Intra‐ and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X‐linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.     

Characterisation and Interpopulation Variability of a Complex HpaI Satellite DNA of Drosophila seriema (repleta Group)

A HpaI satellite DNA has been isolated and characterised from the genome of Drosophila seriema, a cactus-breeding species endemic to the rock fields of the Espinhaço Range in Brazil. The monomer sequences are slightly A + T rich (66%) and there is a significant variation of repetition length (343–391 bp). The length variability is mainly due to a 22 bp indel in some repeats and the presence of a highly variable region characterised by several DNA rearrangements, including indels, inversions and duplications of small sequence segments. The retarded mobility of monomers observed after gel electrophoresis suggests DNA curvature. Thirty satDNA repeats were analysed in samples from five populations which cover D. seriema geographical distribution. Previous studies showed that these populations present low levels of chromosomal divergence in contrast to high levels of mtDNA divergence. The variability among the 30 repeats is pretty low, on average 2%. The results showed that the satDNA sequences are rather homogeneous on both intra and interpopulational levels, presenting no specific feature(s) that could discriminate a particular population or groups of geographically close populations. Possible factors responsible for such homogeneity are discussed.     

Ecology of body size in Drosophila buzzatii: untangling the effects of temperature and nutrition

Abstract.

• 1 A method of separating the effects of two important determinants of body size in natural populations, temperature of larval development and level of larval nutrition, by making measurements of thorax length and wing length of adult flies is investigated.
• 2 I show that at any given time variation in body size of Drosophila buzzatii from two sites in eastern Australia is determined primarily by variation in the quality of nutrition available to larvae.
• 3 Throughout the year adult flies are consistently at least 25% smaller in volume than predicted for optimal nutrition at their predicted temperature of larval development.
• 4 Nutritional stress is therefore a year-round problem for these flies.
• 5 Measurements of adult flies emerging from individual breeding substrates (rotting cactus cladodes) show that there is substantial variation among these substrates in the nutrition available to larvae.
• 6 This method will allow study of spatial and temporal variation in the temperature of larval substrates and in the nutritional resources available to flies in natural populations.

     

Ecological genetics of the Adh-1 locus of Drosophila buzzatii

The Adh-1 polymorphism of Drosophila buzzatii was studied in terms of the effects of the larval substrate (laboratory food and Opuntia stricta tissue inoculated with live yeast species) on larval survival, development time and subsequent adult size, using the three common Adh-1 genotypes. Studies with laboratory food yielded mixed results, but in general the Adh-1^b/Adh-1^b genotype was superior to Adh-1^c/Adh-1^c with the heterozygote intermediate. Studies with the natural substrate (Opuntia stricta tissue) using mono- and bicultures of four associated yeast species also showed that the Adh-1^b/Adh-1^b genotype develops faster and survives better than Adh-1^c/Adh-1^c. There were no genotype-by-yeast interactions which might explain the maintenance of the polymorphism. Drosophila buzzatii larvae develop faster and attain larger adult sizes when raised on bicultures of yeasts as compared with the corresponding monocultures.     

Nucleotide sequence of a major satellite DNA element isolated from a saltwater fishSillago japonica

A member of satellite repetitive DNA was isolated and sequenced from a saltwater fishSillago japonica (Percoidei). This sequence consists of several oligo-dA/dT tracts and two inverted repeats which resemble each other. Dot blot hybridization analysis using a satellite DNA clone pSJ2 among the species in the suborder Percoidei revealed that the pSJ2 sequence was amplified at least after the family Sillaginidae had been derived.     

Divergence in wing morphology among sibling species of the Drosophila buzzatii cluster

The Drosophila buzzatii species cluster consists of the sibling species D. buzzatii, D. koepferae, D. serido, D. borborema, D. seriema, D. antonietae and D. gouveai, all of which breed exclusively in decaying cactus tissue and, except for D. buzzatii (a colonizing subcosmopolitan species), are endemic to South America. Using a morphometric approach and multivariate analysis of 17 wing parameters, we investigated the degree of divergence in wing morphology among the sibling species of this cluster. Significant differences were obtained among the species and discriminant analysis showed that wing morphology was sufficiently different to allow the correct classification of 98.6% of the 70 individuals analysed. The phenetic relationships among the species inferred from UPGMA cluster analysis based on squared Mahalanobis distances (D²) were generally compatible with previously published phylogenetic relationships. These results suggest that wing morphology within D. buzzatii cluster is of phylogenetic importance.     

Methylation status of Sillago japonica satellite DNA examined by bisulfite modification

A member of Sillago japonica satellite DNA contained internal subrepeats in its 174 bp unit. S. Japonica genomic DNA isolated from liver tissue was subjected to bisulfite modification, and the DNA sequences of about 40 bp flanked by both subrepeats were amplified by polymerase chain reaction (PCR). This protocol, combination of bisulfite reaction and PCR, converts cytosines in the genomic DNA to thymines in the amplified DNA, whereas 5-methylcytosines in the genomic DNA remain as cytosines. Sequence analysis of the amplified DNA fragments revealed that most of the cytosine residues at CpG were methylated in this region.     

The evolutionary history of Drosophila buzzatii. XXXV. Inversion polymorphism and nucleotide variability in different regions of the second chromosome

Inversions are portions of a chromosome where the gene order is reversed relative to a standard reference orientation. Because of reduced levels of recombination in heterokaryotypes, inversions have a potentially important effect on patterns of nucleotide variability in those genomic regions close to, or included in, the inverted fragments. Here we report sequence variation at three anonymous regions (STSs) located at different positions in relation to second-chromosome inversion breakpoints in 29 isochromosomal lines derived from an Argentinean population of Drosophila buzzatii. In agreement with previous findings in Drosophila, gene flux (crossing over and/or gene conversion) between arrangements seems to appreciably increase as we approach the middle sections of inversion 2j, and patterns of nucleotide variability within, as well as genetic differentiation between chromosome arrangements, are comparable to those observed at the molecular marker outside the inverted fragments. On the other hand, nucleotide diversity near the proximal breakpoint of inversion 2j is reduced when contrasted with that found at the other regions, particularly in the case of derived inverted chromosomes. Using the data from the breakpoint, we estimate that the inversion polymorphism is approximately 1.63 N generations old, where N is the effective population size. An excess of low-frequency segregating polymorphisms is detected; mostly in the ancestral 2st arrangement and probably indicating a population expansion that predates the coalescent time of inversion 2j. Heterogeneity in mutation rates between the markers linked to the inversions may be sufficient to explain the different levels of nucleotide diversity observed. When considered in the context of other studies on patterns of variation relative to physical distance to inversion breakpoints, our data appear to be consistent with the conclusion that inversions are unlikely to be "long-lived" balanced polymorphisms.     

Genetic variation in original and colonizing Drosophila buzzatii populations analysed by microsatellite loci isolated with a new PCR screening method

A new polymerase chain reaction-based screening method for microsatellites is presented. Using this method, we isolated 12 microsatellite loci from Drosophila buzzatii, two of which were X-linked. We applied the other 10 microsatellite loci to the analysis of genetic variation in five natural populations of D. buzzatii. Two populations were from the species' original distribution in Argentina, whereas the other three were from Europe (two) and Australia that were colonized 200 and 65 years ago, respectively. Allelic variation was much larger in the original populations than in the colonizing ones and there was a tendency to decreased heterozygosity in the colonizing populations. We used three different statistical procedures for detecting population bottlenecks. All procedures suggested that the low variability in the populations in the Old World was not the result of the recent population decline, but was due to a founder effect followed by a population expansion. In fact, one procedure which detects population expansions and declines based on the genealogical history of microsatellite data suggested that an expansion had taken place in all the colonized populations.     

The foldback-like transposon Galileo is involved in the generation of two different natural chromosomal inversions of Drosophila buzzatii

Chromosomal inversions are the most common type of genome rearrangement in the genus Drosophila. Although the potential of transposable elements (TEs) for generating inversions has been repeatedly demonstrated in the laboratory, little is known on their role in the generation of natural inversions, which are those effectively contributing to the adaptation and/or evolution of species. We have cloned and sequenced the two breakpoints of the polymorphic inversion 2q7 of D. buzzatii. The sequence analysis of the breakpoint regions revealed the presence in the inverted chromosomes of large insertions, formed by complex assemblies of transposons, that are absent from the chromosomes without the inversion. Among the transposons inserted, the Foldback-like element Galileo, that was previously found responsible of the generation of the widespread inversion 2j of D. buzzatii, is present at both 2q7 breakpoints and is the most likely inducer of the inversion. A detailed study of the nucleotide and structural variation in the breakpoint regions of six chromosomal lines with the 2q7 inversion detected no nucleotide differences between them, which suggests a monophyletic and recent origin. In contrast, a remarkable degree of structural variation was observed in the same six chromosomal lines. It thus appears that the two breakpoints of the inverted chromosomes have become genetically unstable hotspots, as was previously found for the 2j inversion breakpoints. The possibility that this instability is caused by structural properties of Foldback elements is discussed.     

Population genetic study of allozyme variation in natural populations of Drosophila antonietae (Insecta, Diptera)

Drosophila antonietae is an endemic South American cactophilic species found in relictual xerophytic vegetation, mostly associated with Cereus hildmaniannus cactus. Low differentiation among populations of this species has been detected using several markers. In this work, we performed an allozyme genetic variability analysis of 11 natural populations of D. antonietae and included a discussion about the possible influences of several evolutionary processes that might be acting to maintain the pattern observed. The genetic variability of 14 isoenzyme loci was analysed and showed a high genetic diversity (average observed heterozygosity = 0.319) and a moderate genetic differentiation among populations ( F statistics = 0.0723). A correlation between genetic and geographical and ecological distances was detected among pairs of populations and the regional equilibrium analysis was thus applied. This analysis resulted in Nm (number of migrants) of approximately 3.21, indicating that moderate levels of both gene flow and genetic drift occur in this species, with gene flow overlapping genetic drift. However, considering ecological features of drosophilids, we propose a hypothesis to explain the moderate differentiation encountered as a result of three different processes, or a combination of them: (1) gene flow; (2) a short period of differentiation, i.e. maintenance of ancestral polymorphism; and (3) action of natural selection. Moreover, if gene flow is present, the high genetic diversity compared with other cactophilic and non-cactophilic species could be due to differential selection in different populations followed by gene exchange among them. These factors are discussed in the light of D. antonietae 's historical and evolutionary association with the host cactus.     

Allelic variation,fragment length analyses and population genetic models: a case study on Drosophila microsatellites*

The allelic variation of 16 microsatellite loci from selected species of the Drosophila melanogaster and D. obscura group was determined. Intra‐ and interspecific sequence comparisons allowed discrimination of mutations affecting the repetitive microsatellite from those affecting the flanking regions. The hypotheses that slippage needs a minimum number of repeats in order to become efficient with respect to microsatellite variability, and of an increased mutation rate with increased length of the microsatellite are supported by the results of our analyses. There is in particular at the interrupted complex microsatellite locus BICOID in the species of the D. obscura group, extensive variation in the flanking regions in addition to length and sequence variation of the repetitive microsatellite. The allelic variation at this locus can hardly be explained by slippage alone. Estimates of microsatellite variability by fragment length analyses will pick up only a minor fraction of allelic variation at such loci, and conclusions that are based on the stepwise mutation model will not hold.     

Remating and sperm displacement in a natural population of Drosophila buzzatii inferred from mother-offspring analysis of microsatellite loci

Prospects for estimation of parameters of models of sperm competition from field data have improved recently with the development of methods that employ multilocus genotype data from brood-structured samples. Sperm competition in Drosophila buzzatii is of special interest because it is possible to directly observe the breeding behaviour of this species in its natural habitat of rotting cactus. Previous laboratory experiments showed that this species exhibits an unusual pattern of frequent remating and sperm partitioning. This paper reports the first attempt to estimate the frequency of female remating and sperm competition in natural populations of D. buzzatii. For the Australian population studied, the mean remating frequency was lower (alpha = 2.12-2.20) than previously estimated in laboratory experiments with the same population, whereas mean sperm displacement (beta = 0.69-0.71) fell within the limits of previous laboratory results. The evolution of the D. buzzatii mating system is discussed.     

Satellite DNA and speciation: A species specific satellite DNA of Drosophila guanchel

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.     

Characterization of an <Emphasis Type="Italic">Eco</Emphasis>RI family of satellite DNA from two species

We have cloned and sequenced a 321bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321bp which is composed of two subunits of 160bp, suggested by the existence of a 160bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.     

Nucleotide sequence of BamHI family satellite DNA and its unit length polymorphism in bluegill sunfish Lepomis macrochirus

We have isolated and sequenced a member of tandem repetitive DNA containing BamHI site (BamHI family satellite DNA) from bluegill sunfish Lepomis macrochirus. PCR amplification with specific primers was performed to define the size of unit length repeat of the BamHI family satellite DNA, revealing that there were two distinct size of DNA fragments (0.9 kb and 1.3 kb) in the PCR products. The longer fragment (1.3 kb) consisted of internal sub-duplication of shorter fragment (0.9 kb). We have compared the size of PCR products among four fish populations, and found that both fragments co-existed in one population whereas the longer fragment was dominant in other three populations. The results may reflect ongoing homogenization of satellite DNA type over a short evolutionary time scale.     

Cloning and characterization of KM190, a specific satellite DNA family of Drosophila kitumensis and D. microlabis.

The nucleotide sequences of nine clones, pKA191/l-4 from Drosophila kitumensis and pMR.190/1–5 from D. microlabis, were determined. They represent a tandemly arranged and highly repetitive satellite DNA family, KM190, which is specific for the two species.     

果蝇DNA甲基化研究进展

DNA甲基化是表观遗传调控的重要机制,但果蝇很久以来被认为是一种缺乏甲基化的模式生物。近年来才证实果蝇基因组中有5′-甲基胞嘧啶残基的存在,其DNA甲基化水平在胚胎发育早期达到最高,总体水平低于脊椎动物及植物。果蝇拥有一个包含dDNMT2和dMBD2/3的简单甲基化修饰系统,其分别与哺乳动物中的DNMT2家族及MBD2/MBD3蛋白高度同源。果蝇DNA甲基化模式和特点可能随果蝇种类不同而不同。文章对果蝇DNA甲基化特点及其功能研究进展进行了综述。     

Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity

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Recent radiation of endemic Caribbean Drosophila of the dunni subgroup inferred from multilocus DNA sequence variation

Studies of island endemism provide a unique opportunity to elucidate fundamental mechanisms of speciation. Here we examine intra- and interspecific DNA sequence variation at four unlinked genetic loci among populations of the Drosophila dunni subgroup to provide a detailed genealogical portrait of the process of speciation among these island endemic species. Our data indicate two major rounds of diversification that have shaped the D. dunni subgroup. The first occurred 1.6-2.6 million years ago and separated three major lineages, one in Puerto Rico and the Virgin Islands, a second in the northern Lesser Antilles and Barbados, and a third in St. Vincent and Grenada. A second round of diversification occurred in the last 96,000 years in the northern Lesser Antilles and Barbados. The four distinct species that resulted from this recent round of diversification maintain relatively high amounts of genetic variation, similar to that of a closely related mainland species, and share extensive ancestral polymorphism. These data suggest a minimal role for population bottlenecks linked to founder events in the history of the D. dunni subgroup. Further, the recent divergence of these island populations highlights the extremely rapid development of reproductive isolation and distinct patterns of abdominal pigmentation that has occurred in these species.