Comparative evolutionary analysis of rDNA ITS regions in Drosophila

The internal transcribed spacer (ITS) of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence. We have analyzed the ITS regions of five species from the Drosophila melanogaster subgroup, two Drosophila species from outside this group (D. pseudoobscura and D. virilis), as well as from the more distantly related dipteran fly Musca domestica. The sequence comparisons show a distinctive conservation/divergence pattern, indicating that some regions are more conserved than others. Moreover, secondary-structure calculations indicate several conserved structural elements within the ITS regions. On the other hand, a statistical test that allows us to estimate the fraction of sites that are not under selective constraint suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila. The ITS sequences can be used to derive a molecular phylogeny for the species under study. We find that the ITS tree is largely in line with the so-far-known phylogeny of this group of species, with one difference. The species most distant within the D. melanogaster subgroup is D. yakuba, rather than D. orena, as is normally assumed.      

Reproductive Isolation and Morphogenetic Evolution in Drosophila Analyzed by Breakage of Ethological Barriers

This article reports the breaking of ethological barriers through the constitution of soma-germ line chimeras between species of the melanogaster subgroup of Drosophila, which are ethologically isolated. Female Drosophila yakuba and D. teissieri germ cells in a D. melanogaster ovary produced functional oocytes that, when fertilized by D. melanogaster sperm, gave rise to sterile yakuba-melanogaster and teissieri-melanogaster male and female hybrids. However, the erecta-melanogaster and orena-melanogaster hybrids were lethal, since female D. erecta and D. orena germ cells in a D. melanogaster ovary failed to form oocytes with the capacity to develop normally. This failure appears to be caused by an altered interaction between the melanogaster soma and the erecta and orena germ lines. Germ cells of D. teissieri and D. orena in a D. melanogaster testis produced motile sperm that was not stored in D. melanogaster females. This might be due to incompatibility between the teissieri and orena sperm and the melanogaster seminal fluid. A morphological analysis of the terminalia of yakuba-melanogaster and teissieri-melanogaster hybrids was performed. The effect on the terminalia of teissieri-melanogaster hybrids of a mutation in doublesex, a regulatory gene that controls the development of the terminalia, was also investigated.     

HeT-A_pi1, a piRNA target sequence in the Drosophila telomeric retrotransposon HeT-A, is extremely conserved across copies and species

The maintenance of the telomeres in Drosophila species depends on the transposition of the non-LTR retrotransposons HeT-A, TAHRE and TART. HeT-A and TART elements have been found in all studied species of Drosophila suggesting that their function has been maintained for more than 60 million years. Of the three elements, HeT-A is by far the main component of D. melanogaster telomeres and, unexpectedly for an element with an essential role in telomere elongation, the conservation of the nucleotide sequence of HeT-A is very low. In order to better understand the function of this telomeric retrotransposon, we studied the degree of conservation along HeT-A copies. We identified a small sequence within the 3' UTR of the element that is extremely conserved among copies of the element both, within D. melanogaster and related species from the melanogaster group. The sequence corresponds to a piRNA target in D. melanogaster that we named HeT-A_pi1. Comparison with piRNA target sequences from other Drosophila retrotransposons showed that HeT-A_pi1 is the piRNA target in the Drosophila genome with the highest degree of conservation among species from the melanogaster group. The high conservation of this piRNA target in contrast with the surrounding sequence, suggests an important function of the HeT-A_pi1 sequence in the co-evolution of the HeT-A retrotransposon and the Drosophila genome.     

Local recombination and mutation effects on molecular evolution in Drosophila.

I studied the cause of the significant difference in the synonymous-substitution pattern found in the achaete-scute complex genes in two Drosophila lineages, higher codon bias in Drosophila yakuba, and lower bias in D. melanogaster. Besides these genes, the functionally unrelated yellow gene showed the same substitution pattern, suggesting a region-dependent phenomenon in the X-chromosome telomere. Because the numbers of A/T --> G/C substitutions were not significantly different from those of G/C --> A/T in the yellow noncoding regions of these species, a AT/GC mutational bias could not completely account for the synonymous-substitution biases. In contrast, we did find an approximately 14-fold difference in recombination rates in the X-chromosome telomere regions between the two species, suggesting that the reduction of recombination rates in this region resulted in the reduction of the efficacy of selection in D. melanogaster. In addition, the D. orena yellow showed a 5% increase in the G + C content at silent sites in the coding and noncoding regions since the divergence from D. erecta. This pattern was significantly different from those at the orena Adh and Amy loci. These results suggest that local changes in recombination rates and mutational pressures are contributing to the irregular synonymous-substitution patterns in Drosophila.     

Evolution of the Transposable Element Mariner in Drosophila Species

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.     

Vertical Transmission of the Retrotransposable Elements R1 and R2 during the Evolution of the Drosophila Melanogaster Species Subgroup

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.     

Identical satellite DNA sequences in sibling species of Drosophila

The evolution of simple satellite DNAs was examined by DNA-DNA hybridization of ten Drosophila melanogaster satellite sequences to DNAs of the sibling species, Drosophila simulans and Drosophila erecta. Seven of these repeat types are present in tandem arrays in D. simulans and each of the ten sequences is repeated in D. erecta. In thermal melts, six of the seven satellite sequences in D. simulans and seven of the ten sequences in D. erecta melted within 1 deg.C of the corresponding values in D. melanogaster. The remaining sequences melted within 3 deg.C of the homologous hybrids. Therefore, there is little or no alteration in those satellite sequences held in common, despite a period of about ten million years since the divergence of D. melanogaster and D. simulans from a common ancestor. Simple satellite sequences appear to be more highly conserved than coding regions of the genome, on a per nucleotide basis. Since multiple copies of three satellite sequences could not be detected in D. simulans yet are present in D. erecta, a species more distantly related to D. melanogaster than is D. simulans, these sequences show discontinuities in evolution. There were major quantitative variations between species, showing that satellite DNAs are prone to massive amplification or diminution events over timespans as short as those separating sibling species. In D. melanogaster, these sequences amount to 21% of the genome but only 5% in D. simulans and 0.4% in D. erecta. There was a general trend of lower abundance with evolutionary distance for most satellites, suggesting that the amounts of different satellite sequences do not vary independently during evolution.     

Distribution and conservation of mobile elements in the genus Drosophila

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.      

Evolutionary relationships to parasitism by seven species of the Drosophila melanogaster subgroup

Parasitic wasps are an important component of the niche of Drosophila species. The susceptibility to the Cynipid Leptopilina boulardi was estimated in the seven sibling species of Drosophila belonging to the melanogaster subgroup. Three categories of Hies can be distinguished, according to the level of cellular immune reaction and success of parasitism. Drosophila melanogaster and D. mauritiana belong to the category 1, specified by no encapsulative reaction and a high rate of successful parasitism. Category 2, characterized by a moderate encapsulation rate and a high mortality include D. simulans.5, D. erecta and D. orena . Category 3, with D. yakuba and D. tcissien , is specified by a very low rate or absence of successful parasitism due to a highly efficient immune cellular reaction. This classification parallels the phylogenic relationship based upon polytene chromosome banding sequences. Such specific ditferences in susceptibility to parasites may plan an important role in the competition between these species in Africa.     

Local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes

I present here evidence of remarkable local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes. The substitution pattern at 10 loci in the telomeric region of the X chromosome was studied for four species of the Drosophila melanogaster species subgroup. Drosophila orena and Drosophila erecta are clearly the most closely related species pair (the erecta complex) among the four species studied; however, the overall data at the 10 loci revealed a clear dichotomy in the silent substitution patterns between the AT-biased- substitution melanogaster and erecta lineages and the GC-biased-substitution yakuba and orena lineages, suggesting two or more independent changes in GC/AT substitution biases. More importantly, the results indicated a between- loci heterogeneity in GC/AT substitution bias in this small region independently in the yakuba and orena lineages. Indeed, silent substitutions in the orena lineage were significantly biased toward G and C at the consecutive yellow, lethal of scute, and asense loci, but they were significantly biased toward A and T at sta. The substitution bias toward G and C was centered in different areas in yakuba (significantly biased at EG:165H7.3, EG:171D11.2, and suppressor of sable). The similar silent substitution patterns in coding and noncoding regions, furthermore, suggested mutational biases as a cause of the substitution biases. On the other hand, previous study reveals that Drosophila yakuba has about 20-fold higher crossover frequencies in the telomeric region of the X chromosome than does D. melanogaster; this study revealed that the total genetic map length of the yakuba X chromosome was only about 1.5 times as large as that of melanogaster and that the map length of the X-telomeric y-sta region did not differ between Drosophila yakuba and D. erecta. Taken together, the data strongly suggested that an approximately 20- fold reduction in the X-telomeric crossover frequencies occurred in the ancestral population of D. melanogaster after the melanogaster-yakuba divergence but before the melanogaster-simulans divergence.     

Phylogenetic incongruence in the Drosophila melanogaster species group

Drosophila melanogaster and its close relatives are used extensively in comparative biology. Despite the importance of phylogenetic information for such studies, relationships between some melanogaster species group members are unclear due to conflicting phylogenetic signals at different loci. In this study, we use twelve nuclear loci (eleven coding and one non-coding) to assess the degree of phylogenetic incongruence in this model system. We focus on two nodes: (1) the node joining the Drosophila erecta-Drosophila orena, Drosophila melanogaster-Drosophila simulans, and Drosophila yakuba-Drosophila teissieri lineages, and (2) the node joining the lineages leading to the melanogaster, takahashii, and eugracilis subgroups. We find limited evidence for incongruence at the first node; our data, as well as those of several previous studies, strongly support monophyly of a clade consisting of D. erecta-D. orena and D. yakuba-D. teissieri. By contrast, using likelihood based tests of congruence, we find robust evidence for topological incongruence at the second node. Different loci support different relationships among the melanogaster, takahashii, and eugracilis subgroups, and the observed incongruence is not easily attributable to homoplasy, non-equilibrium base composition, or positive selection on a subset of loci. We argue that lineage sorting in the common ancestor of these three subgroups is the most plausible explanation for our observations. Such lineage sorting may lead to biased estimation of tree topology and evolutionary rates, and may confound inferences of positive selection.     

Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.     

Divergence and heterogeneity of the histone gene repeating units in the Drosophila melanogaster species subgroup

The repeating units of the histone gene cluster containing the H1, H2A, H2B and H4 genes were amplified by PCR from the Drosophila melanogaster species subgroup, i.e., D. yakuba, D. erecta, D. sechellia, D. mauritiana, D. teissieri and D. orena. The PCR products were cloned and their nucleotide sequences of about 4.6-4.8kbp were determined to elucidate the mechanism of molecular evolution of the histone gene family. The heterogeneity among the histone gene repeating units was 0.6% and 0.7% for D. yakuba and D. sechellia, respectively, indicating the same level of heterogeneity as in the H3 gene region of D. melanogaster. Divergence of the genes among species even in the most closely related ones was much greater than the heterogeneity among family members, indicating a concerted mode of evolution for the histone gene repeating units. Among the species in the D. melanogaster species subgroup, the histone gene regions as well as 3rd codon position of the coding region showed nearly the same GC contents. These results suggested that the previous conclusion on analysis of the H3 gene regions, the gene family evolution in a concerted fashion, holds true for the whole histone gene repeating unit.     

Microevolutionary divergence pattern of the segmentation gene hunchback in Drosophila

To study the microevolutionary processes shaping the evolution of the segmentation gene hunchback (hb) from Drosophila melanogaster, we cloned and sequenced the gene from 12 isofemale lines representing wild-type populations of D. melanogaster, as well as from the closely related species Drosophila sechellia, Drosophila orena, and Drosophila yakuba. We find a relatively low degree of sequence variation in D. melanogaster (theta = 0.0017), which is, however, consistent with its chromosomal location in a region of low recombination. Tests of neutrality do not reject a neutral-evolution model for the whole region. However, pairwise tests with different subregions indicate that there is a relative excess of polymorphic sites in the leader and the intron. Codon usage pattern analysis shows a particularly biased codon usage in the highly conserved regions, which is in line with the hypothesis that selection on translational accuracy is the driving force behind such a bias. A comparison of the expression pattern of hb in different sibling species of D. melanogaster reveals some regulatory changes in D. yakuba, which could be interpreted as changes in the timing of secondary expression domains.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.