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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.

Centromeres are the basic unit for chromosome inheritance, but their evolutionary dynamics is poorly understood. We generate high-quality reference genomes for multipleDrosophila obscuragroup species to reconstruct karyotype evolution. All chromosomes in this lineage were ancestrally telocentric and the creation of metacentric chromosomes in some species was driven byde novoseeding of new centromeres at ancestrally gene-rich regions, independently of chromosomal rearrangements. The emergence of centromeres resulted in a drastic size increase due to repeat accumulation, and dozens of genes previously located in euchromatin are now embedded in pericentromeric heterochromatin. Metacentric chromosomes secondarily became telocentric in thepseudoobscurasubgroup through centromere repositioning and a pericentric inversion. The former (peri)centric sequences left behind shrunk dramatically in size after their inactivation, yet contain remnants of their evolutionary past, including increased repeat-content and heterochromatic environment. Centromere movements are accompanied by rapid turnover of the major satellite DNA detected in (peri)centromeric regions.

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.

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 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.

Insects are host to a diverse range of vertically transmitted micro-organisms, but while their bacterial symbionts are well-studied, little is known about their vertically transmitted viruses. We have found that two sigma viruses (Rhabdoviridae) recently discovered in Drosophila affinis and Drosophila obscura are both vertically transmitted. As is the case for the sigma virus of Drosophila melanogaster, we find that both males and females can transmit these viruses to their offspring. Males transmit lower viral titers through sperm than females transmit through eggs, and a lower proportion of their offspring become infected. In natural populations of D. obscura in the United Kingdom, we found that 39% of flies were infected and that the viral population shows clear evidence of a recent expansion, with extremely low genetic diversity and a large excess of rare polymorphisms. Using sequence data we estimate that the virus has swept across the United Kingdom within the past ∼11 years, during which time the viral population size doubled approximately every 9 months. Using simulations based on our lab estimates of transmission rates, we show that the biparental mode of transmission allows the virus to invade and rapidly spread through populations at rates consistent with those measured in the field. Therefore, as predicted by our simulations, the virus has undergone an extremely rapid and recent increase in population size. In light of this and earlier studies of a related virus in D. melanogaster, we conclude that vertically transmitted rhabdoviruses may be common in insects and that these host–parasite interactions can be highly dynamic.

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.

We describe three new species of the Drosophila obscura species group, D. hypercephala, D. hideakii and D. quadrangula Gao & Toda, spp. nov., all discovered from high altitudes (>1500 m) on Mt Kinabalu, Sabah, Malaysia. These are the southernmost distribution records of the D. obscura group in the Oriental Region. The three new species are each morphologically similar to different species of the Old World D. obscura species subgroup. This suggests that they are descendants of different founder species that independently colonized the tropics from the subtropical or temperate zones during the glacial ages. The new species D. hypercephala is hypercephalic not only in males but also, although less distinct, in females, unlike other hypercephalic drosophilid species in which only males are hypercephalic. This new finding of a hypercephalic species from the subgenus Sophophora, of which species are relatively easy to culture in the laboratory and include the model species such as D. melanogaster and D. pseudoobscura, will promote genetic analyses on the development and the evolution of hypercephaly.

Unable to form cellular capsules around large foreign bodies, the species Drosophila subobscura Collin in Gordon, 1936 was previously shown devoid of lamellocytes, the capsule-forming hemocytes in Drosophila melanogaster Meigen, 1830. This unusual case of deficiency in encapsulation ability was remarkable enough to motivate further investigations in phylogenetically related species of the obscura group. Like D. subobscura, the species Drosophila azteca Sturtevant and Dobzhansky, 1936, Drosophila bifasciata Pomini, 1940, Drosophila guanche Monclus, 1976, Drosophila miranda Dobzhansky, 1935, Drosophila persimilis Dobzhansky and Epling, 1944, and Drosophila pseudoobcura Frovola and Astaurov, 1929 were found to be unable to encapsulate large foreign bodies and also to lack lamellocytes. Surprisingly, Drosophila affinis Sturtevant, 1916, Drosophila tolteca Patterson and Mainland, 1944, and Drosophila obscura Fallen, 1823 were capable of mounting cellular capsules, although their encapsulation abilities remained weak. These three species were free of lamellocytes but possessed small pools of never before described “atypical hemocytes” present in the hemolymph when capsules were formed.

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.

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.

Three new and two known species of the Drosophila (Sophophora) obscura species-group are reported from Yunnan Province, southern China. The sinobscura species-subgroup is newly established by D. sinobscura, D. hubeiensis and D. luguensis sp. nov. Geographic distribution of the obscura group in and around China is discussed, and a key to 10 Chinese species of the obscura group is provided.

Five morphometrical traits (wing and thoraxlength, 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/thoraxratio, which may be related to flight capacity, showed little variation. Ovariole number was highly variabl e (range 27–53) both within and between species, and was positively correlated with the wing/thoraxratio, 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 thoraxwith a median darkening was observed in sixspecies. 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.

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.