Xanthine dehydrogenase (XDH): episodic evolution of a "neutral" protein.

We investigated the evolution of xanthine dehydrogenase (Xdh) in 34 species from the three multicellular kingdoms, including one plant, two fungi, and three animal phyla, two classes of vertebrates, four orders of mammals, and two orders of insects. We adopted a model-based maximum-likelihood framework of inference. After accounting for among-site rate variation and heterogeneous nucleotide composition of the sequences using the discrete gamma distribution, and using nonhomogeneous nonstationary representations of the substitution process, the rate of amino acid replacement is 30.4 x 10(-10)/site/year when Drosophila species are compared but only approximately 18 x 10(-10)/site/year when comparisons are made between mammal orders, between insect orders, or between different animal phyla and approximately 11 x 10(-10)/site/year when comparisons are made between birds and mammals, between fungi, or between the three multicellular kingdoms. To account for these observations, the rate of amino acid replacement must have been eight or more times higher in some lineages and at some times than in others. Spastic evolution of Xdh appears to be related to the particularities of the genomes in which the locus is embedded.

Erratic overdispersion of three molecular clocks: GPDH, SOD, and XDH.

The neutrality theory predicts that the rate of neutral molecular evolution is constant over time, and thus that there is a molecular clock for timing evolutionary events. It has been observed that the variance of the rate of evolution is generally larger than expected according to the neutrality theory, which has raised the question of how reliable the molecular clock is or, indeed, whether there is a molecular clock at all. We have carried out an extensive investigation of three proteins, glycerol-3-phosphate dehydrogenase (GPDH), superoxide dismutase (SOD), and xanthine dehydrogenase (XDH). We have observed that (i) the three proteins evolve erratically through time and across lineages and (ii) the erratic patterns of acceleration and deceleration differ from locus to locus, so that one locus may evolve faster in one than another lineage, whereas the opposite may be the case for another locus. The observations are inconsistent with the predictions made by various subsidiary hypotheses proposed to account for the overdispersion of the molecular clock.

Shared nucleotide composition biases among species and their impact on phylogenetic reconstructions of the Drosophilidae.

Compositional changes are a major feature of genome evolution. Overlooking nucleotide composition differences among sequences can seriously mislead phylogenetic reconstructions. Large compositional variation exists among the members of the family Drosophilidae. Until now, however, base composition differences have been largely neglected in the formulations of the nucleotide substitution process used to reconstruct the phylogeny of this important group of species. The present study adopts a maximum-likelihood framework of phylogenetic inference in order to analyze five nuclear gene regions and shows that (1) the pattern of compositional variation in the Drosophilidae does not match the phylogeny of the species; (2) accounting for the heterogeneous GC content with Galtier and Gouy's nucleotide substitution model leads to a tree that differs in significant aspects from the tree inferred when the nucleotide composition differences are ignored, even though both phylogenetic hypotheses attain strong nodal support in the bootstrap analyses; and (3) the LogDet distance correction cannot completely overcome the distorting effects of the compositional variation that exists among the species of the Drosophilidae. Our analyses confidently place the Chymomyza genus as an outgroup closer than the genus Scaptodrosophila to the Drosophila genus and conclusively support the monophyly of the Sophophora subgenus.

A long-term study on seasonal changes of gametic disequilibrium between allozymes and inversions in Drosophila subobscura.

Seasonal variation (spring, early summer, late summer, and autumn) of gametic disequilibrium between gene arrangements (OST and O3+4) of the O chromosome and Lap, Pept-1, and Acph allozyme loci, located inside these inversions, has been recorded in a natural population of Drosophila subobscura during seven years over a 15-year period. The length of the study allowed us to investigate the temporal variation of the allozyme-inversion associations by statistical methods of time series analysis. Cyclic seasonal changes of allozyme-inversion associations for both Lap and Pept-1 are detected in the natural population. In both cases, the patterns of seasonal change are due to the seasonal change of frequency of Lap and Pept-1 allozymes occurring exclusively within the OST gene arrangement. In contrast, the allozyme frequencies at these loci within the O3+4 gene arrangement are stable along seasons. The patterns of temporal variation of allozyme-inversion associations for Lap and Pept-1 in the natural population are contrasted with those previously published that correspond to gene arrangements of the O chromosome and nucleotide polymorphism at the rp49 region located inside these inversions, suggesting that natural selection is operating on these allozyme-inversion associations.

Evidence for a high ancestral GC content in Drosophila.

Study of the nucleotide composition in Drosophila, focusing on the saltans and willistoni groups, has revealed unanticipated differences in nucleotide composition among lineages. Compositional differences are associated with an accelerated rate of nucleotide substitution in functionally less constrained regions. These observations have been set forth against the extended opinion that the pattern of point mutation has remained constant during the evolution of the genus. A crucial assumption has been that the most recent common ancestor of the subgenus Sophophora had an elevated GC content. Until now, this assumption has been supported by indirect arguments, consisting of extrapolations from closely related outgroups and limited by the robustness of mathematical descriptions concerning the extensive nucleotide composition differences among sequences. The present study seeks to test the assumption of a high ancestral GC content using realistic representations of the nucleotide substitution process to account for potential biases induced by the heterogeneous GC content of the taxa. The analysis of eight nuclear genes unambiguously corroborates that the common ancestor of Sophophora had an elevated GC content.

Tree rooting with outgroups when they differ in their nucleotide composition from the ingroup: the Drosophila saltans and willistoni groups, a case study.

Rooting is frequently the most precarious step in any phylogenetic analysis. Outgroups can become useless for rooting if they are too distantly related to the ingroup. Specifically, little attention has been paid to scenarios where outgroups have evolved different nucleotide frequencies from the ingroup. We investigate one empirical example that arose seeking to determine the phylogenetic relationship between the saltans and the willistoni groups of Drosophila (subgenus Sophophora). We have analyzed 2085 coding nucleotides from the xanthine dehydrogenase (Xdh) gene in 14 species, 6 from the saltans group and 8 from the willistoni group. We adopt a two-step strategy: (1) we investigate the phylogeny without outgroups, rooting the network by the midpoint method; (2) we reinvestigate the rooting of this phylogeny using predefined outgroups in both a parsimony- and a model-based maximum-likelihood framework. A satisfactory description of the substitution process along the Xdh region calls for six substitution types and substitution rate variation among codon positions. When the ingroup sequences are considered alone, the phylogeny obtained using this description corroborates the known relationships derived from anatomical criteria. Inclusion of the outgroups makes the root unstable, apparently because of differences between ingroups and outgroups in the substitution processes; these differences are better accounted for by a simplified model of evolution than by more complex, realistic descriptions of the substitution process.

Molecular evolution and phylogeny of the buzzatii complex (Drosophila repleta group): a maximum-likelihood approach.

The buzzatii complex of the mulleri subgroup (Drosophila repleta group) consists of three clusters of species whose evolutionary relationships are poorly known. We analyzed 2,085 coding nucleotides from the xanthine dehydrogenase (XDH:) gene in the 10 available species of the complex and Drosophila mulleri and Drosophila hydei. We adopted a statistical model-fitting approach within the maximum-likelihood (ML) framework of phylogenetic inference. We first modeled the process of nucleotide substitution using a tree topology which was reasonably accurate. Then we used the most satisfactory description so attained to reconstruct the evolutionary relationships in the buzzatii complex. We found that a minimally realistic description of the substitution process of XDH: should allow six substitution types and different substitution rates for codon positions. Using this description we obtained a strongly supported, fully resolved tree which is congruent with the already-known (yet few) relationships. We also analyzed published data from three mitochondrial cytochrome oxidases (CO I, II, and III). In our analyses, these relatively short DNA sequences failed to discriminate statistically among alternative phylogenies. When the data of these three gene regions are combined with the XDH: sequences, the phylogenetic signal emerging from XDH: becomes reinforced. All four of the gene regions evolve faster in the buzzatii and martensis clusters than in the stalkeri cluster, paralleling the amount of chromosomal evolution.

Fluctuating mutation bias and the evolution of base composition in Drosophila.

The idea that the pattern of point mutation in Drosophila has remained constant during the evolution of the genus has recently been challenged. A study of the nucleotide composition focused on the Drosophila saltans group has evidenced unsuspected nucleotide composition differences among lineages. Compositional differences are associated with an accelerated rate of amino acid replacement in functionally less constrained regions. Here we reassess this issue from a different perspective. Adopting a maximum-likelihood estimation approach, we focus on the different predictions that mutation and selection make about the nonsynonymous-to-synonymous rate ratio. We investigate two gene regions, alcohol dehydrogenase (Adh) and xanthine dehydrogenase (Xdh), using a balanced data set that comprises representatives from the melangaster, obscura, saltans, and willistoni groups. We also consider representatives of the Hawaiian picture-winged group. These Hawaiian species are known to have experienced repeated bottlenecks and are included as a reference for comparison. Our results confirm patterns previously detected. The branch ancestral to the fast-evolving willistoni/saltans lineage, where most of the change in GC content has occurred, exhibits an excess of synonymous substitutions. The shift in mutation bias has affected the extent of the rate variation among sites in Xdh.

Disparate evolution of paralogous introns in the Xdh gene of Drosophila.

Drosophila nuclear introns are commonly assumed to change according to a single rate of substitution, yet little is known about the evolution of these non-coding sequences. The hypothesis of a uniform substitution rate for introns seems to be at odds with recent findings that the nucleotide composition of introns varies at a scale unknown before, and that their base content variation is correlated with that of the adjacent exons. However, no direct attempt at comparing substitution rates in introns seems to have been addressed so far. We have studied the rate of nucleotide substitution over a region of the Xdh gene containing two adjacent short, constitutively spliced introns, in several species of Drosophila and related genera. The two introns differ significantly in base composition and substitution rate, with one intron evolving at least twice as fast as the other. In addition, the substitution pattern of the introns is positively associated with that of the surrounding coding regions, evidencing that the molecular evolution of these introns is impacted by the region in which they are embedded. The observed differences cannot be attributed to selection acting differently at the level of the secondary structure of the pre-mRNA. Rather, they are better accounted for by locally heterogeneous patterns of mutation.

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.

Molecular evolution and phylogeny of the Drosophila saltans species group inferred from the Xdh gene.

The Drosophila saltans group of the subgenus Sophophora consists of five species subgroups whose phylogenetic relationships are poorly known. We have analyzed 2085 coding nucleotides from the xanthine dehydrogenase (Xdh) gene in six species, at least one from each subgroup. We follow a model-based maximum likelihood framework. We first model the substitution process using a tree topology that is approximately accurate. Then we evaluate several candidate tree topologies using a working model of nucleotide substitution. We found that a minimally realistic description of the substitution process along the Xdh region should allow two transition and four transversion rate parameters and different fixed rates for codon positions, which are distributed statistically according to different gamma distributions. The phylogeny obtained using this description differs in significant respects from a phylogeny based on anatomical criteria. We have also analyzed data from five additional (three nuclear and two mitochondrial) gene regions. In our analysis, these relatively short DNA sequences, either separately or jointly, fail to discriminate statistically among alternative phylogenies. When the data for these five gene regions are combined with the Xdh sequences, the strong phylogenetic signal emerging from Xdh becomes somewhat diluted rather than reinforced. The phylogeny of the species and biogeographical considerations suggest that the D. saltans group originated in the tropics of the New World, similarly as the closely related D. willistoni 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.

Eradication thresholds in epidemiology, conservation biology and genetics.

A simple model has been used to describe metapopulation dynamics, the spread of an infectious disease, and the dynamics of transposable elements (TEs). This suggests underlying common dynamics despite the different nature of the systems. Eradication thresholds are derived from the common model and they are interpreted for each system. TEs have been viewed as intragenomic parasites. Thus, some ideas derived from epidemiology, and in particular the existence of such eradication thresholds, can be used to explain some evolutionary puzzles such as the strange distribution of TE families within the phylogeny of host species.

New Drosophila introns originate by duplication.

We have analyzed the phylogenetic distribution of introns in the gene coding for xanthine dehydrogenase in 37 species, including 31 dipterans sequenced by us. We have discovered three narrowly distributed novel introns, one in the medfly Ceratitis capitata, the second in the willistoni and saltans groups of Drosophila, and the third in two sibling species of the willistoni group. The phylogenetic distribution of these introns favors the "introns-late" theory of the origin of genes. Analysis of the nucleotide sequences indicates that all three introns have arisen by duplication of a preexisting intron, which is pervasive in Drosophila and other dipterans (and has a homologous position as an intron found in humans and other diverse organisms).

Time-series analysis of seasonal changes of the O inversion polymorphism of Drosophila subobscura.

We have studied seasonal variation (spring, early summer, last summer and autumn) of inversion polymorphisms of the O chromosome of Drosophila subobscura in a natural population over 15 years. The length of the study allowed us to investigate the temporal behavior (short-term seasonal changes and long-term directional trends) of the O arrangements by the powerful statistical method of time series analysis. It is shown that the O inversion polymorphisms varied on two different time scales: short-term seasonal changes repeated over the years superimposed on long-term directional trends. All the common arrangements (O3+4+7, Osr, O3+4+8) showed significant cyclic seasonal changes, and all but one of these arrangements (O3+4+7) showed significant long-term trends. Moreover, the degree of seasonality was different for different arrangements. Thus, O3+4+7 and OST showed the highest seasonality, which accounted for approximately 61 and 47% of their total variances, respectively. The seasonal changes in the frequencies of chromosome arrangements were significantly associated with the seasonal variation of the climate (temperature, rainfall, humidity and insolation). In particular, O3+4+7 and OST, the arrangements with the greatest seasonal component, showed the strongest association with all climatic factors investigated, especially to the seasonal changes of extreme temperature and humidity.