Heliconius possess a unique ability among butterflies to feed on pollen. Pollen feeding significantly extends their lifespan, and is thought to have been important to the diversification of the genus. We used RNA sequencing to examine feeding-related gene expression in the mouthparts of four species of Heliconius and one non-pollen feeding species, Eueides isabella. We hypothesized that genes involved in morphology and protein metabolism might be upregulated in Heliconius because they have longer proboscides than Eueides, and because pollen contains more protein than nectar. Using de novo transcriptome assemblies, we tested these hypotheses by comparing gene expression in mouthparts against antennae and legs. We first looked for genes upregulated in mouthparts across all five species and discovered several hundred genes, many of which had functional annotations involving metabolism of proteins (cocoonase), lipids and carbohydrates. We then looked specifically within Heliconius where we found eleven common upregulated genes with roles in morphology (CPR cuticle proteins), behavior (takeout-like), and metabolism (luciferase-like). Closer examination of these candidates revealed that cocoonase underwent several duplications along the lineage leading to heliconiine butterflies, including two Heliconius-specific duplications. Luciferase-like genes also underwent duplication within lepidopterans, and upregulation in Heliconius mouthparts. Reverse-transcription PCR confirmed that three cocoonases, a peptidase and one luciferase-like gene are expressed in the proboscis with little to no expression in labial palps and salivary glands. Our results suggest pollen feeding, like other dietary specializations, was likely facilitated by adaptive expansions of pre-existing genes—and that the butterfly proboscis is involved in digestive enzyme production.
Annotation file of open reading frames from 5 butterfly species
This is an annotation file for open reading frames (ORFs) from five species of heliconiine butterflies. The file includes ORFs from all five species, top BLAST hits, and the ortholog group the ORF belongs to. Column headers are as follows: "Row#" (row number), "Seq_ID" (sequence ID, including: 3 letter species code [Eis = Eueides isabella, Hdo = Heliconius doris, Her = Heliconius erato, Hmel = Heliconius melpomene, Hsa = Heliconius sara], Trinity 'gene' contig ID [e.g., compXXXX_cX], and ORF coordinates within contig), "H-ID" (hit ID), "H-Eval" (hit e-value), "H-Desc" (hit description), "H-Species" (hit species), "H-Type" (hit type), "H-Tag" (hit database), "H-Len" (hit length), and "OM" (orthocluster membership). NOTE: The program TCW produces two BLAST hits: 1. Best e-value, 2. best e-value with best evidence. The ones presented here are number 2.
Annotation_file_of_ORFs_from_5_species_incl_speciesID_and_orthocluster_membership.txt
Eueides isabella Trinity assembly
This is a fasta format file containing all mRNA contigs for Eueides isabella. The assembly was performed in Trinity version "20130216" and is a raw output that includes all assembled contigs (see Table 1 for contig counts). Assembly was carried out as described in the Methods section of the publication.
Eueides_isabella_Trinity_assembly_Dryad.fasta
Heliconius doris Trinity assembly
This is a fasta format file containing all mRNA contigs for Heliconius doris. The assembly was performed in Trinity version "20130216" and is a raw output that includes all assembled contigs (see Table 1 for contig counts). Assembly was carried out as described in the Methods section of the publication.
Heliconius_doris_Trinity_assembly_Dryad.fasta
Heliconius erato Trinity assembly
This is a fasta format file containing all mRNA contigs for Heliconius erato. The assembly was performed in Trinity version "20130216" and is a raw output that includes all assembled contigs (see Table 1 for contig counts). Assembly was carried out as described in the Methods section of the publication.
Heliconius_erato_Trinity_assembly_Dryad.fasta
Heliconius melpomene Trinity assembly
This is a fasta format file containing all mRNA contigs for Heliconius melpomene. The assembly was performed in Trinity version "20130216" and is a raw output that includes all assembled contigs (see Table 1 for contig counts). Assembly was carried out as described in the Methods section of the publication.
Heliconius_melpomene_Trinity_assembly_Dryad.fasta
Heliconius sara Trinity assembly
This is a fasta format file containing all mRNA contigs for Heliconius sara. The assembly was performed in Trinity version "20130216" and is a raw output that includes all assembled contigs (see Table 1 for contig counts). Assembly was carried out as described in the Methods section of the publication.
Heliconius_sara_Trinity_assembly_Dryad.fasta
Eueides isabella RSEM mapped count data
File is count data (expression level) output of Trinity contigs from RSEM for Eueides isabella. Column headers contain species name, sex (M or F), specimen ID (see Table S1 for details), and tissue type ("ant" = antennae, "leg" = legs, and "mou" = mouthparts).
Eueides_isabella_RSEMmapped_count_data_3tissues_2sexes.txt
Heliconius doris RSEM mapped count data
File is count data (expression level) output of Trinity contigs from RSEM for Heliconius doris. Column headers contain species name, sex (M or F), specimen ID (see Table S1 for details), and tissue type ("ant" = antennae, "leg" = legs, and "mou" = mouthparts).
Heliconius_doris_RSEMmapped_count_data_3tissues_2sexes.txt
Heliconius erato RSEM mapped count data
File is count data (expression level) output of Trinity contigs from RSEM for Heliconius erato. Column headers contain species name, sex (M or F), specimen ID (see Table S1 for details), and tissue type ("ant" = antennae, "leg" = legs, and "mou" = mouthparts).
Heliconius_erato_RSEMmapped_count_data_3tissues_2sexes.txt
Heliconius melpomene RSEM mapped count data
File is count data (expression level) output of Trinity contigs from RSEM for Heliconius melpomene. Column headers contain sex (M or F), tissue type ("ant" = antennae, "leg" = legs, and "mou" = mouthparts), and sample number. See Table S1 for specimen IDs. Samples labelled "1" are the result of merging two technical replicates (see Methods section of paper for details).
Heliconius_melpomene_RSEMmapped_count_data_3tissues_2sexes.txt
Heliconius sara RSEM mapped count data
File is count data (expression level) output of Trinity contigs from RSEM for Heliconius sara. Column headers contain species name, sex (M or F), specimen ID (see Table S1 for details), and tissue type ("ant" = antennae, "leg" = legs, and "mou" = mouthparts).
Heliconius_sara_RSEMmapped_count_data_3tissues_2sexes.txt