Supplementary Methods and Materials.

Gene expression variation is pervasive across all levels of organismal organization, including development. Few studies, however, have examined variation in developmental transcriptional dynamics among populations, or how it contributes to phenotypic divergence. Indeed, the evolution of gene expression dynamics when both the evolutionary and temporal timescale are comparatively short remains relatively uncharacterized. Here, we examined coding and non-coding gene expression in the fat body of an ancestral African and a derived European Drosophila melanogaster population across three developmental stages spanning ten hours of larval development. Between populations, expression divergence was largely stage-specific. We detected higher expression variation during the late wandering stage, which may be a general feature of this stage. During this stage, we also detected higher and more extensive lncRNA expression in Europe, suggesting that lncRNA expression may be more important in derived populations. Interestingly, the temporal breadth of protein-coding and lncRNA expression became more restricted in the derived population. Taken together with the signatures of potential local adaptation that we detected at the sequence level in 9–25% of candidate genes (those showing evidence of expression divergence between populations), this finding suggests that gene expression becomes more developmental stage-specific during adaptation to new environments. We further used RNAi to identify several candidate genes that likely contribute to known phenotypic divergence between these populations. Our results shed light on the evolution and dynamics of expression variation over short developmental and evolutionary timescales, and how this variation contributes to population and phenotypic divergence.</div

Top 20 most differentially expressed genes between a Zambian and a Dutch population.

Colors represent differentially expressed (DE) genes shared among stages (white: DE in all; blue: DE in 2 stages, orange: unique) (XLSX)</p

Interpopulation expression variation between a Dutch (NL) and a Zambian (ZI) population across three developmental stages.

A) Principal component analysis of expression variation in the Dutch (blue) and the Zambian (grey) population during the early (circles) and late (triangles) wandering larvae and prepupal (square) stages. B) Upset plot showing differentially expressed genes and their overlap between the Netherlands and Zambia during the three examined stages. Black, horizontal bars indicate the total number of differentially expressed genes that were significantly up-regulated in one population versus the other population during each stage. A black circle indicates that a stage and population combination is included in an intersection class. Intersection classes with a single circle are comprised of genes significantly up-regulated in a single population and stage. Circles connected by a line indicate intersection classes comprised of multiple stage and population combinations. Vertical bars correspond to the number of genes in each intersection class. Colored, non-black, vertical bars indicate groups of genes up-regulated in a single stage and population (yellow: early; green: late; red: prepupal) or up-regulated across all stages in one of the populations (blue: the Netherlands; grey: Zambia).</p

Distribution of genes across expression bins.

All analyzed genes were binned in each stage and population combination according to their expression in TPM (very low ≤ 1, low ≤ 10, moderate ≤ 25, high ≤ 50, very high >50). A) All analyzed genes expressed in the Netherlands (NL) or Zambia (ZI) in early, late, and/or pupal stages binned according to their expression level. B) All genes differentially expressed between the Netherlands and Zambia during early, late, or prepupal stages binned according to their expression level. Shown are the number of genes expressed in each stage and population. Blue colors represent genes up-regulated in the Netherlands and grey colors genes up-regulated in Zambia during each respective stage. C) All genes differentially expressed between a given stage and any other stage in either the Dutch or the Zambian population. (PDF)</p

High <i>F</i>_<i>ST</i> outlier SNPs in genes differentially expressed at all stages and population-associated modules.

High FST outlier SNPs in genes differentially expressed at all stages and population-associated modules.</p

lncRNA and protein coding gene expression in a Dutch (NL) and a Zambian (ZI) population.

Heatmaps of A) lncRNA and B) protein coding gene log2 expression in a Dutch and Zambian population during the early, late, and prepupal (prepup) stages. Each row represents a gene. Populations were clustered separately based on mean gene expression using k-means clustering (k = 5). Mean C) lncRNA and D) protein coding gene expression for the Netherlands (NL, blue) and Zambia (ZI, grey) during the examined stages. E) Developmental stage specificity, as measured by tau, τ, for lncRNA and protein coding (PC) genes in the Netherlands and Zambia. Significant differences between groups were determined using a t-test with a Bonferroni multiple test correction. * P P P −5. B and D) Because it was not expressed in the NL early stage, CG16931 was excluded from these analyses.</p

Effect of single nucleotide polymorphism (SNP) variation at positions 1174 and 1063 on adult body size and wing loading in F2 offspring of reciprocal crosses between 2 cosmopolitan “AT” variant isofemale lines and a sub-Saharan “CG” variant line from a Rwandan population.

<p>Effect of single nucleotide polymorphism (SNP) variation at positions 1174 and 1063 on adult body size and wing loading in F2 offspring of reciprocal crosses between 2 cosmopolitan “AT” variant isofemale lines and a sub-Saharan “CG” variant line from a Rwandan population.</p

Connectivity and differentially expressed (DE) genes in population (pop.)- and age-associated modules.

Shown are the number (num.) of genes in each module; the mean module connectivity (connect.); any significant positive (+) or negative (-) correlations with age or population; genes DE between populations at any stage, across all stages, or number of the 5 most highly connected genes DE between populations during all stages; number of genes DE between stages in any population or both populations and number of the 5 most highly connected genes DE between any two stages; and the number of genes with a significant interaction between developmental stage and population as tested with DESeq2. (XLSX)</p

Distribution of mapping bias.

Dutch (blue) and Zambian (yellow) mapping bias for all genes in A) early, B) late, and C) prepupal samples and for only noncoding genes in D) early, E) late, and F) prepupal samples. (PDF)</p

Results of WGCNA for Dutch and Zambian samples.

A) Size of identified gene co-expression modules. Each module is identified by a color. The grey module contains all genes whose expression did not correlate with any other genes. B) Heatmap of Pearson’s correlation between co-expression modules and the traits developmental stage and population. Significant differences between groups were determined using the student asymptotic P-value from the Pearson’s correlation. * P P P −5.</p