Nonrecombining genes in a recombination environment: the Drosophila "dot" chromosome

Rate of recombination is a powerful variable affecting several aspects of molecular variation and evolution. A nonrecombining portion of the genome of most Drosophila species, the "dot" chromosome or F element, exhibits very low levels of variation and unusual codon usage. One lineage of Drosophila, the willistoni/saltans groups, has the F element fused to a normally recombining E element. Here, we present polymorphism data for genes on the F element in two Drosophila willistoni and one D. insularis populations, genes previously studied in D. melanogaster. The D. willistoni populations were known to be very low in inversion polymorphism, thus minimizing the recombination suppression effect of inversions. We first confirmed, by in situ hybridization, that D. insularis has the same E + F fusion as D. willistoni, implying this was a monophyletic event. A clear gradient in codon usage exists along the willistoni F element, from the centromere distally to the fusion with E; estimates of recombination rates parallel this gradient and also indicate D. insularis has greater recombination than D. willistoni. In contrast to D. melanogaster, genes on the F element exhibit moderate levels of nucleotide polymorphism not distinguishable from two genes elsewhere in the genome. Although some linkage disequilibrium (LD) was detected between polymorphic sites within genes (generally <500 bp apart), no long-range LD between F element loci exists in the two willistoni group species. In general, the distribution of allele frequencies of F element genes display the typical pattern of expectations of neutral variation at equilibrium. These results are consistent with the hypothesis that recombination allows the accumulation of nucleotide variation as well as allows selection to act on synonymous codon usage. It is estimated that the fusion occurred ～20 Mya and while the F element in the willistoni lineage has evolved "normal" levels and patterns of nucleotide variation, equilibrium may not have been reached for codon usage.     

Evolutionary dynamics of wAu-like Wolbachia variants in neotropical Drosophila spp

Wolbachia bacteria are common intracellular symbionts of arthropods and have been extensively studied in Drosophila. Most research focuses on two Old Word hosts, Drosophila melanogaster and Drosophila simulans, and does not take into account that some of the Wolbachia associations in these species may have evolved only after their fast global expansion and after the exposure to Wolbachia of previously isolated habitats. Here we looked at Wolbachia of Neotropical Drosophila species. Seventy-one lines of 16 Neotropical Drosophila species sampled in different regions and at different time points were analyzed. Wolbachia is absent in lines of Drosophila willistoni collected before the 1970s, but more recent samples are infected with a strain designated wWil. Wolbachia is absent in all other species of the willistoni group. Polymorphic wWil-related strains were detected in some saltans group species, with D. septentriosaltans being coinfected with at least four variants. Based on wsp and ftsZ sequence data, wWil of D. willistoni is identical to wAu, a strain isolated from D. simulans, but can be discriminated when using a polymorphic minisatellite marker. In contrast to wAu, which infects both germ line and somatic tissues of D. simulans, wWil is found exclusively in the primordial germ line cells of D. willistoni embryos. We report on a pool of closely related Wolbachia strains in Neotropical Drosophila species as a potential source for the wAu strain in D. simulans. Possible evolutionary scenarios reconstructing the infection history of wAu-like Wolbachia in Neotropical Drosophila species and the Old World species D. simulans are discussed.     

Evolution of exon 1 of the dopamine D4 receptor (DRD4) gene in primates

The dopamine D4 receptor (DRD4) gene exhibits a large amount of expressed polymorphism in humans. To understand the evolutionary history of the first exon of DRD4-which in humans contains a polymorphic 12bp tandem duplication, a polymorphic 13bp deletion, and other rare variants-we examined the homologous exon in thirteen other primate species. The great apes possess a variable number of tandem repeats in the same region as humans, both within and among species. In this sense, the 12bp tandem repeat of exon 1 is similar to the 48bp VNTR of exon 3 of DRD4, previously shown to be polymorphic in all primate species examined. The Old World monkeys show no variation in length, and a much higher conservation of amino acid sequence than great apes and humans. The New World monkeys show interspecific differences in length in the region of the 12bp polymorphism, but otherwise show the higher conservation seen in Old World monkeys. The different patterns of variation in monkeys compared to apes suggest strong purifying selective pressure on the exon in these monkeys, and somewhat different selection, possibly relaxed selection, in the apes.     

Evolution of the Adh locus in the Drosophila willistoni group: the loss of an intron, and shift in codon usage

We report here the DNA sequence of the alcohol dehydrogenase gene (Adh) cloned from Drosophila willistoni. The three major findings are as follows: (1) Relative to all other Adh genes known from Drosophila, D. willistoni Adh has the last intron precisely deleted; PCR directly from total genomic DNA indicates that the deletion exists in all members of the willistoni group but not in any other group, including the closely related saltans group. Otherwise the structure and predicted protein are very similar to those of other species. (2) There is a significant shift in codon usage, especially compared with that in D. melanogaster Adh. The most striking shift is from C to U in the wobble position (both third and first position). Unlike the codon-usage-bias pattern typical of highly biased genes in D. melanogaster, including Adh, D. willistoni has nearly 50% G + C in the third position. (3) The phylogenetic information provided by this new sequence is in agreement with almost all other molecular and morphological data, in placing the obscura group closer to the melanogaster group, with the willistoni group farther distant but still clearly within the subgenus Sophophora.      

Enzyme Variability in the DROSOPHILA WILLISTONI Group. IV. Genic Variation in Natural Populations of DROSOPHILA WILLISTONI

We describe allelic variation at 28 gene loci in natural populations of D. willistoni. Seventy samples were studied from localities extending from Mexico and Florida, through Central America, the West Indies, and tropical South America, down to South Brazil. At least several hundred, and often several thousand, genomes were sampled for each locus. We have discovered a great deal of genetic variation. On the average, 58% loci are polymorphic in a given population. (A locus is considered polymorphic when the frequency of the most common allele is no greater than 0.95). An individual fly is heterozygous, on the average, at 18.4% loci.-Concerning the pattern of the variation, the most remarkable finding is the similarity of the configuration of allelic frequencies from locality to locality throughout the distribution of the species. Our observations support the conclusion that balancing natural selection is the major factor responsible for the considerable genetic variation observed in D. willistoni.     

Mitochondrial pseudogenes in the nuclear genomes of Drosophila

Mitochondrial pseudogenes in nuclear chromosomes (numts) have been detected in the genomes of a diverse range of eukaryotic species. However, the numt content of different genomes and their properties is not uniform, and study of these differences provides insight into the mechanisms and dynamics of genome evolution in different organisms. In the genus Drosophila, numts have previously only been identified on a genome-wide scale in the melanogaster subgroup. The present study extends the identification to 11 species of the Drosophila genus. We identify a total of 302 numts and show that the numt complement is highly variable in Drosophilids, ranging from just 4 in D. melanogaster to 67 in D. willistoni, broadly correlating with genome size. Many numts have undergone large-scale rearrangements in the nucleus, including interruptions, inversions, deletions and duplications of sequence of variable size. Estimating the age of the numts in the nucleus by phylogenetic tree reconstruction reveals the vast majority of numts to be recent gains, 90% having arisen on terminal branches of the species tree. By identifying paralogs and counting duplications among the extant numts we estimate that 23% of extant numts arose through post-insertion duplications. We estimate genus average rates of insertion of 0.75 per million years, and a duplication rate of 0.010 duplications per numt per million years.     

Horizontal transfer and selection in the evolution of P elements

The roles of selection and horizontal transfer in the evolution of the canonical subfamily of P: elements were studied in the saltans and willistoni species groups of the genus Drosophila (subgenus Sophophora). We estimate that the common ancestor of the canonical P: subfamily dates back 2-3 Myr at the most, despite the much older age (more than 40 Myr) of the P: family as a whole. The evolution of the canonical P: subfamily is characterized by weak selection at nonsynonymous sites. These sites have evolved at three quarters the rate of synonymous sites, in which no selective constraints were detected. Their recent horizontal transfer best explains the high degree of similarity among canonical P: elements from the saltans and willistoni species groups. These results are consistent with a model of P:-element evolution in which selective constraints are imposed at the time of horizontal transfer. Furthermore, it is estimated that the spread and diversification of the canonical subfamily involved a minimum of 11 horizontal transfer events among the 18 species surveyed within the past 3 Myr. The presence of multiple P: subfamilies in the saltans and willistoni species groups is likely to be the result of multiple invasions that have previously swept through these taxa in a succession of horizontal transfer events. These results suggest that horizontal transfer among eukaryotes might be more common than anticipated.     

Rapid divergence of microsatellite abundance among species of Drosophila

Among major taxonomic groups, microsatellites exhibit considerable variation in composition and allele length, but they also show considerable conservation within many major groups. This variation may be explained by slow microsatellite evolution so that all species within a group have similar patterns of variation, or by taxon-specific mutational or selective constraints. Unfortunately, comparing microsatellites across species and studies can be problematic because of biases that may exist among different isolation and analysis protocols. We present microsatellite data from five Drosophila species in the Drosophila subgenus: D. arizonae, D. mojavensis, and D. pachea (three cactophilic species), and D. neotestacea and D. recens (two mycophagous species), all isolated at the same time using identical protocols. For each species, we compared the relative abundance of motifs, the distribution of repeat size, and the average number of repeats. Dimers were the most abundant microsatellites for each species. However, we found considerable variation in the relative abundance of motif size classes among species, even between sister taxa. Frequency differences among motifs within size classes for the three cactophilic species, but not the two mycophagous species, are consistent with other studied Drosophila. Frequency distributions of repeat number, as well as mean size, show significant differences among motif size classes but not across species. Sizes of microsatellites in these five species are consistent with D. virilis, another species in the subgenus Drosophila, but they have consistently higher means than in D. melanogaster, in the subgenus Sophophora. These results confirm that many aspects of microsatellite variation evolve quickly but also are subject to taxon-specific constraints. In addition, the nature of microsatellite evolution is dependent on temporal and taxonomic scales, and some variation is conserved across broad taxonomic levels despite relatively high rates of mutation for these loci.     

Nucleotide and repeat length variation at the nonA gene of the Drosophila virilis group species and its effects on male courtship song

Genes found to affect male courtship song characters in Drosophila melanogaster are good candidates when tracing genes responsible for species-specific songs in other Drosophila species. It has previously been shown that Thr–Gly repeat length variation at the period gene affects song traits in D. melanogaster, which gives the repetitive regions a special interest. In this work, we have characterised the patterns of nucleotide variation for gene regions containing two Gly and one Gln–Ala repeat in another D. melanogaster song gene, no-on-transient A, in D. virilis group species. The levels of nucleotide variability in D. virilis nonA were similar to those found for other genes of the species, and the gene sequences showed no signs of deviation from neutrality. The Gly 2 repeat preceding the central domain of the gene exhibited length variation, which did not, however, correlate with song variation either within D. virilis or between the species of D. virilis group. The Gly 3 repeat located on the other side of the central domain showed amino acid divergence parallel to the consensus phylogeny of the D. virilis group species. The species of the virilis subgroup having Asn after the first three glycines in this repeat have simple songs with no species-specificity, while the species of the montana subgroup having two Gly or Asn–Ser in this site have unique courtship songs. Amino acid differences between the species in this repeat may, however, reflect species phylogeny rather than have an effect on song divergence per se.     

Vege and Mar: two novel hAT MITE families from Drosophila willistoni

Two novel families of miniature inverted repeat transposable elements (MITEs), Vege and Mar, are described from Drosophila willistoni. Based on their structures, both element families are hypothesized to belong to the hAT superfamily of transposable elements. Both elements have perfect, inverted terminal repeats and 8-bp target site duplications and were found to have inserted within fixed copies of nonautonomous P elements. Vege is present in all studied D. willistoni populations and appears to have a relatively low copy number. Mar was identified in only a single D. willistoni population, and its copy number is presently unknown. Although MITEs occupy relatively large proportions of the genomes of a broad range of organisms, this may be their first unambiguous identification in any species of the genus Drosophila.     

Newly developed polymorphic microsatellite markers for frogs of the genus Ascaphus

Thirteen polymorphic microsatellite loci were identified and developed for the coastal tailed frog, Ascaphus truei, from sites within the Olympic Peninsula of Washington, USA. These tetranucleotide repeat loci were highly variable, averaging 19 alleles per locus and expected heterozygosity of 0.91. In addition, these loci cross-amplify in the sister species, Ascaphus montanus. These markers will prove useful in identifying fine-scale genetic structure, as well as provide insight into the evolution and conservation of this group across fragmented landscapes.     

Phylogeny of the subgenus sophophora (Diptera: drosophilidae) based on combined analysis of nuclear and mitochondrial sequences

Sequences from the nuclear (nu) alcohol dehydrogenase gene, the nu 28S ribosomal RNA locus, and the mitochondrial cytochrome oxidase II gene were used both individually and in combined analyses to infer the phylogeny of the subgenus Sophophora (Diptera: Drosophilidae). We used several optimality criteria, including maximum likelihood, maximum parsimony, and minimum evolution, to analyze these partitions to test the monophyly of the subgenus Sophophora and its four largest species groups, melanogaster, obscura, saltans, and willistoni. Our results suggest that the melanogaster and obscura species groups are each monophyletic and form a closely related clade. The Neotropical clade, containing the saltans and willistoni species groups, is also recovered, as previous studies have suggested. While the saltans species group is strongly supported as monophyletic, the results of several analyses indicate that the willistoni species group may be paraphyletic with respect to the saltans species group.     

Macro-evolution of the hairy enhancer in Drosophila species

It has been suggested that many of the changes in the developmental program might be in the cis-acting promoters and enhancer regions. Here I study the macro-evolutionary changes of an enhancer region for the early developmental gene hairy in Drosophila melanogaster, D. simulans, D. pseudoobscura, D. willistoni, D. nebulosa, D. hydei, and D. virilis. The enhancer region is characterized by small, highly conserved blocks interspersed among highly variable regions. Nevertheless, species phylogenies constructed by the enhancer sequences agree with the widely accepted phylogeny of these species. The evolution of the variable regions is consistent with a molecular clock, while the evolution of the conserved blocks is significantly different from a clock. In particular, the D. pseudoobscura lineage shows the highest degree of species-specific change consistent with changes in expression timing reported in an earlier study. It has been suggested that the variation in sequence length between highly conserved blocks may play a role in the coordination of regulatory processes, such as protein-protein interactions; thus, stabilizing selection has been suggested to act on the length variations. Here I develop a test for stabilizing selection on length variation and show that the hairy enhancer does not show statistically significant evidence for stabilizing selection. J. Exp. Zool. (Mol. Dev. Evol.) 291:175-185, 2001.     

Slip-Sliding Away: Serial Changes and Homoplasy in Repeat Number in the Drosophila yakuba Homolog of Human Cancer Susceptibility Gene BRCA2

Several recent studies have examined the function and evolution of a Drosophila homolog to the human breast cancer susceptibility gene BRCA2, named dmbrca2. We previously identified what appeared to be a recent expansion in the RAD51-binding BRC-repeat array in the ancestor of Drosophila yakuba. In this study, we examine patterns of variation and evolution of the dmbrca2 BRC-repeat array within D. yakuba and its close relatives. We develop a model of how unequal crossing over may have produced the expanded form, but we also observe short repeat forms, typical of other species in the D. melanogaster group, segregating within D. yakuba and D. santomea. These short forms do not appear to be identical-by-descent, suggesting that the history of dmbrca2 in the D. melanogaster subgroup has involved repeat unit contractions resulting in homoplasious forms. We conclude that the evolutionary history of dmbrca2 in D. yakuba and perhaps in other Drosophila species may be more complicated than can be inferred from examination of the published single genome sequences per species.     

Molecular Analysis of Circadian Clocks in Drosophila simulans

The Drosophila simulans per gene is polymorphic for the length of a repeat that encodes a series of Thr-Gly pairs. We have examined the circadian behaviour of flies derived from isofemale lines that carry the major variants, and find some significant differences in the way that the clock responds to temperature challenge, that might relate to the observed frequencies of these alleles in nature. We also observe that circadian thermal behaviour is also predictably influenced by subtle differences in the temperature of the locality from which these flies have been originally collected. There appear to be species-specific differences in the circadian locomotor patterns of D. melanogaster and D. simulans and in the way they may respond to temperature. Using chimeric per transgenes which carry the different species Thr-Gly fragments, we have been able to identify components of the behaviour that are modulated by this region of the PER protein.     

Evolution of P elements in natural populations of Drosophila willistoni and D. sturtevanti

To determine how population structure of the host species affects the spread of transposable elements and to assess the strength of selection acting on different structural regions, we sequenced P elements from strains of Drosophila willistoni and Drosophila sturtevanti sampled from across the distributions of these species. Elements from D. sturtevanti exhibited considerable sequence variation, and similarity among them was correlated to geographic distance between collection sites. By contrast, all D. willistoni elements sampled were essentially identical (pi < 0.2%) and exhibited patterns typical of a recent population expansion. While the canonical P elements sampled from D. sturtevanti appear to be long-time residents in that species, a rapid expansion of a very young canonical P-element lineage is suggested in D. willistoni, overcoming barriers such as large geographical distances and moderate levels of population subdivision. Between-species comparisons reveal selective constraints on P-element evolution, as indicated by significantly different substitution rates in noncoding, silent, and replacement sites. Most remarkably, in addition to replacement sites, selection pressure appears to be strong in the first and third introns and in the 3' and 5' flanking regions.     

Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks

Drosophila melanogaster has been a canonical model organism to study genetics, development, behavior, physiology, evolution, and population genetics for nearly a century. Despite this emphasis and the completion of its nuclear genome sequence, the timing of major speciation events leading to the origin of this fruit fly remain elusive because of the paucity of extensive fossil records and biogeographic data. Use of molecular clocks as an alternative has been fraught with non-clock-like accumulation of nucleotide and amino-acid substitutions. Here we present a novel methodology in which genomic mutation distances are used to overcome these limitations and to make use of all available gene sequence data for constructing a fruit fly molecular time scale. Our analysis of 2977 pairwise sequence comparisons from 176 nuclear genes reveals a long-term fruit fly mutation clock ticking at a rate of 11.1 mutations per kilobase pair per Myr. Genomic mutation clock-based timings of the landmark speciation events leading to the evolution of D. melanogaster show that it shared most recent common ancestry 5.4 MYA with D. simulans, 12.6 MYA with D. erecta+D. orena, 12.8 MYA with D. yakuba+D. teisseri, 35.6 MYA with the takahashii subgroup, 41.3 MYA with the montium subgroup, 44.2 MYA with the ananassae subgroup, 54.9 MYA with the obscura group, 62.2 MYA with the willistoni group, and 62.9 MYA with the subgenus Drosophila. These and other estimates are compatible with those known from limited biogeographic and fossil records. The inferred temporal pattern of fruit fly evolution shows correspondence with the cooling patterns of paleoclimate changes and habitat fragmentation in the Cenozoic.