Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Phylogeny of Drosophila and related genera: conflict between molecular and anatomical analyses

Drosophila species are extensively used in biological research; yet, important phylogenetic relationships within the genus and with related genera remain unresolved. The combined data for three genes (Adh, Sod, and Gpdh) statistically resolves outstanding issues. We define the genus Drosophila inclusively so as to include Scaptomyza and Zaprionus (considered distinct genera in the taxonomy of Wheeler, 1981) but excluding Scaptodrosophila. The genus Drosophila so defined is monophyletic. The subgenus Sophophora (including the melanogaster, obscura, and willistoni groups) is monophyletic and the sister clade to all other Drosophila subgenera. The Hawaiian Drosophila (including Scaptomyza) is a monophyletic group, but the subgenus Drosophila is not monophyletic, because the immigrans group is more closely related to the subgenus Hirtodrosophila than to other species of the subgenus Drosophila, such as the virilis and repleta groups.     

The Drosophila subobscura Adh genomic region contains valuable evolutionary markers.

We have sequenced 4 kb of the genomic region comprising the Adh (Alcohol dehydrogenase) gene of Drosophila subobscura. In agreement with other species which belong to the same subgenus, two structural genes, Adh and Adh-dup, are contained in this region. The main features of these two genes of D. subobscura have been inferred from the sequence data and compared with the homologous region of D. ambigua and D. pseudoobscura. Drosophila subobscura Adh and Adh-dup differ from those of D. ambigua at a corrected estimation of 10.1% and 12.5%, respectively, while from those of D. pseudoobscura they differ by 9.5% and 8.1%, respectively. Our data suggest that Adh and Adh-dup are evolving independently, showing a species-specific pattern. Moreover, particular features of some regions of these genes make them valuable evolutionary hallmarks. For instance, replacement substitutions in the third exon of Adh may indicate the branching of the melanogaster-obscura groups, whereas replacement substitutions in the third exon of the Adh-dup could be used to assess speciation within the obscura group.     

Switch in codon bias and increased rates of amino acid substitution in the Drosophila saltans species group.

We investigated the nucleotide composition of five genes, Xdh, Adh, Sod, Per, and 28SrRNA, in nine species of Drosophila (subgenus Sophophora) and one of Scaptodrosophila. The six species of the Drosophila saltans group markedly differ from the others in GC content and codon use bias. The GC content in the third codon position, and to a lesser extent in the first position and the introns, is higher in the D. melanogaster and D. obscura groups than in the D. saltans group (in Scaptodrosophila it is intermediate but closer to the melanogaster and obscura species). Differences are greater for Xdh than for Adh, Sod, Per, and 28SrRNA, which are functionally more constrained. We infer that rapid evolution of GC content in the saltans lineage is largely due to a shift in mutation pressure, which may have been associated with diminished natural selection due to smaller effective population numbers rather than reduced recombination rates. The rate of GC content evolution impacts the rate of protein evolution and may distort phylogenetic inferences. Previous observations suggesting that GC content evolution is very limited in Drosophila may have been distorted due to the restricted number of genes and species (mostly D. melanogaster) investigated.     

The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr

To gain insights into the relationship between codon bias, mRNA secondary structure, third-codon position nucleotide distribution, and gene expression, we predicted secondary structures in two related drosophilid genes, Adh and Adhr, which differ in degree of codon bias and level of gene expression. Individual structural elements (helices) were inferred using the comparative method. For each gene, four types of randomization simulations were performed to maintain/remove codon bias and/or to maintain or alter third-codon position nucleotide composition (N3). In the weakly expressed, weakly biased gene Adhr, the potential for secondary structure formation was found to be much stronger than in the highly expressed, highly biased gene Adh. This is consistent with the observation of approximately equal G and C percentages in Adhr ( approximately 31% across species), whereas in Adh the N3 distribution is shifted toward C (42% across species). Perturbing the N3 distribution to approximately equal amounts of A, G, C, and T increases the potential for secondary structure formation in Adh, but decreases it in Adhr. On the other hand, simulations that reduce codon bias without changing N3 content indicate that codon bias per se has only a weak effect on the formation of secondary structures. These results suggest that, for these two drosophilid genes, secondary structure is a relatively independent, negative regulator of gene expression. Whereas the degree of codon bias is positively correlated with level of gene expression, strong individual secondary structural elements may be selected for to retard mRNA translation and to decrease gene expression.     

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.      

Isolation and characterization of the genomic region from Drosophila kuntzei containing the Adh and Adhr genes

The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among DROSOPHILA: This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2-20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8-44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity (pi) was 0.0139, which is two times higher than that in D. melanogaster.