Evolution and population biology of theperiodgene

The clock gene period (per), originally identified inDrosophila, evolves relatively quickly within the insects; probably for this reason, no convincingperhomologues have been identified outside this class. However antibodies toDrosophilaPER have labelled neural pacemakers in other organisms, including mammals. Conserved regions, such as the PAS dimerization domain, reflect its functional importance, but the long Thr–Gly repeat encoded withinperis not conserved outside theDrosophila. The repeat appears to be a component of the temperature compensation system in the fly. This is reflected in the population structure of natural Thr–Gly length variants ofD. melanogaster. Patterns of nucleotide variation withinDrosophila perhave been used to examine the selective forces that have shaped the evolution of this gene.     

Molecular Phylogeny of the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Species Subgroup

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. Present address (Ryan M. David): University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, USA     

Temporal analysis of tone pulses within the courtship songs of two siblingDrosophila species,their interspecific hybrid,and behavioral mutants ofD. melanogaster (Diptera: Drosophilidae)

Temporal analyses were applied to the tone pulses within the courtship songs of Drosophila melanogaster, D. simulans,their interspecific hybrid, and behavioral mutants of D. melanogaster.Linear regression was performed on various parameters of the song pulses (cycles per pulse, absolute peak amplitude, intrapulse frequency, number of peaks in fast Fourier transform, width of the primary frequency peak, and interpulse interval), as a function of their positions within pulse trains. Significant differences in the slope values of these two species and of the mutant genotypes allowed for discriminative quantification of temporal changes within trains. These results are discussed in relation to previous kinds of temporal analyses of Drosophilacourtship songs and also with regard to the mechanisms of song production.     

Drosophila virilis histone gene clusters lacking H1 coding segments

Summary Approximately 30–40% ofDrosophila virilis DNA complementary to clonedDrosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as theD. melanogaster core histone genes in the plasmid cDm500: . Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30–40% ofD. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution ofDrosophila. The ancestors of modernDrosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in thevirilis andmelanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes ofD. virilis andD. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently,D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes ofD. virilis andD. melanogaster are >25% divergent. Our estimate of sequence divergence in the H1 genes ofD. virilis andD. melanogaster seems high until one considers that the coding sequences of cloned H1 genes from the closely related speciesD. melanogaster andD. simulans are 5% divergent.     

Courtship songs and genetic control of their acoustic characteristics in sibling species of the Drosophila melanogaster subgroup

Six sibling species of the melanogaster subgroup differ in their wing displays and in the acoustic characteristics of their courtship songs. D. melanogaster, D. simulans and D. mauritiana have distinct but similar courtship songs. D. mauritiana, which is allopatric to the others, produces, an ambivalent song with sine song frequency and intrapulse frequency like simulans and modal interpulse interval like melanogaster. These three species appear to be behaviourally more closely similar to each other than to the three African species D. yakuba, D. teissieri and D. erecta. The acoustic characteristics of the songs of interspecific hybrids, indicate that the interpulse interval and intrapulse frequency are quantitatively controlled by genes located on the autosomes. The ability to generate sine song may be controlled by one or more genes located on the X-chromosome, but an alternative possibility, of autosomal dominant inheritance, cannot be excluded.     

The Effect of Male Competition on the Courtship Song of Drosophila melanogaster

The courtship song of Drosophila has been extensively used as a model system for studies of sexual selection and species recognition. Traditionally, the courtship song has been recorded from males placed individually with a female. However, under natural conditions females are exposed to multiple courting males, and the effect of their joint signal on mate recognition by the female is not yet understood. Here, we recorded the courtship behavior of D. melanogaster males singing either individually to a female lpar;1:1) or in the presence of an additional male lpar;2:1). We compared the structure of the male song in the two experimental designs. Our results show that when two males courted a female their songs could overlap each other. Males produced a significantly different signal in the presence of competition; the duration of each song component was significantly shorter and the rate of singing was markedly lower. The present study demonstrates that male competition can dramatically alter the acoustic signal detected by the female.     

Behavioral Sequence Leading to Sexual Isolation Between <Emphasis Type="Italic">Drosophila ananassae</Emphasis> and <Emphasis Type="Italic">D. pallidosa</Emphasis>

Drosophila ananassae and D. pallidosa are closely related, sympatric species that lack postmating isolation. Sexual isolation has been considered important in maintaining them as independent species. To clarify the behavioral processes leading to sexual isolation, we analyzed behavioral sequences and examined the effect of courtship song on mating success and on behaviors of both sexes by surgically removing male wings (song generators), female aristae (song receivers), or female wings (means of fluttering). We found that heterospecific courtship songs evoked female wing fluttering, whereas conspecific courtship song did not. Furthermore, female wing fluttering made courting males discontinue courtship. These findings suggest that strong sexual isolation is achieved through the following behavioral sequence: heterospecific song→female wing fluttering→courtship discontinuation.     

Variation and repeatability of courtship song characters among wild-caught and laboratory-rearedDrosophila montana andD. littoralis males (Diptera: Drosophilidae)

InDrosophila montana andD. littoralis (species of theD. virilis group), females use male courtship song in their mate choice in wild preferring males which produce short and dense sound pulses (Aspi and Hoikkala, 1995). In the present study these song characters were found to be repeatable among overwintered males. Male progenies of wild-caught flies reared in the laboratory, and inD. montana also the males collected in wild before overwintering, exhibited very little variation between males in these characters. Contrary to pulse characters, pulse train characters measured forD. montana song varied significantly between laboratory-reared males. Our findings suggest that inD. montana andD. littoralis song characters playing a part in sexual selection in the wild are more condition dependent than song characters which are not the direct targets of female choice.     

Variation in male wing song characters inDrosophila plantibia (Hawaiian picture-wingedDrosophila group)

The males of most species of the HawaiianDrosophila planitibia group produce songs when vibrating their wings during courtship. In four of the most recently evolved species,D. differens, D. planitibia, D. heteroneura, andD. silvestris, these songs are simple in structure and possess a higher and more variable carrier frequency than the songs of the more ancestral species of the group. In the present paper, we studied the variation in wing song production and song characters in aD. planitibia population. Some males vibrated their wings at a very low amplitude or slow speed, producing no detectable sound. Other males produced sound bursts varying in carrier frequency and burst length. The carrier frequency of the song depended mainly on the wing posture of the male during wing vibrations and was consistent for individual males. Variation between males of different isofemale progenies was not significant in any measured song trait. Songs of the males recorded in the present study differed, however, from songs of the males of a laboratory strain recorded earlier.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

The function of courtship song rhythms in Drosophila

The courtship songs of Drosophila are produced by the male's wing vibration and consist of a series of pulses, with an inter-pulse interval (IPI) of 34 ms for D. melanogaster and 48 ms for D. simulans. The IPI's are not constant in length during courtship, but oscillate sinusoidally with 55-s cycles in the former species and 35-s cycles in the latter. We have stimulated D. melanogaster females with artificially generated courtship songs, and have observed that they mate fastest when the song incorporates a 55-s oscillation superimposed on a 34-ms IPI. D. simulans females, on the other hand, mate fastest with a 48-ms IPI and a 35-s oscillation period. Consequently these newly-discovered song cycles produce significant mating enhancement in these species, with the females showing a preference for songs which carry both the species-specific IPI and the species-specific IPI rhythm.     

Adaptation to a Starch Environment and Regulation of alpha-Amylase in Drosophila

The adaptation to glucose and starch foods insix species, D. melanogaster, D.virilis, D. saltans, D. funebris,D. levanonensis and D. americana, wasstudied by measuring productivity. D.melanogaster and D. virilis adapted more to thestarch environment than to the glucose environment,while D. saltans adapted more to the glucoseenvironment than to the starch environment. D.funebris, D. levanonensis, and D. americana did not distinctlyadapt to either environment. In addition, the regulationof amylase in the six species was investigated bymeasuring the levels of amylase activity with glucoseand starch food environments. The levels of amylaseactivity in D. levanonensis and D.saltans were substantially low, indicating thatthese species cannot utilize starch as a carbon source.The starch-adapted species, D. melanogaster and D.virilis, showed higher levels of amylase activitywith the starch environment and higher inducibility.These results suggest that changing the regulation ofamylase is important for the adaptation to a starch environment inDrosophila.     

Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa

Sexual isolation has been considered one of the primary causes of speciation and its genetic study has the potential to reveal the genetics of speciation. In Drosophila, the importance of courtship songs in sexual isolation between closely related species has been well investigated, but studies analysing the genetic basis of the difference in the courtship songs associated with sexual isolation are less well documented. Drosophila ananassae and Drosophila pallidosa are useful for studies of sexual isolation, because of their sympatric distribution and absence of postmating isolation. Courtship songs are known to play a crucial role in sexual isolation between these two species, and the female discrimination behaviour against the courting male has been revealed to be controlled by a very narrow region on the second chromosome. In this study we investigated the genetic basis controlling the song differences associated with their sexual isolation, using intact and wingless males with chromosomes substituted between species. The results obtained from F₁ hybrid males between these species indicate the dominance of the song characters favoured by D. pallidosa females. In addition, the results obtained from backcross F₂ males indicate that chromosome 2 had a major effect on the control of the song characters associated with sexual isolation.     

Evolutionary divergence of the cytochrome b5 gene of Drosophila

Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b₅ (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b₅ locus encodes an essential product of the Drosophila system.Correspondence to: C.E. Rozek     

Gypsy homologous sequences inDrosophila subobscura (gypsyDS)

Summary Characterization of sequences homologous to theDrosophila melanogaster gypsy transposable element was carried out inDrosophila subobscura (gypsyDS). They were found to be widely distributed among natural populations of this species. From Southern blot and in situ analyses, these sequences appear to be mobile in this species.GypsyDS sequences are located in both euchromatic and heterochromatic regions. A completegypsyDS sequence was isolated from aD. subobscura genomic library, and a 1.3-kb fragment which aligns with the ORF2 of theD. melanogaster gypsy element was sequenced. Comparisons of this sequence in three species (D. subobscura, D. melanogaster, and D. virilis) indicate that there is greater similarity between theD. subobscura-D. virilis sequences than betweenD. subobscura andD. melanogaster. Molecular divergence ofgypsy sequences betweenD. virilis andD. subobscura is estimated at 16 MY, whereas the most likely divergence time of these two species is more than 60 MY. These data strongly suggest thatgypsy sequences have been horizontally transferred between these species.Offprint requests to: T.M. Alberola     

Evidence for interspecific transfer of the transposable element mariner betweenDrosophila andZaprionus

Summary The transposable element mariner occurs widely in themelanogaster species group ofDrosophila. However, in drosophilids outside of themelanogaster species group, sequences showing strong DNA hybridization with mariner are found only in the genusZaprionus. the mariner sequence obtained fromZaprionus tuberculatus is 97% identical with that fromDrosophila mauritiana, a member of themelanogaster species subgroup, whereas a mariner sequence isolated fromDrosophila tsacasi is only 92% identical with that fromD. mauritiana. BecauseD. tsacasi is much more closely related toD. mauritiana than isZaprionus, the presence of mariner inZaprionus may result from horizontal transfer. In order to confirm lack of a close phylogenetic relationship between the genusZaprionus and themelanogaster 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 betweenZ. tuberculatus andD. mauritiana, as compared with 90% identical betweenD. tsacasi andD. 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 theZaprionus mariner sequence, which clusters with mariner fromDrosophila teissieri andDrosophila yakuba in themelanogaster species subgroup. These results strongly suggest horizontal transfer.     

Association Between Levels of Coding Sequence Divergence and Gene Misregulation in <Emphasis Type="Italic">Drosophila</Emphasis> Male Hybrids

Previous studies have shown widespread conservation of gene expression levels between species of the Drosophila melanogaster subgroup as well as a positive correlation between coding sequence divergence and expression level divergence between species. Meanwhile, large-scale misregulation of gene expression level has been described in interspecific sterile hybrids between D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. Using data from gene expression analysis involving D. simulans, D. melanogaster, and their hybrids, we observed a significant positive correlation between protein sequence divergence and gene expression differences between hybrids and their parental species. Furthermore, we demonstrate that underexpressed misregulated genes in hybrids are evolving more rapidly at the protein sequence level than nonmisregulated genes or overexpressed misregulated genes, highlighting the possible effects of sexual and natural selection as male-biased genes and nonessential genes are the principal gene categories affected by interspecific hybrid misregulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Carlo G. Artieri and Wilfried Haerty contributed equally to this publication.     

Courtship inDrosophila sechellia: Its structure,functional aspects,and relationship to those of other members of theDrosophila melanogaster species subgroup

The courtship behavior of Drosophila sechelliais described. Male wing displays are mainly vibration and scissoring, with low levels of rowing. As courtship proceeds the proportion of courtship spent in male wing vibration and licking increases, whereas female movement decreases. The male courtship song of sechelliacontains pulse song but no sine song. This species also shows a distinctive copulation song associated with mounting and copulation. The main cuticular hydrocarbon in females is 7,11-heptacosadiene. The number of copulations increased when flies were placed in the presence of food. Visual and acoustic stimuli appear to be important for mating. A multidimensional comparison was used to compare members of the melanogaster species subgroup, based upon courtship behavior, song characteristics, and cuticular hydrocarbons. A multidimensional comparison of courtship sequences in sechellia, melanogaster, simulans,and mauritianashowed differences in variability between the two island species as compared to the two cosmopolitan species. The courtship song of D. orenais described: it shows both sine and pulse song; there is also a copulation song in this species.     

Positive and Negative Selection in the β-Esterase Gene Cluster of the Drosophila melanogaster Subgroup

We examine the pattern of molecular evolution of the β-esterase gene cluster, including the Est-6 and ψEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For ψEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of ψEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of ψEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the β-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function. [Reviewing Editor: Dr. Martin Kreitman]     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. 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 ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster 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 themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.