Molecular evolution of Drosophila cuticular protein genes

Abstract

Several multigene families have been described that together encode scores of structural cuticular proteins in Drosophila, although the functional significance of this diversity remains to be explored. Here I investigate the evolutionary histories of several multigene families (CPR, Tweedle, CPLCG, and CPF/CPFL) that vary in age, size, and sequence complexity, using sequenced Drosophila genomes and mosquito outgroups. My objective is to describe the rates and mechanisms of 'cuticle-ome' divergence, in order to identify conserved and rapidly evolving elements. I also investigate potential examples of interlocus gene conversion and concerted evolution within these families during Drosophila evolution. The absolute rate of change in gene number (per million years) is an order of magnitude lower for cuticular protein families within Drosophila than it is among Drosophila and the two mosquito taxa, implying that major transitions in the cuticle proteome have occurred at higher taxonomic levels. Several hotspots of intergenic conversion and/or gene turnover were identified, e.g. some gene pairs have independently undergone intergenic conversion within different lineages. Some gene conversion hotspots were characterized by conversion tracts initiating near nucleotide repeats within coding regions, and similar repeats were found within concertedly evolving cuticular protein genes in Anopheles gambiae. Rates of amino-acid substitution were generally severalfold higher along the branch connecting the Sophophora and Drosophila species groups, and 13 genes have Ka/Ks significantly greater than one along this branch, indicating adaptive divergence. Insect cuticular proteins appear to be a source of adaptive evolution within genera and, at higher taxonomic levels, subject to periods of gene-family expansion and contraction followed by quiescence. However, this relative stasis is belied by hotspots of molecular evolution, particularly concerted evolution, during the diversification of Drosophila. The prominent association between interlocus gene conversion and repeats within the coding sequence of interacting genes suggests that the latter promote strand exchange.

Figure 1. Schematic representing the estimation of ω, the ratio of nonsynonymous to synonymous substitutions, along branches of the Drosophila phylogeny.

A. Based on initial pairwise calculations of Ka/Ks, three distinct ω parameters were estimated for each set of orthologous genes. One ω was estimated for the Sophophora species group, one for the Drosophila species group, and a third ω was estimated for the branch connecting the two species groups. The branches of the tree labeled with each ω class are indicated by colored boxes. B. To test whether the estimated ω for the branch between the Sophophora and Drosophila groups is significantly greater than 1 for a particular set of orthologous genes, ω is recalculated for that branch with all other branches assigned to a single background ω class.

Figure 2. The array of CPR cuticular protein genes located approximately at band 44C of D. melanogaster chromosome 2R and the orthologous regions in six other Drosophila species.

A. Schematic of the organization of genes in the array, with colored boxes matching colored symbols in the phylogeny according to the legend at left. Names at top are of D. melanogaster genes; plus and minus symbols indicate relative orientation. Genes with dark outlines are predicted to be intronless. B. Neighbor-joining phylogeny (see Methods) of predicted amino acid sequence with bootstrap support indicated.

Figure 3. The array of CPR cuticular protein genes located approximately at band 65A of D. melanogaster chromosome 3L and the orthologous regions in six other Drosophila species.

A. Schematic of the organization of genes in the array, with colored boxes matching colored symbols in the phylogeny according to the legend at left. Numbered positions in the array correspond to numbered clades in the phylogeny of part B. Names at top are of D. melanogaster genes; plus and minus symbols indicate relative orientation. B. Neighbor-joining phylogeny (see Methods) of predicted amino acid sequence. Arrow indicates the clade within which genes cluster as paralogs rather than as orthologs (see text for details). Bootstrap support is not shown for clarity, but is greater than 90% for all numbered groups and generally low outside these groups.

Figure 4. Gene organization and polymorphism patterns within the 67F array of Drosophila CPR genes.

A. Gene organization in the 12 Drosophila genomes. The arrow indicates the melanogaster species group. Gray boxes indicate highly similar paralogs. B. Number of polymorphic sites within a sliding window of 50 bases across aligned Cpr67Fa1 and Cpr67Fa2 alleles (step of 25 bases). Polymorphism between paralogs is much lower than between orthologs across the entire coding region. The part of the X axis corresponding to the R&R Consensus is shaded.

Figure 5. Gene organization and divergence patterns within the 84A array of Drosophila CPR genes.

Note that genes in this array have the historical names Edg84A and Ccp84Aa-g and thus diverge from nomenclature for other arrays. A. Gene organization in ten Drosophila genomes. D. persimilis and D. pseudoobscura were excluded due to the presence of gaps in the genome sequence. Pairs of genes that cluster by species rather than with orthologs are shaded corresponding shades of gray. B. Graph of the number of polymorphic sites within a sliding window of 50 bases between aligned Ccp84Aa and Ccp84Ab alleles (step of 25 bases). Polymorphism is much lower between paralogs than between orthologs in the central region of the gene, which includes the R&R Consensus, but tends to increase at the 5′ and 3′ ends of coding sequence, approaching the level of orthologs. The part of the X axis corresponding to the R&R Consensus is shaded.

Figure 6. Graph of the number of polymorphic sites within a sliding window of 50 bases between aligned Ccp84Ad and Ccp84Ad′/Cpr5C alleles (step of 25 bases).

Note that Cpr5C is the presumed ortholog of Ccp84Ad′ based on amino-acid sequence phylogeny, but occurs on the X chromosome (see text). Polymorphism is lower between paralogs than between orthologs for all comparisons. However, the level of paralog polymorphism is lowest for those species (solid lines) that have Ccp84Ad′. Species (dotted lines) that lack Ccp84Ad′ but instead have Cpr5C show intermediate levels of divergence. The part of the X axis corresponding to the R&R Consensus is shaded.

Figure 7. Dot plots of the 84A array in two Drosophila species that illustrate sequence repetition (indicated by red circles) within the coding regions of those genes evolving concertedly.

Independently evolving genes in the array lack this sequence repetition. Dot plots of this region for the other ten Drosophila species are shown in Text S4.

Figure 8. Alignment of Ccp84Aa and Ccp84Ab sequence identified as repetitive in dot plots.

Nucleotide sequence was aligned with ClustalW and then trimmed around the most conserved repeat unit, although genes typically have more than two such units at varying degrees of conservation. Brackets indicate two copies of a repeated sequence that is well conserved among all species. Other repeats can be seen that are found in only a subset of species.

Figure 9. Concerted evolution of Drosophila Tweedle genes within an array at 97C.

A. Neighbor-joining phylogeny of predicted proteins. B. Dot plot of array.

Figure 10. Dot plots of the co-orthologous regions of the D. melanogaster 97C Tweedle array from D. pseudoobscura and D. willistoni, with the region around Dwil5471 and Dwil6460 enlarged.

Figure 11. Dot plots of concertedly evolving cuticular protein genes of An. gambiae. (see , for details).

Additional dot plots are shown in Text S4.

Figure 12. Graphs showing the range of ω estimates for orthologous ‘single-copy’ genes of each cuticular protein gene family, as described in the text.

CPR genes occurring in tandem arrays or as isolated genes (‘singletons’) are shown separately. For each gene, the values in the Sophophora (S) and the Drosophila (D) species groups are shown separately, connected by a line to more clearly illustrate the trends among genes. The red lines indicate the mean values.