Adaptation and Conservation throughout the Drosophila melanogaster Life-Cycle

Abstract

Previous studies of the evolution of genes expressed at different life-cycle stages of Drosophila melanogaster have not been able to disentangle adaptive from nonadaptive substitutions when using nonsynonymous sites. Here, we overcome this limitation by combining whole-genome polymorphism data from D. melanogaster and divergence data between D. melanogaster and Drosophila yakuba. For the set of genes expressed at different life-cycle stages of D. melanogaster, as reported in modENCODE, we estimate the ratio of substitutions relative to polymorphism between nonsynonymous and synonymous sites (α) and then α is discomposed into the ratio of adaptive (ωa) and nonadaptive (ωna) substitutions to synonymous substitutions. We find that the genes expressed in mid- and late-embryonic development are the most conserved, whereas those expressed in early development and postembryonic stages are the least conserved. Importantly, we found that low conservation in early development is due to high rates of nonadaptive substitutions (high ωna), whereas in postembryonic stages it is due, instead, to high rates of adaptive substitutions (high ωa). By using estimates of different genomic features (codon bias, average intron length, exon number, recombination rate, among others), we also find that genes expressed in mid- and late-embryonic development show the most complex architecture: they are larger, have more exons, more transcripts, and longer introns. In addition, these genes are broadly expressed among all stages. We suggest that all these genomic features are related to the conservation of mid- and late-embryonic development. Globally, our study supports the hourglass pattern of conservation and adaptation over the life-cycle.

Keywords: DFE-alpha; adaptation; conservation; evo-devo; hourglass hypothesis; natural selection.

Fig. 1.

—Temporal pattern of the four selection statistics estimated with DFE-alpha (ω_a, α, ω_na, and ω). (A) ω_a, the rate of adaptive nonsynonymous substitutions relative to the mutation rate. (B) α, the proportion of base substitutions fixed by natural selection. (C) ω_na, the rate of nonadaptive nonsynonymous substitutions relative to the mutation rate. (D) ω, the rate of nonsynonymous substitutions relative to the mutation rate. Each boxplot (A–E, 100 bootstrap replicates per stage) in a plot is calculated for a randomly drawn sample of the set of genes expressed in a stage with replacement. The solid line going through the boxplot is inferred by LOESS. For the male and female stages, the line is simply a linear regression. The dashed line shows the mean value of each statistic for the genes that are expressed in all stages (again with 100 bootstrap replicates). The embryonic stages are named by the hour’s intervals (from 0 to 24 h), the larval stages are the first instar (L1), second instar (L2), and third instar (L3). The L3 stages are subdivided into the first 12 h (L3-12h) and several puff stages (L3-PS1 to L3-PS7). WPP is the white prepupae stage. The pupal stages with RNA-seq are phanerocephalic pupa, 15 h (P5), 25.6 h pupa (P6), yellow pharate, 50.4 h (P8), amber eye-pharate, 74.6 h (P9–10), and green meconium pharate, 96 h (P15). Adult stages are 1, 5, and 30 days after eclosion (1, 5, and 30 days). Number of genes analyzed is in supplementary table S8, Supplementary Material online. The earliest stages show more variation in the selection statistics because they have less genes.

Fig. 2.

—Clusters of temporal profiles of expression of embryonic development genes and their ω_a, α, ω_na, and ω estimated using DFE-alpha. (A) Temporal expression profile for all the genes belonging to each cluster. (B) ω for each cluster. (C) ω_a sampling for each cluster. (D) ω_na for each cluster. (E) α for each cluster. Each point in the plots in (B)–(E) is calculated for a randomly drawn sample of the set of genes in each cluster with replacement (100 bootstrap replicates per cluster). Asterisks represent the significance by a permutation test, the color indicates whether the value was higher (red) or lower (blue) than expected (•, 0.1–0.05, *<0.05, **<0.01, and ***<0.001). Number of genes analyzed in supplementary table S1, Supplementary Material online. Permutation P values are shown in supplementary table S12, Supplementary Material online.

Fig. 3.

—Nine genomic features over developmental stages. Lines and stages as in figure 1. (A) Size is the coding sequences length of a gene in base pairs. (B) Number of exons is the number of exons for the genes expressed in a stage. (C) Number of transcripts is the number of different isoforms of each gene expressed in a stage. (D) Fop is a measure of codon usage bias: the ratio of optimal codons to synonymous codons. (E) Average intron length is the average distance, in bases, between the exons of a gene. (F) The expression bias is a measure of how much the expression of a gene is restricted to one or few stages estimated as equation (1) (see Materials and Methods). (G) Recombination rate is estimated in 100-kb nonoverlapping windows. (H) CG content of each gene. (I) PPIs are estimated as the number of PPIs of each gene. Mean sampling distribution was obtained by resampling 100 times with replacement the genes from each stage. See supplementary table S8, Supplementary Material online, for the genes analyzed. The same patterns are found when using 4-fold data, see supplementary figure S19, Supplementary Material online.

Fig. 4.

—Selection statistics (ω_a, ω, ω_na, and α) for maternal, maternal–zygotic and zygotic genes. Maternal genes are those genes whose mRNA are laid by the mother in the egg and are never zygotically transcribed, maternal–zygotic are those genes whose mRNA is present in the egg but that are also transcribed by the zygote. Zygotic genes are genes whose mRNA is not laid in the egg by the mother. (A) ω_a is not statistically different between these gene categories. (B) ω is significantly higher in maternal genes than the other two gene categories (P = 0.024). (C) ω_na is significantly higher in maternal genes than in the other two gene categories (P value = 0.003). (D) α is marginally lower in maternal genes compared with the other two categories. Each point in a plot (100 bootstrap replicates per group) is calculated for a randomly drawn sample of the set of genes in each gene category. Asterisks represent the significance by a permutation test, the color indicates whether the value was higher (red) or lower (blue) than expected (•, 0.1–0.05, *<0.05, **<0.01, and ***<0.001). The number of genes analyzed is shown in supplementary table S6, Supplementary Material online. P values in supplementary table S20, Supplementary Material online.