short sequence of the element from several species of the obscura group. The obtained phylogram agrees with the phylogeny of the species, which suggests vertical transmission of the element.

P elements of two different subfamilies designated as M- and O-type are thought to have invaded host species in the Drosophila obscura group via horizontal transmission from external sources. Sequence comparisons with P elements isolated from other species suggested that the horizontal invasion by the O-type must have been a rather recent event, whereas the M-type invasion should have occurred in the more distant past. To trace the phylogenetic history of O-type elements, additional taxa were screened for the presence of O- and M-type elements using type-specific PCR primers. The phylogeny deduced from the sequence data of a 927-bp section (14 taxa) indicate that O-type elements have undergone longer periods of regular vertical transmission in the lineages of the saltans and willistoni groups of Drosophila. However, starting from a species of the D. willistoni group they were transmitted horizontally into other lineages. First the lineage of the D. affinis subgroup was infected, and finally, in a more recent wave of horizontal spread, species of three different genera were invaded by O-type elements from the D. affinis lineage: Scaptomyza, Lordiphosa, and the sibling species D. bifasciata/D. imaii of the Drosophila obscura subgroup. The O-type elements isolated from these taxa are almost identical (sequence divergence

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.

A new Neotropical member of the Drosophila obscura species group from El Salvador and Honduras, Drosophila maya, is described. This species belongs to the New World pseudoobscura subgroup and, based on a variety of internal and external morphological characters, is most closely related to D. lowei. This species is distinguished from other members of the pseudoobscura subgroup by the shape of the hypandrium and by having fewer teeth on the sex combs. Ecologically, D. maya inhabits cloud forests at elevations around 2,000 m in El Salvador and Honduras. A key to eight members of the obscura species group which have been recorded in and near the Neotropical region is presented.

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 α-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. subsilvestris 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.

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.

The nematodes Parasitylenchus diplogenus and Howardular aoronymphium (Allantonematidae) are parasites of drosophilids (Diptera). Nematodes were found in 3/14 drosophilid species sampled in Danish woodlands: Drosophila phalerata Meigen (3.5% parasitism) was parasitized by H. aoronymphium and D. obscura Fallen (0.5%) and D. subobscura Collin (2.1%) by P. diplogenus. Parasitism was generally rare, and few drosophilid species parasitized, compared to elsewhere in Europe. Parasitism was positively correlated with relative host abundance both within Denmark and across communities, suggesting that species diversity is promoted by a disproportionately high parasitism of more common host species. The prevalence of H. aoronymphium, but not P. diplogenus, parasitism is positively related to temperature across communities.

Males of many species exhibit sperm heteromorphism, in which multiple morphologies of sperm are produced in a common testis (see references in [1]). Polymegaly, a form of sperm heteromorphism, is found in the Drosophila obscura group and is characterized by the production of two size classes of nucleated sperm that differ only in head and tail lengths [1,2]. Both the length and the ratio of sperm types produced is species-specific [1,3–5], and each sperm type develops in its own bundle deriving from a single stem cell [4]. Previous studies suggested that both types of sperm are fertilization-competent on the basis of similar ultrastructure [6], DNA content [6], nuclear protein transition during spermatogenesis [7], and storage in females [3–5]. However, a previous study demonstrated that only long sperm fertilize eggs [3]. Here, we extend this study to examine fertilization in six obscura group species using anti-sperm antibodies and digital deconvolution microscopy. Consistent with the previous study, we found that all eggs were fertilized by only the long sperm type, even in polyspermic eggs. Moreover, sperm entry and position during and following fertilization were similar to other Drosophila groups [3,8–10]. Thus, polymegaly and its maintenance appear to have arisen independently of fertilization processes per se .

Summary Males of the Drosophila obscura species group develop sperm of more than one length class. Also the lengths of the nuclei are different. When the total sperm length and the nucleus length were subject to a cluster analysis, it was shown that in D. subobscura and in D. affinis two classes of sperm exist. In D. azteca three classes were found. In the analyzed species the lengths of short sperm are more similar than the lengths of long ones. For example, the short sperm of D. subobscura and D. affinis have the same length while the long sperm of the two species are significantly different. This fact supports the idea that in evolution there is a tendency towards an increase in the length of sperm. But according to our data, in species with sperm heteromorphism preferentially the length of the long class is increased. In the two species D. subobscura and D. affinis, the size of the sperm nuclei corresponded to the total length of sperm. Long sperm had longer nuclei than short sperm. In D. azteca only ...

The 5S rDNA repeats of ten species of the Drosophila obscura group were amplified by PCR and characterized. In the coding regions of the 5S rDNA genes, only very few differences were detected between the species. The non-coding intergenic spacers of the sibling species D. subobscura, D. madeirensis and D. guanche , and D. bifaciata and D. imaii , respectively, are almost identical, but differ substantially between other more distantly related species. For the most part, short insertions/deletions are the main source of interspecific sequence variability. Consequently, an alignment of the intergenic 5S rDNA spacers of distantly related species is almost impossible. The value of phylogenetic networks based on the 5S rDNA spacer sequences appears to be rather limited compared to those derived from other data with too few informative differences between closely related species and too many between those slightly more distantly related.

The Drosophila obscura group consists of several dozen Nearctic and Palearctic species, with a phylogeny that remains largely unresolved in spite of numerous morphological, cytogenetic, and molecular investigations. We have partially sequenced two genes, Gpdh (about 1000 bp) and Sod (about 700 bp) in 12-13 species and 1 subspecies in order to settle the issues. Difficulties in resolving the phylogeny emanate from the rapid sequence of successive radiations. Nevertheless, the following conclusions are warranted: (1) The Palearctic species include two monophyletic subgroups, subobscura (which may be the most ancient clade of the whole group) and obscura, plus two other species with unresolved phylogenetic positions, D. bifasciata and D. subsilvestris; (2) the Nearctic species form a monophyletic group consisting of two monophyletic sister clades, the affinis subgroup and the pseudoobscura subgroup. The Palearctic radiation may have resulted from adaptation to expanding temperate forests in the Old World. A second radiation occurred during the colonization of the deciduous forests of the New World by the descendants of a single lineage that soon split into the affinis and pseudoobscura subgroups.

We report on a form of sperm polymorphism, termed polymegaly, that occurs in species of the Drosophila obscura group. Individual males of species in this group characteristically produce more than one discrete length of nucleated, motile sperm. Hypotheses suggested to explain the evolutionary significance of sperm polymorphism have been either nonadaptive or adaptive, with the latter focusing on sperm competition or nutrient provisioning. These hypotheses assume all sperm types fertilize eggs; however, no data have been gathered to test this assumption. We found that two size classes of sperm are produced and transferred to females in approximately equal numbers by the male; only long sperm persist in significant numbers in female sperm storage organs. Furthermore, we used a DNA-specific dye (bisbenzimide) and sperm-specific antibodies to ask if both sperm types fertilize eggs in Drosophila pseudoobscura. Confocal microscopy and immunofluorescent analyses of fertilized eggs using anti-sperm polyclonal antisera demonstrated that only long sperm participate in fertilization. These data falsify those hypotheses in which all sperm types are assumed to be functionally equivalent (fertilize eggs). Any remaining or new hypotheses for the evolutionary significance of polymegaly must incorporate these findings. Several new areas of research are suggested.

Thirteen species of the Drosophila obscura group that were investigated consistently exhibited the remarkable phenomenon of having basically two kinds of fertilizing eusperm: short and long. They are produced synchronously by one single male gonad and transferred to the female. Sperm heteromorphism therefore appears to be a phylogenetic trait that evolved uniquely in the obscura group. The sperm heteromorphic pattern differs among males of the same species mainly because the long class is broad and comprises an assortment of sperm varying from one male to another. However, the short class distribution is narrow and similar regardless of the male. The range of the distribution is of the same order of magnitude regardless of the male or population concerned. Moreover, the between-population matches the between-male variation. The implications on sperm competition are discussed.

Homologies of the sections of the polytene chromosomes of element C, among several species of the obscura group of the genus Drosophila, were established according to the similarity of their banding patterns. The information gathered was used to construct an unrooted phylogenetic tree based on qualitative criteria. This tree is compared to three other similar trees derived from independent information provided by the study of chromosomal elements B, D and E. The general congruence of the patterns of these trees proves the well-foundedness of this approach. A single exception to this congruence is discussed. Finally a consensus tree, encapsulating information from all chromosomal elements, is presented and its topology is compared to those derived from electrophoretic data.

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.Key words: Drosophila obscura group, phylogeny, chromosomal inversions, element D, chromosomal gene arrangements.

Between species, variation in sperm size has been related to male-female coevolution and male-male competition. In contrast, variation within species is poorly understood. A particular case of intraspecific sperm-size variation occurs in sperm-heteromorphic species, where males produce distinct sperm morphotypes, usually only one of which is fertile. This allows to investigate sperm size variation under different selection regimes. Nonfertile morphotypes, whose role is aside from fertilization, may have other functions, and this may be reflected by changes in develop- mental processes and a different phenotype compared to fertile sperm. We show that the intra- specific coefficient of variation in sperm length is up to four times lower for fertile than nonfertile morphotypes across 150 sperm-heteromorphic species (70 butterfly, 71 moth, 9 diopsid fly species). This is in agreement with a previous study on 11 species in the Drosophila obscura group. Sig- nificantly lower variation in fertile than nonfertile sperm morphometry may result from fertiliza- tion-related selection for optimal sperm size, novel functions of nonfertile sperm, or from tighter control of fertile sperm development. More data are needed to clarify the consequences and adaptive significance of within-morph variation, and its consistent pattern across sperm-hetero- morphic insects.

The evolution of heteromorphic sex chromosomes creates a genetic condition favoring the invasion of sex-ratio meiotic drive elements, resulting in the biased transmission of one sex chromosome over the other, in violation of Mendel's first law. The molecular mechanisms of sex-ratio meiotic drive may therefore help us to understand the evolutionary forces shaping the meiotic behavior of the sex chromosomes. Here we characterize a sex-ratio distorter on the X chromosome (Dox) in Drosophila simulans by genetic and molecular means. Intriguingly, Dox has very limited coding capacity. It evolved from another X-linked gene, which also evolved de nova. Through retrotransposition, Dox also gave rise to an autosomal suppressor, not much yang (Nmy). An RNA interference mechanism seems to be involved in the suppression of the Dox distorter by the Nmy suppressor. Double mutant males of the genotype dox; nmy are normal for both sex-ratio and spermatogenesis. We postulate that recurrent bouts of sex-ratio meiotic drive and its subsequent suppression might underlie several common features observed in the heterogametic sex, including meiotic sex chromosome inactivation and achiasmy., Author Summary Mendel's first law of genetics states that two alleles of a heterozygote are transmitted to the next generation at an equal ratio. The cornerstone of population genetics, this law states that the evolutionary fate of genetic variants is solely governed by their contribution to the good of their carriers. However, meiotic drive genes—which skew transmission in their own favor—can evolve under certain circumstances, even though they cause harm to the genome as a whole. Meiotic drive elements are often enriched on the two sex chromosomes (i.e., the X and the Y) because of a lack of recombination between them. Here we describe the genetic and molecular characterization of a meiotic drive distorter on the X chromosome in Drosophila simulans. This distorter apparently formed de nova from yet another new gene. To fight back against this harmful distorter, the D. simulans genome has evolved an ingenious mechanism based on DNA sequence homology. We postulate that repeated meiotic drive invasion and its suppression could be a major mechanism for genome evolution, underlying the ultimate cause for the inactivation of sex chromosome during meiosis and the occasional loss of recombination (achiasmy), which is observed only in the heterogametic (XY) sex., X-linked genes gain transmission advantage by killing Y-bearing sperm. The first case iof this class of rogue genes, also called sex-ratio distorters, has been identified in Drosophila simulans.