Research ArticleEVOLUTIONARY BIOLOGY

Parallel evolution of Batesian mimicry supergene in two Papilio butterflies, P. polytes and P. memnon

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Science Advances  18 Apr 2018:
Vol. 4, no. 4, eaao5416
DOI: 10.1126/sciadv.aao5416
  • Fig. 1 Phenotypes and genome information for P. memnon.

    (A) P. memnon and its model species, A. polyeuctes. Males and nonmimetic females (f. agenor) of P. memnon do not have tails on hind wings, whereas mimetic females (f. achates) have tails. (B) Comparison of main genome data among Papilio butterflies. (C) Phylogenetic tree of the concatenated protein-coding gene sequences for five Papilio butterflies. Multiple alignments of the 4163 ortholog groups were concatenated, and a phylogenetic tree was constructed by using 1,698,871 amino acid sites. Bootstrap values are shown on the branches.

  • Fig. 2 A long dsx-HDR is associated with the mimicry trait.

    (A) Comparison of the mimetic and nonmimetic genome sequences. For each 50-kb segment in the mimetic draft genome, sequence identity to the nonmimetic genome (vertical axis) and mapped position to the chromosomes of B. mori (horizontal axis) were calculated (see Materials and Methods) and plotted as a point. Red points correspond to segments whose identity is <95%. The sequence data were obtained from a nonmimetic female (nonmimetic 1, genotype: aa) and a mimetic female (mimetic 1, genotype: AA). (B) Harr plot for the three HDRs on chromosomes 9 (scaffold position, 4000 to 8000 kb), 23 (scaffold position, 4400 to 4800 kb), and 25 (scaffold position, 0 to 400 kb). Horizontal axis, allele a; vertical axis, allele A. The color of each dot indicates the homology level between the sequences of the a and A alleles as shown at the bottom of each figure. The HDRs on chromosomes 9, 23, and 25 (dsx-HDR) are shown with red arrowheads. (C) Estimation of the zygosity of the A-type dsx-HDR and inverted-type HDRs for 11 butterflies based on the normalized coverage depths. The coverage depth of HDR in chromosomes 9, 23, and 25 of 11 butterflies was normalized by the value corresponding to the homozygous regions (see Materials and Methods). Whole-genome sequencing of 11 butterflies revealed that only the dsx-HDR in chromosome 25 was completely associated with mimicry phenotypes. Individuals used: four nonmimetic females (nonmimetic 1 to 4, genotype: aa), six mimetic females (mimetic 1 to 2, genotype: AA and mimetic 4 to 7, genotype: Aa), and one male (mimetic 3, genotype: AA). (D) Genetic divergence (Fst) between mimetic and nonmimetic populations. Each point corresponds to a 10-kb window in the P. memnon draft genome, and windows with Fst > 0.5 are shown as red points. All windows are mapped on B. mori chromosomes. (E) A 4-Mb region on two P. memnon scaffolds that contain the highest-Fst windows and dsx genes. The order of the two scaffolds is determined based on links of mate pairs and synteny with other Papilio species.

  • Fig. 3 Detailed structure of dsx-HDR in P. memnon.

    (A) Comparison of the detailed structure of dsx-HDR on chromosome 25 between H and h in P. polytes and between A and a in P. memnon. The direction of the dsx-HDR is reversed between h and H of P. polytes, but is the same between a and A of P. memnon. Putative BPs in P. polytes or BSs in P. memnon between the heterozygous and homozygous regions are indicated by red or turquoise dotted lines, respectively. Graphical overview of the homology between heterozygous regions is also shown. Mimetic (H/A) and nonmimetic (h/a) sequences were aligned using the LAGAN program and visualized with VISTA (see Materials and Methods). Exon regions are shown in blue. (B) Marked change in the A/a identity across the BS from the homozygous to heterozygous region. The upper panels represent zoom of regions around right and left BPs (P. polytes) or BSs (P. memnon) in (A). The locations of UXT, dsx, and Nach-like are also shown. Vertical lines (bold or thin) indicate the exon region in each gene, and horizontal lines between exons indicate introns. Red and turquoise dotted lines indicate the BSs in P. memnon and the BPs in P. polytes, respectively. The lower panels show 2-kb sequence comparisons between a and A of P. memnon in the upper panels. (C) Percentages of the various types of repetitive sequences in the whole genome and within dsx-HDR. The proportion of repetitive sequences in scaffolds longer than 500 bp was calculated by using RepeatMasker (see Materials and Methods).

  • Fig. 4 Dimorphic structure and expression of genes in dsx-HDR.

    (A) Dimorphic structure of dsx [F3 isoform reported previously (8)] and Nach-like between nonmimetic (a and h; blue) and mimetic (A and H; red) alleles. Amino acid changes of Dsx and Nach-like are shown above (a/h) and below (A/H) each line representing the exon structure. Conserved amino acid residues in Lepidoptera are shown in red above the amino acid changes; “X” indicates the absence of a conservative amino acid sequence. (B) Phylogenetic tree of three genes (dsx, UXT, and Nach-like). Bootstrap values are shown on the branches. Four sequences of P. polytes– and P. memnon–associated allele types of the dsx-HDR are attached to the species names. (C) Phylogenetic tree of dsx-HDR sequences (maximum-likelihood method using RAxML and GTR+Γ model). Bootstrap values are shown on the branches. (D) Gene expression levels in females (mimetic and nonmimetic) and males at day 2 of the pupal stage (P2) in P. memnon. Three samples for each of the mimetic and nonmimetic pupae were analyzed by quantitative reverse transcription PCR. *P < 0.05 by Student’s t test. The expression level of RpL3 was used as an internal control. The red and black bars indicate A and a allele genes, respectively. PCR primers to discriminate the heterozygous sequence in UXT and Nach-like could not be designed, and hence, we used homozygotes (AA and aa) for the assay. HW, hind wing; Ov, ovary; T, testis. (E) Gene expression levels in hind wings of A/a females and males at stage P7 (n = 4 for mimetic female; n = 3 for male) estimated by RNA-seq (table S1). The red and black bars indicate genes for A and a alleles, respectively. The mean FPKM (fragments per kilobase of transcript per million mapped reads) values are shown with SD.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaao5416/DC1

    fig. S1. Neighbor-joining tree based on average Ks value matrix.

    fig. S2. Harr plot view of the 2-Mb region around dsx-HDRs on chromosome 25 between P. memnon and P. polytes.

    fig. S3. The normalized coverage depth of each region (1-kb window) in the dsx-HDR.

    fig. S4. Relation between LD and genomic distance for dsx-HDR.

    fig. S5. Comparison of detailed and dimorphic structures of dsx-HDR between the A/a locus in P. memnon and the H/h locus in P. polytes.

    fig. S6. Sequence homology of dsx-HDR among A, a, H, h, and the P. xuthus corresponding regions.

    fig. S7. Verification of the BS junction without inversion by mate-pair mapping.

    fig. S8. PCR-based verification of junction sites for the left and right BSs in dsx-HDR of P. memnon.

    fig. S9. Nucleotide sequence comparison of the left and right BSs of dsx-HDR between the A/a locus of P. memnon and the H/h locus of P. polytes.

    fig. S10. Repetitive sequences around BPs or BSs in the A/a locus.

    fig. S11. Amino acid sequence substitutions in Dsx and Nach-like.

    fig. S12. Fst between mimetic and nonmimetic P. memnon at CDSs or UTRs related to dsx-HDR.

    table S1. Statistics of sequenced read data.

    table S2. Statistics of draft genome.

    table S3. Statistics of protein-coding genes.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Neighbor-joining tree based on average Ks value matrix.
    • fig. S2. Harr plot view of the 2-Mb region around dsx-HDRs on chromosome 25 between P. memnon and P. polytes.
    • fig. S3. The normalized coverage depth of each region (1-kb window) in the dsx-HDR.
    • fig. S4. Relation between LD and genomic distance for dsx-HDR.
    • fig. S5. Comparison of detailed and dimorphic structures of dsx-HDR between the A/a locus in P. memnon and the H/h locus in P. polytes.
    • fig. S6. Sequence homology of dsx-HDR among A, a, H, h, and the P. xuthus corresponding regions.
    • fig. S7. Verification of the BS junction without inversion by mate-pair mapping.
    • fig. S8. PCR-based verification of junction sites for the left and right BSs in dsx-HDR of P. memnon.
    • fig. S9. Nucleotide sequence comparison of the left and right BSs of dsx-HDR between the A/a locus of P. memnon and the H/h locus of P. polytes.
    • fig. S10. Repetitive sequences around BPs or BSs in the A/a locus.
    • fig. S11. Amino acid sequence substitutions in Dsx and Nach-like.
    • fig. S12. Fst between mimetic and nonmimetic P. memnon at CDSs or UTRs related to dsx-HDR.
    • table S1. Statistics of sequenced read data.
    • table S2. Statistics of draft genome.
    • table S3. Statistics of protein-coding genes.

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