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. 2021 Jul 19;10:e68549. doi: 10.7554/eLife.68549

Figure 2. Differential expression of genes at Chromosome 15 implicate cortex as most likely candidate driving yellow bar differences.

(a) Hindwing tissue from co-mimetic morphs of H. melpomene and H. erato were collected at three developmental stages fifth-instar caterpillar, Day 1 Pupae (36hAPF) and Day 2 Pupae (60hAPF). For pupal tissue, hindwing tissue was dissected using the wing vein landmarks shown, corresponding to the future adult position of the hindwing yellow bar (dissection scheme based on Hanly et al., 2019). (b) Relative abundance of transcripts corresponding to the genes cortex, domeless-truncated, domeless/washout throughout developmental stages. (c) Log2FoldChange for the genes cortex, domeless-truncated (dome-t), domeless/washout (dome-wash) across developmental stages. Comparisons are for whole wing discs (Larvae, L) and across wing sections differing in the presence of a yellow bar in pupal wings (D1 and D2; see Figure 2—figure supplement 2: for depiction of contrasts analysed). *Adjusted p<0.05; n/s = not significant. N = 3 for each bar plot.

Figure 2—source data 1. RNA-seq samples were genotyped relative to protein-coding WGS SNPs from individuals from the source populations in Panama.
Both SNPs were contained in the protein-coding sequence of the gene Cortex. Individuals from the RNA-seq experiment match the genotype of the source populations.
Figure 2—source data 2. This was repeated for the H. erato samples; here, only one informative protein-coding SNP was found, in the gene parn.
Once again, all individuals match the expected genotype.
Figure 2—source data 3. Primers used for qPCR experiments for housekeeping genes and cortex are shown below.
Figure 2—source data 4. Gene IDs in the H. melpomene Yb locus and their corresponding IDs in the H. erato genome.

Figure 2.

Figure 2—figure supplement 1. qPCR confirms direction of differential expression in Heliconius erato.

Figure 2—figure supplement 1.

(a) Cortex log2 fold change relative to H. e. hydara using delta CT. Data were normalised against the geometric average CT of three housekeeping genes eF1a, rpL3, and polyABP. (b) Cortex deltaCT in H. e. hydara and H. e. demophoon. Error bars represent the confidence interval for n = 3 (p=0.0444).
Figure 2—figure supplement 2. DGE analysis shows cortex and dome/wash are consistently differentially expressed between colour pattern races and pupal wing sections.

Figure 2—figure supplement 2.

Differential expression across the cortex locus in H. erato, shown as the negative log of the adjusted p-value (-log(padj)). Top: larvae, middle: Day 1 pupae, bottom, Day 2 pupae. See Figure 2—source data 4 for gene IDs and homology with H. melpomene. The red shading highlights the genes cortex, dome-T, and Dome-Wash. The horizontal line indicates the cutoff for significance, at padj = 0.1. Colours are used for each of the contrasts, depicted in Figure 3. In this analysis, genes were differentially expressed in contrast E and C (depictions of these contrasts are provided). E gives genes differentially regulated in black posterior compartment, and C gives genes differentially regulated in yellow anterior compartment.
Figure 2—figure supplement 3. Differential expression across the cortex locus in H. melpomene, shown as the negative log of the adjusted p-value (–log(padj)).

Figure 2—figure supplement 3.

Top: larvae, middle: day one pupae, bottom, day two pupae. See Figure 2—source data 4 for gene IDs and homology with H. erato. Red bars highlight the genes cortex, Dome-T, and Dome-Wash. The horizontal line indicates the cutoff for significance, at padj = 0.1. Colours are used for each of the contrasts, depicted in Figure 2—figure supplement 4. In this analysis, genes were differentially expressed in contrast E, C, and F (depictions of these contrasts are provided). F is the difference between races, E gives genes differentially regulated in black posterior compartment, and C gives genes differentially regulated in yellow anterior compartment (these contrasts are depicted in cartoon form).
Figure 2—figure supplement 4. Depiction of contrasts.

Figure 2—figure supplement 4.

Dark blue represents the yellow races, H. m. rosina and H. e. demophoon from Panama. Light blue represents the black races, H. m. melpomene and H. e. hydara from Colombia.
Figure 2—figure supplement 5. analysis of the cdc20/cdh1 family reveals Cortex is a derived and insect-specific derivative of cdc20.

Figure 2—figure supplement 5.

Full-length protein homologs retrieved from TBLASTN searches were used to generate a curated alignment with MAFFT/Guidance2 with a column threshold of 0.5. TBLASTN searches against arthropod genome and transcriptome NCBI repositories did not recover Cortex homologues outside of the Neoptera lineage. The maximum-likelihood tree was constructed with W-IQ-TREE with the ‘Auto’ function to find a best-fit model of substitution. Colour circles indicate the scores of SH-like approximate likelihood ratio tests (SH-aLRT) computed over 1000 replicates, with numeric values for scores > 80. Scale bar indicates amino-acid substitutions per site. Abbreviations: fzy, fizzy; fzr, fizzy-related; rap, retina aberrant pattern (syn. fzr).
Figure 2—figure supplement 6. Full Cdc20 family protein alignments (see legend in Figure 2—figure supplement 5 for abbreviations).

Figure 2—figure supplement 6.

The conserved C-box motif, necessary for interaction with the APC/C and the IR are highlighted.