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During precopulatory courtship, male Drosophila typically produce wing vibration to generate species-specific songs before mounting females. Three species in the lini clade of the montium species subgroup have been found to produce species-specific sine song only after mounting and during copulation. Here we investigated and analyzed the courtship behavior of 29 species in the montium subgroup from video and song recordings and measured the duration of wing vibration. We describe a great diversity of courtship behavior in the montium subgroup. The courtship patterns can be categorized into four types in the montium subgroup: 1) type P/C, species with both precopulatory and copulatory courtship, such as D. parvula and D. nikananu, 2) type P-/C, species with sporadic precopulatory and mainly copulatory courtship, such as D. auraria and D. triauraria. 3) type C, species with only copulatory courtship, such as D. tani and D. pectinifera, 4) type C-, species with only very brief copulatory courtship, such as D. rufa and D. asahinai. According to a phylogenetic tree based on sequences of mitochondrial COI and COII, and the nuclear Adh, both precopulatory courtship and copulatory courtship were present in the most basal species D. parvula. Each of two branches in the montium subgroup contains four types of courtship behavior. Type C is present in each sub-branch. These results suggest that the courtship behavior initially involved both precopulatory and copulatory courtship, but that subsequently precopulatory courtship has gradually been lost in the montium subgroup. We suggest reasons why precopulatory behavior might come to be lost in the montium subgroup.

Analyses of the nucleotide sequences of the duplicatedAmy genes in the eight species of theDrosophila melanogaster species subgroup have revealed concerted evolution of the coding regions and divergent evolution between the duplicated genes of the 5’-flanking regions. Homogenization between the duplicated genes in the coding region is maintained by frequent genetic exchange in various portions of the coding region. On the other hand, such genetic exchange seems to produce a large amount of DNA sequence variation and protein polymorphism at the two loci within a species. The puzzling observation that concerted evolution is restricted to the coding regions seems to be explained by not only adaptive evolution of the AMY proteins in speciation but also adaptive fixation of selectively advantageous mutations in the intergenic region that differentiate expression of the twoAmy genes. We review molecular work on theAmy gene system inDrosophila, including evidence from biochemical characterization of the AMY proteins and molecular characterization of the cis regulatory elements.

The nucleotide sequences of the 5'-flanking regions of the duplicated Amy genes in eight sibling species belonging to the melanogaster species subgroup are analyzed. In Drosophila melanogaster, a region of about 450 bp immediately upstream of the translation initiation site of the two paralogous genes (the proximal and distal genes) has sequence similarities. However, we could not detect any significant sequence similarity in the region more upstream than -450. This result indicates that the coding regions of the ancestral Amy gene were duplicated together with 450 bp of the 5'-flanking region as one unit. Multiple alignment of these 450-bp sequences in the proximal and distal genes of all eight species revealed a mosaic pattern of highly conserved and divergent regions. The conserved regions included almost all the putative regulatory elements identified in previous analyses of the sequences. A phylogenetic analysis of the aligned sequences shows that these 450-bp sequences are clustered into the proximal and the distal groups. As a whole, the divergence between groups in this region is very large in contrast to that in the coding regions. Based on the divergence between groups, the 450-bp region is divided into two subregions. We found that the ratios of the divergence between groups to that within groups differ in the two subregions. From these observations, we discuss a possibility of positive selection acting on the subregion immediately upstream of the Amy coding region to cause divergence of regulatory elements of the paralogous genes.

From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a "specialist" species for host plants and probably also occurs in the process of speciation in general.

Drosophila leontia (warm adapted) has been considered as a sister species of Drosophila kikkawai (sub-cosmopolitan) with a very similar morphology. We found divergent strategies for coping with desiccation stress in these two species of montium subgroup. Interestingly, in contrast to clinal variation for body melanization in D. kikkawai, cuticular lipid mass showed a positive cline in D. leontia across a latitudinal transect. On the basis of isofemale line analysis, within population trait variability in cuticular lipid mass per fly is positively correlated with desiccation resistance and negatively correlated with cuticular water loss in D. leontia. A comparative analysis of water budget of these two species showed that higher abdominal melanization, reduced rate of water loss and greater dehydration tolerance confer higher desiccation resistance in D. kikkawai while the reduced rate of water loss is the only possible mechanism to enhance desiccation tolerance in D. leontia. The use of organic solvents has supported water proofing role of cuticular lipids in D. leontia but not in D. kikkawai. Thus, we may suggest that body melanization and cuticular lipids may represent alternative strategies for coping with dehydration stress in melanic versus non-melanic drosophilids. In both these species, carbohydrates were utilized under desiccation stress but a higher level of stored carbohydrates was evident in D. kikkawai. Further, we found increase desiccation resistance in D. kikkawai through acclimation while D. leontia lacks such a response. Thus, species specific divergence in water balance related traits in these species are consistent with their adaptations to wet and dry habitats.

Among subtropical species of the Drosophila takahashii species subgroup, temperature adaptations were related to altitudinal distribution; D. trilutea was adapted to lower temperature and occurred at high altitudes in September in Taiwan, D. takahashii was adapted to higher temperature and occurred mainly at low altitudes, and D. prostipennis was adapted to an intermediate range of temperature and occurred at mid to high altitudes. A temperate species, D. lutescens, was adapted to a wider range of temperature than the subtropical species: it was more cold-hardy than all three subtropical species and more heat-tolerant than D. prostipennis and D. trilutea. The temperature ranges encountered in the temperate region far exceed the range in subtropical areas, and this is likely to be reflected in the temperature adaptations of these temperate and subtropical species. D. lutescens has a photoperiodically controlled reproductive diapause. No latitudinal cline was observed on cold-hardiness of D. takahashii. By ...

A review of the native Hawaiian leafhopper genus Nesophrosyne (Hemiptera: Cicadellidae: Deltocephalinae) is presented. Specimens were examined from across the entire Hawaiian Archipelago, Nesophrosyne is redescribed, and the subgenus Nesoreias is synonymized with Nesophrosyne. Eight new species associated with the widespread host plant species Broussaisia arguta are described: N. heopoko sp. n. from Kaua’i; N. makaihe sp. n. from O’ahu; N. magnaccai sp. n. from Moloka’i; N. broussaisiai sp. n., N. ogradyi sp. n., and N. kaupoi sp. n. from Maui; and, N. aakokohaikea sp. n. and N. kanawao sp. n. from Hawai’i Island. Morphological and molecular characters were employed to delineate new species. Populations associated with B. arguta on different islands, individual volcanic mountains, and discrete geographic areas represent reciprocally monophyletic species. A monophyletic complex of five sibling species, morphologically cryptic on individual islands, were identified from Maui and Hawai’i Island. The kanawao species group is erected for these species and is further subdivided into two species subgroups based on monophyly, island endemicity, and morphology: broussaisiai species subgroup containing N. broussaisiai, N. ogradyi, and N. kaupoi on Maui; and, aakokohaikea species subgroup containing N. aakokohaikea and N. kanawao on Hawai’i Island.

The present study analyzed the drosophilid assemblages in different levels of urbanization in the city of Porto Alegre, Rio Grande do Sul, Brazil. Collections were carried out in 2008 in three different environments: a highly urbanized area—“Jardim Botânico,” a forested area with intermediary urbanization—“Parque Gabriel Knijnik,” and in a relatively well-preserved forested area, although threatened by the urban growth—“Morro Santana.” In Jardim Botânico, 36 species belonging to four genera were found, with high abundance of exotic species as Drosophila simulans Sturtevant and Zaprionus indianus (Gupta). In Parque Gabriel Knijnik, 33 species that belonged to four genera were found, with higher abundances of native species belonging to the Drosophila tripunctata species group and Drosophila willistoni species subgroup, and lower abundance of exotic species. As for Morro Santana, 32 species and three genera were found, with higher abundances of native groups, low representativeness of exotic species, and absence of Zaprionus indianus. The analysis of the Jaccard index showed higher similarity in the species composition between samples collected in summer and autumn, and between samples collected in winter and spring. On the other hand, the Morisita index differentiated Jardim Botânico from the other two studied sites. Our results show that Morro Santana is an important area of native biodiversity, reinforcing, therefore, the inclusion of this area in the project for the creation of an ecological corridor as proposed by the Ministry of the Environment of Brazil.

Triacylglycerols are the major fuel for basal metabolism during the winter in temperate species of the Drosophila melanogaster species group. Differential scanning calorimetry analysis revealed that the transition temperatures of triacylglycerols were lower in diapausing adults than in reproducing ones, and also lower in species or strains adapted to cooler climates than those adapted to warmer climates. These phenomena were correlated to the fatty acid compositions of the triacylglycerols; the proportion of unsaturated fatty acids in the triacylglycerols was higher in the diapausing individuals, and in the species or strains adapted to cooler climates. Furthermore, in the temperate species of the montium species subgroup (D. subauraria, D. biauraria, D. triauraria, and D. rufa), the amount of saturated triacylglycerols was smaller than the value expected on the assumption that fatty acids are randomly distributed in the triacylglycerols, suggesting the nonrandom distribution of unsaturated fatty acids among triacylglycerols. This may facilitate the lowering of the transition temperature of triacylglycerols, and hence may be related to the ability of Drosophila to cope with temperate climates.

The population biology and molecular evolution of the transposable element mariner has been studied in the eight species of the melanogaster subgroup of the Drosophila subgenus Sophophora. The element occurs in D. simulans, D. mauritiana, D. sechellia, D. teissieri, and D. yakuba, but is not found in D. melanogaster, D. erecta, or D. orena. Sequence comparisons suggest that the mariner element was present in the ancestor of the species subgroup and was lost in some of the lineages. Most species contain both active and inactive mariner elements. A deletion of most of the 3' end characterizes many elements in D. teissieri, but in other species the inactive elements differ from active ones only by simple nucleotide substitutions or small additions/deletions. Active mariner elements from all species are quite similar in nucleotide sequence, although there are some species-specific differences. Many, but not all, of the inactive elements are also quite closely related. The genome of D. mauritiana contains 20-30 copies of mariner, that of D. simulans 0-10, and that of D. sechellia only two copies (at fixed positions in the genome). The mariner situation in D. sechellia may reflect a reduced effective population size owing to the restricted geographical range of this species and its ecological specialization to the fruit of Morinda citrifolia.

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The nine species of the Drosophila melanogaster subgroup are currently one of the best systems for studying the genetic basis of species differences and speciation. However, despite the availability of complete genome sequences from most of these species, no comprehensive accounting of their phenotypic differences has been made. We gathered data from about 200 papers published between 1919 and 2008 that report phenotypic differences between species of the D. melanogaster species subgroup and we compiled 163 morphological, physiological and behavioral traits that differ between at least two species of the D. melanogaster subgroup. Our compilation is available online at www.normalesup.org/~vorgogoz/FlyPhenomics. The FlyPhenomics database provides a useful starting point for Drosophila researchers who aim to identify the mutations underlying phenotypic variation.

The distribution of the transposable element mariner was examined in the genus Drosophila. Among the eight species comprising the melanogaster species subgroup, the element is present in D. mauritiana, D. simulans, D. sechellia, D. yakuba and D. teissieri, but it is absent in D. melanogaster, D. erecta and D. orena. Multiple copies of mariner were sequenced from each species in which the element occurs. The inferred phylogeny of the elements and the pattern of divergence were examined in order to evaluate whether horizontal transfer among species or stochastic loss could better account for the discontinuous distribution of the element among the species. The data suggest that the element was present in the ancestral species before the melanogaster subgroup diverged and was lost in the lineage leading to D. melanogaster and the lineage leading to D. erecta and D. orena. This inference is consistent with the finding that mariner also occurs in members of several other species subgroups within the overall melanogaster species group. Within the melanogaster species subgroup, the average divergence of mariner copies between species was lower than the coding region of the alcohol dehydrogenase (Adh) gene. However, the divergence of mariner elements within species was as great as that observed for Adh. We conclude that the relative sequence homogeneity of mariner elements within species is more likely a result of rapid amplification of a few ancestral elements than of concerted evolution. The mariner element may also have had unequal mutation rates in different lineages.

Based on the specimens collected from Myanmar (Burma), 5 new species of the Drosophila melanogaster species-group, viz., D. flavicauda, D. pyo, D. nyinyii, D. fuyamai, and D. myamaungi, are described, along with collection records of 20 known species of the same species-group, of which 11 species are new to Myanmar. Two new species-subgroups, the D. rhopaloa and the D. longissima subgroups, are established.

Female fruitflies of certain species of Drosophila possess cuticular hydrocarbons which act as contact pheromones, capable of inducing male courtship. In three species of the D. melanogaster species subgroup (D. melanogaster, D. sechellia and D. erecta) adult males and females have hydrocarbons of different lengths and structures. In four other species (D. simulans, D. mauritiana, D. yakuba and D. teissieri) there is no qualitative difference between the sexes. Observations of courtships showed that hydrocarbons act as courtship-inducing pheromones in all the species studied here. Males can be induced to court other flies, regardless of sex or species, if the hydrocarbons of the courted fly correspond to the main female cuticular hydrocarbon of the species of the courting male. Males from species that do not show a hydrocarbon dimorphism responded mainly to the principal hydrocarbon of their species. Males from the dimorphic species tended to respond to a wider range of hydrocarbons, and also showed high levels of courtship with young flies of both sexes and all species, whose hydrocarbons had some structural similarities to those of homotypic adult females.

DROSOPHILA melanogaster has played a central role in genetics research since the Morgan lab in the early years of the previous century. Yet, it has played a lesser role in the study of speciation. This is due to the fact that, until recently, there was only one closely related species, D. simulans, and the hybrids between the two species were sterile. Other groups, such as pseudoobscura, provided better opportunities. This changed recently with the discovery in Africa of several closely related species, making a total of nine. This article describes the discovery of these species and some of their salient features not likely to be known to geneticists.

Sixteen species, of which 13 are new, in the subgenus Hebridosimulium Grenier & Rageau are now known. Fiji has only Simulium laciniatum, which is widely distributed on five islands. Tahiti has only one species, the enigmatic S. anatolicum. The remaining species are in Vanuatu and known from nine islands, ranging the full length and breadth of the archipelago. Hebridosimulium species are divided among two species groups. The callipygium species group is characterized, in part, by a smooth posterior abdomen in the larvae, and all but one species possess dorsal tubercles on the larval abdomen. Simulium callipygium is the type species, with the tuberculate species in a subgroup comprised of S. banksi, S. johnfrumi, S. pankumuense, and S. tuberculum. The second species group has larvae with the posteroventral abdomen markedly expanded, and all species lack dorsal tubercles. Constituents of the steatopygium species group are S. anatolicum, S. jolyi, S. laciniatum, S. lucyae, S. paradisium, S. steatopygium, S. subparadisium, S. supercilium, and S. undulatum. Two other species of this group, S. adsonense and S. spiroi, of which larvae have markedly pigmented heads, are assigned to the adsonense species subgroup. A chromosome map for Hebridosimulium is established for the first time.

Genome size varies considerably among organisms due to differences in the amplification, deletion, and divergence of various kinds of repetitive sequences, including the transposable elements, which constitute a large fraction of the genome. However, while the changes in genome size observed at a wide taxonomic level have been thoroughly investigated, we still know little about the process involved in closely related species. We estimated genome sizes and the reverse transcriptase-related sequence (RTRS) content in the nine species of the Drosophila melanogaster species subgroup. We showed that the species differ with regard to their genome size and that the RTRS content is correlated with genome size for all species except Drosophila orena. The genome of D. orena, which is 1.6-fold as big as that of D. melanogaster, has in fact not undergone any major increase in its RTRS content.

The Odysseus (OdsH) gene was duplicated from its ancestral neuron-expressed gene, unc-4, and then evolved very rapidly under strong positive Darwinian selection as a speciation gene causing hybrid-male sterility between closely related species of the Drosophila simulans clade. Has OdsH also experienced similar positive selection between Drosophila sibling species other than those of the simulans clade? We cloned and sequenced OdsH and unc-4 from two clades of the Drosophila montium species subgroup, the Drosophila lini and the Drosophila kikkawai clades. The ratios of Ka/Ks for OdsH were remarkably low between sibling species of these two clades, suggesting that OdsH has been subjected to strong purifying selection in these two clades.

Parthenogenetic strains of several species have been found in the genus Drosophila. The mode of diploidization in the eggs of females has been found to be post-meiotic nuclear fusion. The genetic basis for this parthenogenesis is not understood but is believed to be under the control of a complex polygenic system. We found parthenogenetic females in an isofemale strain (LAE345) of D. pallidosa-like collected in 1981 at Lae, Papua New Guinea, and established a parthenogenetically reproducing strain. Parthenogenetic strains of D. ananassae and D. pallidosa collected at Taputimu, American Samoa had also been established by Futch (1972). D. ananassae, D. pallidosa and D. pallidosa-like are very closely related species belonging to the ananassae complex of the ananassae species subgroup of the melanogaster species group. Using these three species, we found that more than 80% of females from parthenogenetic strains produced progeny parthenogenetically and that inter-specific hybrid females also produced impaternate progeny. In the present report, we demonstrate that the mode of parthenogenesis of D. ananassae appears to be the post-meiotic nuclear doubling of a single meiotic product, and that a major gene responsible for the parthenogenesis maps to the left arm of the second chromosome of D. ananassae. We also suggest that the genetic basis for parthenogenesis capacity may be identical among the three closely related species. We discuss the function of the gene required for parthenogenesis and its significance for the evolutionary process.

Chromosomal arrangement was a historically important character used for defining taxonomic boundaries. The Drosophila virilis species group exhibits a series of chromosomal rearrangements, and the resulting differences among karyotypes were primary characters originally used to define taxa within the group. However, some chromosomally divergent forms have not been sufficiently resolved in phylogenetic reconstructions of DNA sequences from several nuclear genes. Sequences of mitochondrial regions have the potential for finer-scale resolution of closely related taxa; therefore, sequences of two mitochondrial genes were used to examine phylogenetic relationships within the chromosomally variable virilis subgroup. Sequences were obtained from multiple strains of the Palearctic species, D. virilis and D. lummei, and the Nearctic species, D. novamexicana and two chromosomal forms of D. americana. Analyses support the recent emergence of the different chromosomal forms in North America. However, none of these chromosomally divergent forms exhibit reciprocal monophyly of their mtDNA sequences, which is the requirement for attaining genealogical species status.

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an ∼7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.

The Drosophila melanogaster subgroup has been the focus of numerous studies about evolution. We address the question of how the olfactory code has evolved among the nine sister species. By using in vivo electrophysiological measurements, so called single-cell recordings, we have established the ligand affinity of a defined subset of olfactory receptor neurons (ORNs) across all nine species. We show that the olfactory code as relayed by the investigated subset of ORNs is conserved to a striking degree. Distinct shifts in the code have occurred only within the simulans clade. However, these shifts are restricted to an altered tuning profile of the same single ORN type in all three of the simulans siblings and a more drastic change unique to D. sechellia, involving a complete loss of one sensillum type in favour of another. The alterations observed in D. sechellia may represent a novel host-specific adaptation to its sole host, morinda fruit (Morinda citrifolia). The overall high degree of similarity of the code within the subgroup is intriguing when considering the great variety in distributions as well as in habitat and host choice of the siblings, factors that could greatly affect the olfactory system.

Although molecular and phenotypic evolution have been studied extensively in Drosophila melanogaster and its close relatives, phylogenetic relationships within the D. melanogaster species subgroup remain unresolved. In particular, recent molecular studies have not converged on the branching orders of the D. yakuba–D. teissieri and D. erecta–D. orena species pairs relative to the D. melanogaster–D. simulans–D. mauritiana–D. sechellia species complex. Here, we reconstruct the phylogeny of the melanogaster species subgroup using DNA sequence data from four nuclear genes. We have employed “vectorette PCR” to obtain sequence data for orthologous regions of the Alcohol dehydrogenase (Adh), Alcohol dehydrogenase related (Adhr), Glucose dehydrogenase (Gld), and rosy (ry) genes (totaling 7164 bp) from six melanogaster subgroup species (D. melanogaster, D. simulans, D. teissieri, D. yakuba, D. erecta, and D. orena) and three species from subgroups outside the melanogaster species subgroup [D. eugracilis (eugracilis subgroup), D. mimetica (suzukii subgroup), and D. lutescens (takahashii subgroup)]. Relationships within the D. simulans complex are not addressed. Phylogenetic analyses employing maximum parsimony, neighbor-joining, and maximum likelihood methods strongly support a D. yakuba–D. teissieri and D. erecta–D. orena clade within the melanogaster species subgroup. D. eugracilis is grouped closer to the melanogaster subgroup than a D. mimetica–D. lutescens clade. This tree topology is supported by reconstructions employing simple (single parameter) and more complex (nonreversible) substitution models.

The action spectra of mating activity among the six species of the Drosophila melanogaster species subgroup were compared to understand how light wavelength affects mating activity. The species fell into three groups with respect to the action spectrum of mating activity. We chose one representative species from each of the three types for detailed study: D. melanogaster, D. sechellia and D. yakuba. The mating activities were investigated under three different light intensities of three monochromatic lights stimulus. Each species showed a unique spectral and intensity response. To know the evolutionary meaning of the light wavelength dependency of mating activity, we superimposed the type of action spectrum of mating activity in these six species on a cladogram. Mating inhibition under UV was conserved in evolution among these species. Furthermore we clarified that D. melanogaster showed low mating activity under UV because males courted less under UV.

We determined the nucleotide sequences of two regions in the A+T-rich region of mitochondrial DNA (mtDNA) in the siI and siII types of D. simulans, the maII type of D. mauritiana, and D. sechellia. The sequences were aligned with those of the corresponding regions of siIII of D. simulans and maI of D. mauritiana, D. melanogaster, and D. yakuba. The type I and type II elements and the T-stretches were detected in all eight of the mtDNA types compared, indicating that the three elements are essential in the A+T-rich region of this species subgroup. The alignment revealed several short repetitive sequences and relatively large deletions in the central portions of the region. In the highly conserved sequence elements in the type II elements, the substitution rates were not uniform among lineages and acceleration in the substitution rate might have been due to loss of functional constraint in the stem–loop-forming sequences predicted in the type II elements. Patterns of nucleotide substitutions observed in the A+T-rich region were further compared with those in the coding regions and in the intergenic regions of mtDNA. Substitutions between A and T were particularly repressed in the highly conserved sequence elements and in the intergenic regions compared with those in the A+T-rich region excluding the highly conserved sequence elements and in the fourfold degenerate sites in the coding regions. The functional and structural characteristics of the A+T-rich region that might be involved in this substitutional bias are discussed.

Publisher Summary This chapter reviews a paper in which photoperiodic responses of several species from temperate and subtropical areas are examined. It focuses on the Drosophila montium species subgroup of the melanogaster species group. This subgroup is diversified in tropical regions, but only six species are known from temperate areas indicating that the colonization of this subgroup in temperate areas was rather recent in the evolutionary time scale. Therefore, this subgroup provides an opportunity to study the early stage of evolution of photoperiodism. The results of the study showed that there was no sign of photoperiodic response in the subtropical species from Taiwan, whereas a distinct photoperiodic response was observed in D. rufa from warm-temperate areas. In D. rufa and D, asahinai from islands located at the boundary of subtropical and warm-temperate regions, the effect of photoperiod on cold tolerance was observed when reared at 11°C, but was not at 15°C. It is questionable whether the photoperiodic response of D, asahinai is functional, because mean January (the coldest month) temperature of the islands it occurs is about 15°C. In this subgroup, photoperiodism might have evolved under circumstances cooler than these islands of the present, and D. asahinai might have weakened the photoperiodic response as a result of its adaptation to the present chmatic conditions of the islands.

Molecular evolution of the histone multigene family was studied by cloning and sequencing regions of the histone 3 gene in the Drosophila melanogaster species subgroup. Analysis of the nucleotide substitution pattern showed that in the coding region synonymous changes occurred more frequently to A or T in contrast to the GC-rich base composition, while in the 3′ region the nucleotide substitutions were most likely in equilibrium. These results suggested that the base composition at the third codon position of the H3 gene, i.e., codon usage, has been changing to A or T in the Drosophila melanogaster species subgroup.

In this study, the region corresponding to the Thr–Gly region of the period (per) gene in the Drosophila nasuta subgroup of species was sequenced. The results showed that this region was highly conserved in the D. nasuta subgroup. There were only nine variable sites found in this 300-bp-long region, all located in two small regions highly variable among Drosophila species. No length variation was observed either within this subgroup or in the Yunnan (YN) population of D. albomicans. The deduced amino acid sequences were identical for all 14 taxa in the D. nasuta subgroup, and a stretch of alternating Thr–Gly pairs was not observed in this subgroup. A phylogenetic tree was constructed. The clustering of some species was in general agreement with previous works, but it also raised some question on the phylogenetic relationship between the nasuta species. The data did not implicate the Thr–Gly region playing a role in behavioral isolation in this subgroup of Drosophila.

R2 elements are non-long-terminal-repeat (non-LTR) retrotransposons that insert specifically in the 28S rRNA genes of many insects. Previous reports concerning this element in the genus Drosophila have suggested that R2 elements are absent from many species of this genus, particularly those species from the subgenus Drosophila. In this report, we present an extensive study of the distribution and evolution of R2 elements in Drosophila. A PCR survey of 59 species from 23 species groups of the two major Drosophila subgenera found that R2 elements are present in all but two species of the melanogaster species subgroup. Phylogenetic analysis based on partial nucleotide sequences of R2 elements from 23 species demonstrates that the relationships of R2 elements are congruent with those of the Drosophila species phylogeny, suggesting that these elements have been vertically inherited since the divergence of this genus some 60 MYA. Sequence variation between different copies of R2 elements within each species was less than 0.16%, indicating that these elements are undergoing concerted evolution similar to that of the 28S genes. Several properties of the R2 sequences suggest that these elements depend on retrotransposition in addition to simple recombination to remain within the rDNA locus: the rates of synonymous substitutions averaged 4.8 times the rate of replacement substitutions, 82 of 83 R2 copies partially sequenced contained intact open reading frames, and, finally, length variation associated with the poly(A) 3' tails indicated that many R2 copies are the direct result of retrotransposition.

In contrast to male genitalia that typically exhibit patterns of rapid and divergent evolution among internally fertilizing animals, female genitalia have been less well studied and are generally thought to evolve slowly among closely-related species. As a result, few cases of male-female genital coevolution have been documented. In Drosophila, female copulatory structures have been claimed to be mostly invariant compared to male structures. Here, we re-examined male and female genitalia in the nine species of the D. melanogaster subgroup. We describe several new species-specific female genital structures that appear to coevolve with male genital structures, and provide evidence that the coevolving structures contact each other during copulation. Several female structures might be defensive shields against apparently harmful male structures, such as cercal teeth, phallic hooks and spines. Evidence for male-female morphological coevolution in Drosophila has previously been shown at the post-copulatory level (e.g., sperm length and sperm storage organ size), and our results provide support for male-female coevolution at the copulatory level.

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.

R1 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts into a specific sequence of insect 28S ribosomal RNA genes. We have previously shown that this element has been maintained through vertical transmission in the melanogaster species subgroup of Drosophila. To address whether R1 elements have been vertically transmitted for longer periods of evolutionary time, the analysis has been extended to 11 other species from four species groups of the genus Drosophila (melanogaster, obscura, testecea, and repleta). All sequenced elements appeared functional on the basis of the preservation of their open-reading frames and consistently higher rate of substitution at synonymous sites relative to replacement sites. The phylogenetic relationships of the R1 elements from all species analyzed were congruent with the species phylogenies, suggesting that the R1 elements have been vertically transmitted since the inception of the Drosophila genus, an estimated 50-70 Mya. The stable maintenance of R1 through the germ line appears to be the major mechanism for the widespread distribution of these elements in Drosophila. In two species, D. neotestecea of the testecea group and D. takahashii of the melanogaster group, a second family of R1 elements was also present that differed in sequence by 46% and 31%, respectively, from the family that was congruent with the species phylogeny. These second families may represent occasional horizontal transfers or, alternatively, they could reflect the ability of R1 elements to diverge into new families within a species and evolve independently.

The sequences of the mitochondrial DNA (mtDNA) segment containing the two intergenic regions were determined for six species belonging to theDrosophila immigrans species group and compared to the corresponding segments ofDrosophila species which had been studied previously. We found remarkable differences in the evolutionary rates of the two intergenic regions. The Intergenic I region, which lies between thetRNA gln and thetRNA ile genes, was found to be highly conserved in terms of both size (30 ntp) and nucleotide sequence among the species studied. In contrast, the sequences of the Intergenic II region, which lies between thetRNA f-met and thetRNA ile genes, showed considerable variation. The size of the Intergenic II region ranged from 0 to 88 ntp, and accurate alignment was possible only among sequences from geographical strains or very closely related species in thenasuta species subgroup. The observed differences in conservation of the two mtDNA intergenic regions are discussed in light of functional constraints on mtDNA sequences.

R1 and R2 are non-long-terminal repeat retrotransposable elements that insert into specific sequences of insect 28S ribosomal RNA genes. These elements have been extensively described in Drosophila melanogaster. To determine whether these elements have been horizontally or vertically transmitted, we characterized R1 and R2 elements from the seven other members of the melanogaster species subgroup by genomic blotting and nucleotide sequencing. Each species was found to have homogeneous families of R1 and R2 elements with the exception of erecta and orena, which have no R2 elements. The DNA sequences of multiple R1 and R2 copies from each species indicated nucleotide divergence within each species averaged only 0.48% for R1 and 0.35% for R2, well below the level of divergence among the species. Most copies of R1 and R2 (40 of 47) sequenced from the seven species were potentially functional, as indicated by the absence of premature termination codons or translational frameshifts that would destroy the open reading frame of the element. The sequence relationships of both the R1 and R2 elements from the various members of the melanogaster subgroup closely followed that of the species phylogeny, suggesting that R1 and R2 have been stably maintained by vertical transmission since the origin of this species subgroup 17-20 million years ago. The remarkable stability of R1 and R2, compared to what has been suggested for transposable elements that insert at multiple locations in these same species, may be due to their unique specificity for sites in the rRNA gene locus. Under low copy number conditions, when it is essential for any mobile element to transpose, the insertion specificities of R1 and R2 ensure uniform developmentally regulated target sites that can be occupied with little or no detrimental effect on the host.

A comparative study was done on the enzymological features of alpha-amylase among six sibling species belonging to the Drosophila melanogaster species subgroup. The pH optima were 7.6 for D. melanogaster, D. mauritiana and D. simulans and 7.4 for D. erecta, D. teissieri and D. yakuba. The temperature optima were commonly 37 C in all species studied. However, there are two types of temperature dependence; a strict one as observed in D. melanogaster, D. mauritiana and D. simulans, and a flat one as observed in D. erecta, D. teissieri and D. yakuba. These separations are consistent with the general phylogeny within this species subgroup. The substrate dependence was almost the same in all the species studied. The Vmax and Km were also estimated for each species. We found clear differences in the enzymological features between the species. Thus these differences might reflect an adaptation of the amylase enzyme system to speciation in this species subgroup.

Mitochondrial DNA (mtDNA) restriction-site maps for six species (10 strains) of the Drosophila montium subgroup were established. A total of 50 restriction sites were mapped, corresponding to 1.67% of the mtDNA genome. On the basis of differences in the restriction sites, nucleotide divergence (delta) was calculated for each pair of species (strains), and phylogenetic trees were constructed by using distance-matrix and parsimony methods. Comparison of the resultant phylogenetic trees shows that the sibling species D. auraria and D. quadraria are closely related. At the other extreme, considerable divergence was observed between the two strains of D. serrata and between D. serrata and D. birchii, a finding that contrasts with their grouping within the same species complex. Nevertheless, our data indicate that these six oriental montium species are rather closely related.

The Threonine-Glycine (Thr-Gly) region of the period gene (per) in Drosophila was compared in the eight species of the D. melanogaster subgroup. This region can be divided into a diverged variable-length segment which is flanked by more conserved sequences. The number of amino acids encoded in the variable-length region ranges from 40 in D. teissieri to 69 in D. mauritiana. This is similar to the range found within natural populations of D. melanogaster. It was possible to derive a Thr-Gly “allele” of one species from that of another by invoking hypothetical Thr-Gly intermediates. A phylogeny based on the more conserved flanking sequences was produced. The results highlighted some of the problems which are encountered when highly polymorphic genes are used to infer phylogenies of closely related species.

The transposable element mariner occurs widely in the melanogaster species group of Drosophila. However, in drosophilids outside of the melanogaster species group, sequences showing strong DNA hybridization with mariner are found only in the genus Zaprionus. The mariner sequence obtained from Zaprionus tuberculatus is 97% identical with that from Drosophila mauritiana, a member of the melanogaster species subgroup, whereas a mariner sequence isolated from Drosophila tsacasi is only 92% identical with that from D. mauritiana. Because D. tsacasi is much more closely related to D. mauritiana than is Zaprionus, the presence of mariner in Zaprionus may result from horizontal transfer. In order to confirm lack of a close phylogenetic relationship between the genus Zaprionus and the melanogaster species group, we compared the alcohol dehydrogenase (Adh) sequences among these species. The results show that the coding region of Adh is only 82% identical between Z. tuberculatus and D. mauritiana, as compared with 90% identical between D. tsacasi and D. mauritiana. Furthermore, the mariner gene phylogeny obtained by maximum likelihood and maximum parsimony analyses is discordant with the species phylogeny estimated by using the Adh genes. The only inconsistency in the mariner gene phylogeny is in the placement of the Zaprionus mariner sequence, which clusters with mariner from Drosophila teissieri and Drosophila yakuba in the melanogaster species subgroup. These results strongly suggest horizontal transfer.

We have analyzed approximately 18 kb of DNA in and upstream of the defective chorion-1 (dec-1) locus of the eight known species of the melanogaster species subgroup of Drosophila. The restriction maps of D. simulans, D. mauritiana, D. sechellia, D. erecta, and D. orena are shown to have basically the restriction map of D. melanogaster, whereas the maps of D. teissieri and D. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65-200 bp) is found in a repeated sequence of the central transcribed region of D. melanogaster, D. simulans, and D. erecta. Restriction site mapping indicated that the dec-1 gene is highly conserved in the melanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5' translated region. The 5' flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within the melanogaster species subgroup except that the erecta-orena pair is placed closer to the melanogaster complex than to D. teissieri and D. yakuba.

Mitochondrial DNA (mtDNA) restriction site maps for nine species of theDrosophila obscura subgroup and forDrosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for theobscura subgroup species, one Nearctic, represented byDrosophila pseudoobscura, and four Palearctic, two grouping the related triads of speciesDrosophila subobscura, Drosophila madeirensis, Drosophila guanche, andDrosophila ambigua, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each,Drosophila bifasciata, andDrosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related speciesD. subobscura, D. madeirensis, andD. guanche, the pairD. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for theobscura subgroup species was from 15.5 kb forD. pseudoobscura to 17.1 forD. tristis.

Approximately 100 abundant proteins were analyzed by two-dimensional electrophoresis (2DE) in eighteen species belonging to three taxonomically differentDrosophila groups, theD. melanogaster species subgroup, theD. auraria complex and theD. virilis species group. Genetic distances between these species were calculated from a total number of protein differences in the pairs of species. Based on such data of genetic distances a dendrogram was constructed. The genetic distances or phylogeny obtained by 2DE were significantly correlated with those for one-dimensional (conventional) electrophoresis and also with data on mating success between species. It was discussed on the basis of these studies that a technique of 2DE is a useful adjunctive tool in systematics, including the previous reports with different kinds of data.

The X-linked α subunit of larval serum protein 1 (LSP1-α) is shown to lack dosage compensation in six members of the melanogaster species subgroup, viz., Drosophila melanogaster, D. simulans, D. mauritiana, D. erecta, D. yakuba, and D. teissieri, by quantitative filter hybridization and by electrophoretic and autoradiographic analyses of fat body proteins. These results support the hypothesis that there is little selection pressure on the LSP1-α gene to acquire dosage compensation.

The polytene chromosomes of two new species of Drosophila, D. sechellia and D. orena, both members of the melanogaster species subgroup, are described. The chromosomes of D. sechellia, a species endemic to certain islands in the Seychelles, are homosequential with those of D. simulans and D. mauritiana. The chromosomes of D. orena, a species from the mountains of west Africa, are very similar to those of D. erecta. We discuss the interrelationships of the eight known species of the melanogaster species subgroup, based upon an analysis of their chromosome banding patterns.

[A quantitative comparison and sequence analysis of courtship behaviour of four-day old virgin flies from seven species of the Drosophila melanogaster species subgroup is described. In three species (D. melanogaster, D. simulans and D. mauritiana) courtship was observed until copulation. In four species (D. yakuba, D. teissieri, D. erecta and D. orena) courtships were truncated after a standard period. Wing rowing by the male was seen in four species (simulans, mauritiana, yakuba and teissierz). In both mauritiana and simulans the proportion of time spent in rowing tended to increase as courtship proceeded. The four species showing the greatest proportion of wing vibration in courtship (melanogaster, yakuba, erecta and orena) also showed an increase in the proportion of time spent vibrating as courtship proceeded. The amount of wing vibration and wing extension in yakuba is greater than in any other species in the subgroup. The proportion of time spent in wing extension in yakuba declined whilst vibration increased during courtship. In six species the proportion of courtship time spent licking increased during courtship. The mean lick duration was longer in yakuba than in any other species of the subgroup. When males were following actively moving females the time they spent performing courtship behaviours and the mean bout lengths were shorter than in courtship as a whole. D. simulans males expressed scissoring at a low level when courting moving females. Females of all species preened more than the males, but preening during courtship appeared to have no excitatory or inhibitory effect on the male. The behavioural sequence characteristic of the species subgroup is orientation→wing display→licking→ copulation. In five species wing vibration was the only element of wing display immediately to precede licking, but in simulans licking could also be preceded by scissoring and in yakuba by wing extension. The transition vibration→licking appears to be a characteristic of the subgroup., A quantitative comparison and sequence analysis of courtship behaviour of four-day old virgin flies from seven species of the Drosophila melanogaster species subgroup is described. In three species (D. melanogaster, D. simulans and D. mauritiana) courtship was observed until copulation. In four species (D. yakuba, D. teissieri, D. erecta and D. orena) courtships were truncated after a standard period. Wing rowing by the male was seen in four species (simulans, mauritiana, yakuba and teissierz). In both mauritiana and simulans the proportion of time spent in rowing tended to increase as courtship proceeded. The four species showing the greatest proportion of wing vibration in courtship (melanogaster, yakuba, erecta and orena) also showed an increase in the proportion of time spent vibrating as courtship proceeded. The amount of wing vibration and wing extension in yakuba is greater than in any other species in the subgroup. The proportion of time spent in wing extension in yakuba declined whilst vibration increased during courtship. In six species the proportion of courtship time spent licking increased during courtship. The mean lick duration was longer in yakuba than in any other species of the subgroup. When males were following actively moving females the time they spent performing courtship behaviours and the mean bout lengths were shorter than in courtship as a whole. D. simulans males expressed scissoring at a low level when courting moving females. Females of all species preened more than the males, but preening during courtship appeared to have no excitatory or inhibitory effect on the male. The behavioural sequence characteristic of the species subgroup is orientation→wing display→licking→ copulation. In five species wing vibration was the only element of wing display immediately to precede licking, but in simulans licking could also be preceded by scissoring and in yakuba by wing extension. The transition vibration→licking appears to be a characteristic of the subgroup.]

Phylogenic relationships among 21 members (15 species) of the Drosophila montium species subgroup were investigated by O'Farrell's two-dimensional electrophoresis (2DE) and by starch gel electrophoresis (SDE). Phylogenic positions of two unclassified species [D. punjabiensis-like and D. sp. (Iriomote)] as well as those of two new species (D. bocki and D. leontia) within the D. kikkawai complex could be assigned, and it was evidenced that both electrophoretic techniques are adjunctively useful tools in taxonomy. Although the magnitude of genetic differentiation may be different between protein loci surveyed by 2DE and allozyme loci obtained by SDE, the relationship of genetic distances was highly correlated between two data sets of 2DE and SDE, as shown previously by Aquadro and Avise (1981). The biochemical dendrograms in the subgroup were constructed by the data sets (2DE and SDE) of genetic distances between species. The genetic relationships were consistent with the results of interspecific hybridization among these species.

The melanogaster species subgroup of Drosophila comprises six sibling species. The interrelationship between these species has been studied by analysis of the banding patterns of their polytene chromosomes. The species fall into two groups: (1) melanogaster, simulans and mauritiana and (2) erecta, teissieri and yakuba . The former group are chromosomally closely related, indeed simulans and mauritiana are homosequential. The latter group (all African endemic species) are less closely related although they all share eight autosomal inversions of the standard (i. e. melanogaster ) sequence. From this shared sequence the chromosomes of the three African endemic species have diverged considerably by many paracentric inversions. Both D. teissieri and D. yakuba are polymorphic; we describe nine and four inversion sequences in them respectively. D. erecta is monomorphic although our sample size is very small (only two populations). We discuss both the origin of interspecific inversions, especially the problem of inversion breakpoint coincidence, and the light this study throws upon evolutionary relationships within this group of species.