Toh is responsible for collecting data and writing the code.

This data folder contains all data files, phylogenetic tree needed for analyses, and R scripts to perform all analyses described in the study. This data folder also contains the raw confocal images and label files of the Heliconius brain samples, which was used to estimate the number of glomeruli. There are 14 digital files in total. All files can be opened by the AMIRA software from ThermoFisher Scientific.

Note: Additional detailed references for the three ecological traits used for the analyses in the paper are available in the description of Table S8. For all species name abbreviations, please refer to Table S8, columns 'sp full' and 'sp abb'.

Data files within folder:

"Heliconiini_olfactory_evolution_supplementary_tables.xlsx"

This Excel sheet contains 9 tables. Detailed description for each table can be found under supplementary information for: Evolution of the olfactory system during the radiation of Heliconiini butterflies. Below are short descriptions of each table, an overview of abbreviations/column names found in the xlsx, and a short explanation.

Abbreviations/column names:

Variables (columns) in Table S3 are described as follows:

• "Clade"- Nymphalid clade classification
• "Species"- Species name
• "Locus"- Genomic Locus, genomic region where gene is located
• "OG"- Orthologous Group
• "ExtendedSeq"- Extended Sequence Identifier, unique identifier for the annotated sequence
• "Scf"-Scaffold number
• "Start"- Gene start position, Base-pair position on the scaffold where the gene begins.
• "End"- Strand Orientation, DNA strand on which the gene is encoded: + (forward) or - (reverse).
• "Strand"- Frame or sequence-specific identifier (numeric).
• "GenomicLength"-Length of the gene region in units
• "Nexons"- Exon presence; True if one or more exons were predicted, False otherwise.
• "Psignal"- Signal peptide score
• "SeqLength"- Sequence Length, length of the predicted protein sequence in amino acids.
• "TMH"- Transmembrane Helix Classification
• "PFID"- Protein family (PFam) Domain Identifier, accession number of the matched Pfam protein family/domain.
• "PF_Start"- Pfam Domain Start Position, starting amino acid position of the Pfam domain within the protein sequence.
• "PF_End"- Pfam Domain End Position, ending amino acid position of the Pfam domain within the protein sequence.
• "DomainLength"-Domain completeness indicator; “N” = incomplete at N-terminus, “C” = incomplete at C-terminus, “NC” = incomplete at both termini, “–” = complete domain.
• "Incomplete"-Number of incomplete features in the gene model; 0 indicates a complete gene, higher numbers indicate partially missing regions or exons.
• "bitscore"-Bitscore from sequence alignment; higher scores indicate better match to the domain model.
• "E-value"- Expectation value, corrected bit-score adjusted to the sequence database size.
• "bed1-12"- Full genomic coordinates of the olfactory receptor genes.
• "NTseq"- Nucleotide Sequence
• "AAseq"- Amino Acid Sequence

Variables (columns) in Table S8- S10 are described as follows:

• "genus" - Heliconiini genus classification
• "Heliconius"- whether the individuals are part of the Heliconius genus, y= yes, n=no
• "clade" - Heliconiini clade classification
• "sim.clad"- Heliconiini simplified clade classification
• "sp full"- Full name of species
• "sp abb"- Abbreviated name of species
• "ID"- unique identifier number of each individual
• "sex"- sex of each individual
• "country of origin"- country of origin of each individual
• "AL"- Total Antennal Lobe volume
• "ALH"- Antennal Lobe Hub volume
• "GL"- Glomeruli volume
• "rCBR"- rest of Central Brain volume
• "AT(mean)"- Annual Mean Temperature of the habitats over a species range
• "AP(mean)"- Annual Mean Precipitation volume of the habitats over a species range
• "RH"- Annual Mean Relative Humidity levels of the habitats over a species range
• "Windspeed"- Annual Mean Windspeed of the habitats over a species range
• "PF" - presence of pollen feeding, 1=yes, 0=no
• "No.HP" - host plant use number
• "SR"- degree of social roosting, from 1-4
• "OR no."- number of odourant receptors
• "N50"- Assembly N50 statistic; the length of the shortest contig/scaffold such that 50% of the total assembly length is contained in contigs/scaffolds of this length or longer.
• "BUSCO" -Benchmarking Universal Single-Copy Orthologues score, a method to assess genome completeness by searching for evolutionarily conserved single-copy orthologous genes.
• "BUSCO complete score"- Proportion of expected BUSCO genes that are present and complete in the assembly/annotation; reported as a percentage (higher scores indicate greater completeness).

Descriptions:

• Table S1 A-D: MCMC pairwise comparisons of Heliconiini species
• Table S3: Full annotation of 4658 olfactory receptor genes across Heliconiini
• Table S7: SMATR statistics for comparisons of different olfactory neuropils between wild and insectary individuals in 5 Heliconius species
• Table S8: Full volumetric, environmental and ecological data of wild Heliconiini samples. Presence of pollen feeding, as well as the degree of social roosting was obtained from the file "41467_2023_39618_MOESM5_ESM.xlsx" as part of the supplementary dataset 1-9 of Cuoto et al. (2023). Number of host plant use was obtained from the file "freq_hosts_cicc.xlsx" as part of the supplementary information dataset of McLellan and Montgomery (2024).
• Table S9: Filtered dataset of Heliconiini samples for pairwise comparisons
• Table S10: Full volumetric data of wild and insectary Heliconius species.

"Heliconiini.trees"- Heliconiini phylogenetic tree from Cuoto et al. (2023)

R script files within folder:
This section contains 6 annotated scripts.

Scripts for phylogenetically controlled comparative analysis of neuropil volumes and olfactory receptor numbers of the Heliconiini

a. "MCMCglmm_phylogenetic.R" - tests for broad phylogenetic relationships as well as pairwise comparisons of neuropil volumes in the Heliconiini. This script also contains analyses which tests for the strength of the phylogenetic signal (lambda) for the different neuropils.

b. "MCMCglmm_ecology_and_environment.R"- tests for significant environmental and ecological variables affecting neuropil volume 

c. "MCMCglmm_OR_analysis.R"- tests whether neuropil volumes of each species are affected by the number of olfactory receptors , by using BUSCO scores or N50 scores as a control. The script also contains analyses which tests for significant environmental and ecological variables affecting the number of olfactory receptors for each species.

Likelihood ratio testing of phylogenetic signal lambda

a. "llikelihood_ratio_testing_of_phylogenetic_(lambda)_values.R"- tests for the significance of the phylogenetic signal (lambda) by using a likelihood ratio test method where a model with the observed lambda is compared against one that has no phylogenetic signal.

Scripts to test for differences in neuropil volumes of wild vs insectary individuals of Heliconius butterflies

a. "lmer.R"- This analysis runs a linear mixed model to test for the significant variables affecting variation in neuropil volume

b. "SMATR.R"- This analysis tests for differences in slope between two groups (wild vs insectary) and subsequently the presence of elevational or major-axis shifts if slopes are not significantly different

All quantitative analyses performed using R v. 4.4.1

Digital Image files within folder:

"Ealiphera_no83_Panama2013_glo.labels.am"- label files of individual glomeruli for individual number 83 of Eueides aliphera.

"Ealiphera_no83_Panama2013.am"- original confocal image file for individual number 83 of Eueides aliphera.

"PANAMA_2012_Dphae_154.am"- original confocal image file for individual number 154 of Drydaula phaetusa.

"PANAMA_2012_Dphae_154.labels.am"- label files of individual glomeruli for individual number 154 of Drydaula phaetusa.

"URFimages_Dphae_10_ind-glo.labels.am"- label files of individual glomeruli for individual number 10 of Drydaula phaetusa.

"URFimages_Dphae_10.am"- original confocal image file for individual number 10 of Drydaula phaetusa.

"YiPeng_Ealiphera_Pan161_zoom3_glo.labels.am"- label files of individual glomeruli for individual number 161 of Eueides aliphera.

"YiPeng_Ealiphera_Pan161_zoom3.am"- original confocal image file for individual number 161 of Eueides aliphera.

"YiPeng_Ealiphera_Pan3_zoom34_glo.labels.am" - label files of individual glomeruli for individual number 34 of Eueides aliphera.

"YiPeng_Ealiphera_Pan3_zoom34.am"- original confocal image file for individual number 34 of Eueides aliphera.

"YiPeng_Elybia_Pan18_zoom3.am"- original confocal image file for individual number 18 of Eueides lybia.

"YiPeng_Elybia_Pan18_zoom3_glo.labels.am" - label files of individual glomeruli for individual number 18 of Eueides lybia.

"YiPeng_Elybia_Pan54_zoom3_glo.labels.am" - label files of individual glomeruli for individual number 54 of Eueides lybia.

"YiPeng_Elybia_Pan54_zoom3.am"- original confocal image file for individual number 54 of Eueides lybia.