DOI: 10.5061/dryad.7pvmcvf6h
title: Data from: Evolution of the olfactory system during the radiation of Heliconiini butterflies

description: Sensory system evolution plays a crucial role in shaping species’
 interactions with their environment, yet the extent to which olfactory
 system diversity reflects ecological and evolutionary pressures at a
 macroevolutionary scale remains unclear. Here, we investigate the
 evolution of the olfactory system across the Heliconiini butterfly tribe,
 an ecologically diverse but closely related group. Using a comparative
 approach, we examined variation in antennal lobe morphology and its
 constituent structures, the glomeruli and antennal lobe hub, as well as
 odourant receptor repertoires across species. We found that antennal lobe
 size variation is driven by independent shifts in glomerular and antennal
 lobe hub volumes, with species-specific differences occurring against a
 backdrop of broader phylogenetic stability. While no direct associations
 with ecological traits were observed, certain species showed large
 expansions in total glomerular volume and odourant receptor numbers,
 warranting further investigation into unmeasured ecological or behavioural
 factors. Additionally, comparisons between wild-caught and
 insectary-reared individuals revealed a surprising pattern of
 developmental plasticity, with antennal lobe hub volumes increasing and
 glomeruli volumes decreasing in captivity, highlighting the influence of
 environmental conditions on neural development. These findings suggest
 that olfactory evolution in Heliconiini is shaped by both evolutionary
 divergence and developmental plasticity, emphasizing the need to integrate
 phylogenetic, ecological, and developmental perspectives to fully
 understand sensory system adaptation.
description: # Data from: Evolution of the olfactory system during the radiation of
 Heliconiini butterflies 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.