Expansion of IDSs in genome of H. melpomene

We identified candidates potentially involved in terpene synthesis by searching in the genome of H. melpomene for enzymes in the mevalonate pathway and IDSs using well-annotated Drosophila melanogaster orthologs (S1 Table) [30–32]. We identified reciprocal best basic local alignment search tool (BLAST) hits for all enzymes, except for acetoacetyl-CoA thiolase, which showed the closest similarity to 3 Heliconius genes with no reciprocal best BLAST hit (Fig 1B). There was a clear one-to-one relationship for all enzymes, except for the IDSs which showed evidence for gene duplication. Of these, Heliconius contains 2 putative FPPSs, 4 putative copies of decaprenyl diphosphate synthase (DPPS) subunit 2, and 7 putative geranylgeranyl diphosphate synthases (GGPPSs) (Fig 1A and 1B).

The biggest expansion found was that of the GGPPSs, which are IDSs that catalyse the addition of IPP to farnesyl diphosphate (FPP) to form geranylgeranyl diphosphate (GGPP). One of these, HMEL015484g1, shows 83% amino acid sequence similarity to the GGPPS of the moth Choristoneura fumiferana, which has previously been characterised in vitro to catalyse the production of GGPP from FPP and IPP [33]. HMEL015484g1 is also the best reciprocal BLAST hit with the GGPPS of D. melanogaster (Fig 1A and 1B). The other 6 annotated GGPPSs show less than 50% similarity to the moth GGPPS, such that their function is less clear.

QTL for (E)-β-ocimene production on chromosome 6

In order to determine which of the genes identified above could be important for (E)-β-ocimene production in H. melpomene, we generated genetic mapping families composed of crosses between 2 closely related species that differ in presence/absence of (E)-β-ocimene, H. melpomene, and H. cydno (Fig 2A). These 2 species can hybridise and, although the F1 females are sterile, F1 males can be used to generate backcross hybrids. We bred interspecific F1 hybrid males and backcrossed these with virgin females of both species to generate a set of backcross mapping families. The (E)-β-ocimene phenotype segregated in families backcrossed to H. cydno, and so we focused on these families (S1 Fig). Using quantitative trait locus (QTL) mapping with 114 individuals, we detected a single significant peak on chromosome 6 associated with (E)-β-ocimene quantity (Fig 2B). The QTL peak was at 36.4 cM, and the associated confidence interval spans 16.7 to 45.5 cM, corresponding to a 6.89-Mb region containing hundreds of genes. The percentage of phenotypic variance explained by the peak marker is 16.4%, suggesting that additional loci and/or environmental factors also contribute to the phenotype (S2 Fig).

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Fig 2. QTL and gene expression analyses to identify candidate genes for (E)-β-ocimene production.

(A) The 2 species used in the crosses, H. melpomene which produces (E)-β-ocimene and H. cydno which does not. (B) Genome-wide scan for QTL underlying (E)-β-ocimene production. (C) QTL on chromosome 6 for (E)-β-ocimene production. CIs as well as the positions of candidate genes (subunit 1 of DPPS (PDSS1) and the GGPPS cluster) in the region are marked. Black lines above x-axis represent genetic markers, and horizontal line shows genome-wide significance threshold (alpha = 0.05, LOD = 2.97). (D) HMELOS in H. melpomene shows male abdomen-biased expression (for expression of other genes, see S3 Fig). (E) HMELOS and HMEL037108g1 both show greater male-biased expression in H. melpomene than H. cydno (for expression of other genes, see S4 Fig). Full model statistics in S2 and S3 Tables. N = 5 for each boxplot. Gene expression is given in log2 of normalised counts per million (using the TMM transformation). Sequencing data used to make linkage maps are available from the ENA study PRJEB34160. RNA-seq data of H. cydno and H. melpomene heads and abdomens was obtained from GenBank BioProject PRJNA283415. Processed data and scripts are available from OSF (https://osf.io/3z9tg/). CI, confidence interval; ENA, European Nucleotide Archive; LOD, log odds ratio; QTL, quantitative trait locus; RNA-seq, RNA sequencing; TMM, trimmed mean of M values.

https://doi.org/10.1371/journal.pbio.3001022.g002

Patterns of gene expression identify HMELOS and HMEL037108g1 as candidates

To identify candidate genes for (E)-β-ocimene production, we searched within the confidence interval of the QTL peak. We found that subunit 1 of DPPS as well as all 7 GGPPSs were found in this region (Fig 2C). We then compared the expression levels of the 8 genes found within the QTL using published RNA sequencing (RNA-seq) data [34]. We first analysed data from H. melpomene male and female abdomens and heads, mapped to the H. melpomene reference genome (v2.5). Since (E)-β-ocimene is found in male abdomens in H. melpomene, we hypothesised that its synthase would be highly expressed in this sex and tissue. Only 1 gene showed male abdomen-biased expression: HMELOS (previously HMEL037106g1 from the H. melpomene annotation) (tissue * sex, t = −4.18, adjp = 0.0029; Fig 2D and S2 Table). All other genes did not show a significant bias in this direction (S3 Fig and S2 Table).

We next compared gene expression between H. cydno and H. melpomene abdomens. If HmelOS is synthesising (E)-β-ocimene, we might expect HMELOS expression to be higher in H. melpomene male abdomens than in H. cydno, given that H. cydno does not produce the compound. We generated a reference-guided assembly of H. cydno by aligning an existing H. cydno Illumina trio assembly [35] to the H. melpomene reference, followed by automated gene annotation. We then manually identified H. cydno orthologs for our 7 candidate genes and checked for differential expression between species and sexes. HMELOS and HMEL037108g1 were the only genes showing greater male-biased expression in H. melpomene abdomens than in H. cydno abdomens (HMELOS, species * sex, t = 3.15, adjp = 0.0445; HMEL037108g1, species * sex, t = 3.44, adjp = 0.0259; Fig 2E and S3 Table). No other genes showed a significant bias in this direction (S4 Fig and S3 Table). In summary, HMELOS and to a lesser extent HMEL037108g1 are primary candidate genes from within the QTL region.

Functional characterisation demonstrates the TPS activity of HmelOS and HMEL037108g1

We cloned HMELOS and HMEL037108g1 from H. melpomene, as well as the H. cydno ortholog of HMELOS, HCYDOS, into plasmids and were able to generate heterologous expression of both proteins in E. coli (S5 Fig). We then conducted enzymatic assays with the expressed proteins using precursors from different points in the pathway to characterise their enzymatic function (Figs 1 and 3).

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Fig 3. Functional characterisation of TPS activity of HmelOS and HMEL037108g1 from H. melpomene and HcydOS from H. cydno.

(A) Total ion chromatograms of enzyme products in the presence of different precursor compounds. HmelOS produces high amounts of (E)-β-ocimene in the presence of GPP, with trace amounts found in the treatment with DMAPP + IPP and none with FPP. HMEL037108g1 produces large amounts of linalool with GPP and nerolidol with FPP. HcydOS does not exhibit TPS activity, showing no difference from control treatments (see S6 Fig). 1, (E)-β-Ocimene; 2, Linalool; 3, Geraniol; 4, Nerolidol; 5, Farnesol; IS, internal standard. Abundance is scaled to the highest peak of all treatments per enzyme. For quantification of peaks, see S4–S6 Tables. (B) Confirmation of identity of (E)-β-ocimene by comparison of mass spectra of (E)-β-ocimene produced in experiments and a standard. Chromatograms and mass spectra of all standards can be found in S12 Fig. (C) Pathway of how (E)-β-ocimene and linalool are formed from GPP. Raw data are available from OSF (https://osf.io/3z9tg/). DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; TPS, terpene synthase.

https://doi.org/10.1371/journal.pbio.3001022.g003

First, we carried out assays with DMAPP and IPP, the 2 building blocks at the beginning of the terpene synthesis pathway to test for both IDS and TPS activity, as was seen in Ips pini (Fig 1). HmelOS produced trace amounts of (E)-β-ocimene, linalool (another monoterpene), and nerolidol (a sesquiterpene), in this assay. This presumably occurs via the production of geranyl diphosphate (GPP) and FPP; therefore, HmelOS exhibits residual GPPS and FPPS activity, as well as monoterpene synthase and sesquiterpene synthase activity to convert the GPP and FPP to (E)-β-ocimene, linalool, and nerolidol (Fig 3A, S6 Fig and S4 Table). HMEL037108g1 produced trace amounts of linalool (Fig 3A and S6 Fig) and nerolidol from DMAPP and IPP. Again, this demonstrates residual GPS and FPPS activity to form the GPP and FPP and then both monoterpene and sesquiterpene synthase activity to convert these to linalool and nerolidol (Fig 3A and S5 Table). We found no evidence for activity of HcydOS (Fig 3A, S6 Fig and S6 Table).

We then carried out assays with GPP and IPP, as well as GPP alone to test for monoterpene synthase activity (Fig 1). Assaying with GPP and IPP together also allows us to test for potential FPPS and sesquiterpene synthase activity which could occur via production of FPP. HmelOS showed monoterpene synthase activity, producing (E)-β-ocimene when provided with either GPP and IPP, or GPP alone (Fig 3A, S6 Fig and S4 Table). Small amounts of (Z)-β-ocimene were also produced in treatments where (E)-β-ocimene was produced in large quantities (S4 Table). In contrast to HmelOS, HMEL037108g1 only produced (E)-β-ocimene in very small amounts from GPP (S5 Table). Instead, linalool was produced in large amounts from GPP, suggesting that this enzyme is also acting as a monoterpene synthase but is responsible for production of linalool rather than (E)-β-ocimene (Fig 3A, S6 Fig and S5 Table). HmelOS also produced linalool, albeit in much smaller quantities (Fig 3A, S6 Fig and S4 Table). We found no evidence for monoterpene activity of HcydOS (Fig 3A, S6 Fig and S6 Table).

Finally, we carried out assays with FPP and IPP to test for sesquiterpene synthase activity (Fig 1). Although HmelOS exhibited small amounts of sesquiterpene synthase activity through the trace production of nerolidol when provided with DMAPP and IPP (S4 Table), when provided with FPP and IPP, sesquiterpene synthase activity was not demonstrated, suggesting that it is not the primary enzyme function (Fig 3A, S6 Fig, and S4 Table). In contrast, HMEL037108g1 did exhibit sesquiterpene synthase activity, producing large amounts of nerolidol when FPP was provided as a precursor (Fig 3A, S6 Fig and S5 Table). We found no evidence for sesquiterpene synthase activity of HcydOS (Fig 3A, S6 Fig and S6 Table).

Due to the linalool detected in treatments where (E)-β-ocimene was produced by HmelOS, we tested whether linalool could be a metabolic intermediate between GPP and (E)-β-ocimene. However, HmelOS did not produce (E)-β-ocimene from linalool (S7 Fig and S7 Table). The 2 stereoisomers of linalool, (S)-linalool and (R)-linalool, have different olfactory properties. We confirmed the stereochemistry of linalool produced by both enzymes and found that while HmelOS produced mainly (S)-linalool, HMEL037108g1 produced a racemic mixture (S8 Fig).

In summary, HmelOS is a monoterpene synthase, catalysing the conversion of GPP to (E)-β-ocimene in H. melpomene (Fig 3B and 3C and S8 Table). HMEL037108g1 is a bifunctional monoterpene and sesquiterpene synthase catalysing the conversion of GPP to linalool as well as FPP to nerolidol (Fig 3C and S8 Table). We found no evidence for TPS activity of HcydOS (Fig 3A, S6 Fig and S8 Table).

Functional characterisation demonstrates the residual IDS activity of HmelOS, HcydOS, and HMEL037108g1

In order to better understand the origin of TPS activity in these enzymes, we further investigated their residual IDS activity. While the production of terpenes can be tested by direct gas chromatography/mass spectrometry (GC/MS) analysis of the products of each experiment, this method will not detect isoprenyl diphosphates, potentially missing IDS activity if it is present. In order to test for IDS activity, we repeated the above experiments with DMAPP and IPP, GPP and IPP, and FPP and IPP, followed by treatment with alkaline phosphatase to hydrolyse the isoprenyl diphosphate products to their respective alcohols. These alcohols can then be detected by GC/MS analysis.

No further IDS activity was detected in any enzyme, apart from the residual IDS activity of the H. melpomene enzymes already determined above due to the trace amounts of terpenes produced from DMAPP and IPP. When either enzyme is provided with GPP, geraniol is produced, and when provided with FPP, large amounts of farnesol is produced, as expected from the dephosphorylation of the provided precursors, and this is seen in control conditions, as well all experiments with HcydOS (S9–S11 Figs and S9–S11 Tables). As expected from the previous experiments, (E)-β-ocimene is also produced when HmelOS is provided with GPP, and linalool and nerolidol are produced when HMEL037108g1 is provided with GPP and FPP, respectively. Geranylgeraniol is not produced in any treatments, demonstrating that neither HmelOS, HMEL037108g1 nor HcydOS is a GGPPS, as suggested by their annotation (S9–S11 Figs and S9–S11 Tables). In summary, both HmelOS and HMEL037108g1 only exhibit residual IDS activity, while we did not detect any IDS activity for HcydOS.

Evolutionary history of gene family containing Heliconius TPSs

Lineage-specific expansions of gene families are often correlated with functional diversification and the origin of novel biological functions [36]. We therefore carried out a phylogenetic analysis of GGPPS in Lepidoptera to investigate whether gene duplication could have played a role in the evolution of the TPSs HmelOS and HMEL037108g1. Orthologs of the H. melpomene GGPPSs were identified in H. cydno, Heliconius erato, Bicyclus anynana, Danaus plexippus, Papilio polytes, Pieris napi, Manduca sexta, Bombyx mori, and Plutella xylostella [37]. Expansions of the GGPPS group of enzymes can be seen in Heliconius and in Bicyclus, both groups in which terpenes form part of the pheromone blend [38] (S13 Fig). Expansion of gene families is often followed by an acceleration of the nonsynonymous mutation rate facilitated by a relaxation or loss of selectional constraints [39]. To further investigate whether gene duplication played a role in the evolution of TPSs, we examined patterns of synonymous to non-synonymous evolution in the Heliconius GGPPS/TPS family. Heliconius and Bicyclus clades show higher relative non-synonymous evolution consistent with relaxed selection constraints (ω = 0.22 and 0.19, respectively) compared to the strong purifying selection on the non-expanded lepidopteran GGPPS genes (ω = 0.027, χ2 = 338.2, p < 0.00001).

To focus on the Heliconius-specific duplications, we made a phylogeny using the DNA sequence of transcripts from H. melpomene, H. cydno, and H. erato. H. melpomene and H. cydno belong to the same clade within Heliconius, with an estimated divergence time around 1.5 million years ago [40]. H. erato is more distantly related, belonging to a different Heliconius clade which diverged from the H. melpomene/H. cydno group around 10 million years ago [41]. While (E)-β-ocimene is not found in the genitals of H. cydno, it is found in the genitals of H. erato, at around one-tenth the amount of H. melpomene [18]. We hypothesised that duplications between the H. melpomene and H. erato clades may have resulted in subfunctionalisation and a more efficient H. melpomene enzyme facilitating increased (E)-β-ocimene production. We found that both losses and gene duplications have occurred between the H. melpomene and H. erato clades, while gene copy number is conserved between closely related H. melpomene and H. cydno (S14 Fig). The exact orthology between the H. erato and H. melpomene/H. cydno genes is unclear, but H. melpomene/H. cydno have more genes in this family than H. erato (S14 Fig), and both clades have more genes than the ancestral lepidopteran state of 1 copy.

We also found evidence for the formation of pseudogenes following gene duplication. The amino acid sequences from translations of 1 gene in H. cydno, the ortholog of HMEL037104g1, does not contain a complete functional protein domain. This is also seen for Herato0606.241 in H. erato. Furthermore, more recent pseudogene formation could be seen in the H. cydno ortholog of HMEL22306g3, which contained multiple stop codons, despite being transcribed (S4 Fig).

We have demonstrated that HmelOS of H. melpomene acts as an ocimene synthase, with residual IDS activity, while we find no evidence of these functions in its ortholog in H. cydno, HcydOS. To identify likely functional changes between H. melpomene and H. cydno we first analysed the patterns of synonymous and nonsynonymous mutations between these species. The branch leading up to HMELOS and HCYDOS is under positive selection (ω = 1.33); this ratio is greater than the background ratio of the Heliconius GGPPS genes (ω = 0.19) (χ2 = 6.9, p = 0.012). However, we find no difference between ω ratio for the HMELOS branch and HCYDOS branch (χ2 = 0.36, p = 0.54).

We then predicted the sequence of the last common ancestor (LCA) of H. melpomene HmelOS and H. cydno HcydOS using ancestral sequence reconstruction with a posterior probability of 0.998 (S15 Fig). Six amino acid changes differentiate HmelOS from the LCA, and 3 amino acid changes differentiate HcydOS from the LCA. To identify which of these residues are conserved within species, we aligned amino acid sequences from a set of 20 sequences from 10 individuals of each species (S12 Table). Of the sites which differentiate H. cydno and H. melpomene from the predicted sequence of the LCA, the most intraspecifically conserved sites occur between amino acid residues 100 and 125, suggesting that this region is likely to be important for the function of the enzyme (S13 Table). All 3 mutations in the H. cydno lineage (M109T, V119T, and A123T) and 1 of the mutations in the H. melpomene lineage (R122K) occur in this region. Of the amino acid changes in this region, the mutations in the H. cydno lineage involve a larger change in amino acid chemistry (from either methionine or small hydrophobic residues to a polar residue) than the mutation between 2 positively charged amino acids in the H. melpomene lineage.

In order to determine the number of evolutionary origins of insect and plant TPSs, we carried out a broader phylogenetic analysis, including other known insect and plant IDS and TPS proteins. Similar to the other insect TPSs described, Heliconius TPSs are not found within the same clade as plant ocimene synthases, representing an independent origin of ocimene synthesis in Heliconius and plants. Furthermore, the Heliconius TPSs do not group with known insect TPS enzymes in Hemiptera and Coleoptera (Fig 4). Instead, the Heliconius TPS enzymes group with GPP and GGPP synthases, rather than FPP synthases. The TPS enzymes of Heliconius are therefore of an independent evolutionary origin as compared to other insect TPSs.

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Fig 4. Phylogram of GGPPS, FPPS, and TPS proteins of animals, fungi, and plants.

The phylogeny was constructed by PhyML using LG model of amino acid evolution. Bootstrap (n = 1000) values are illustrated. The tree was rooted with the ocimene synthase of Citrus unshiu. Full species names in S14 Table. Script is available from OSF (https://osf.io/3z9tg/). FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; TPS, terpene synthase.

https://doi.org/10.1371/journal.pbio.3001022.g004

Comparison of the amino acid alignment of known insect TPSs with the H. melpomene enzymes (S16 Fig) demonstrated that residues previously identified as conserved in insect TPSs [28] were not found in the H. melpomene TPSs. No residues were shared between all insect TPSs (including H. melpomene TPS) which were not also shared with the H. melpomene GGPPS. This further indicates independent convergent evolution of TPS function in H. melpomene.

Ethics statement

Butterflies were collected under permits SE/AP-23-15 and SE/AP-11-17 issued by the Ministerio de Ambiente de Panamá.

Analysis of biosynthetic pathway in H. melpomene

To identify genes involved in terpene biosynthesis, we searched the H. melpomene genome (v2.5) on LepBase [37,61] for genes in the mevalonate pathway and IDSs. D. melanogaster protein sequences were obtained from FlyBase and used in BLAST searches (blastp) against all annotated proteins in the H. melpomene genome (S1 Table) [52,62]. We used the BLAST interface on LepBase with default parameters (-evalue 1.0e-10 -num_alignments 25) [37,63]. We then searched these candidate orthologs against the annotated proteins of D. melanogaster using the BLAST interface on FlyBase to identify reciprocal best blast hits. We included in our results other hits with an e-value smaller than 1e-⁸⁰.

Butterfly stocks

Outbred stocks of H. melpomene rosina and H. cydno chioneus were established from wild individuals collected in Gamboa (9°7.4′ N, 79°42.2′ W, elevation 60 m) in the nearby Soberania National Park, San Lorenzo National Park (9°17′ N, 79°58′ W; elevation 130 m), and in Altos de Campana National Park (8°69′ N, 79°92′ W; elevation 900 m). Stocks were maintained under ambient conditions in insectaries at the Smithsonian Tropical Research Institute (STRI) facilities in Gamboa, Panama. Individuals for this study were reared between January 2016 and January 2018. Larvae were reared on Passiflora platyloba. Adult male butterflies were kept in cages with other males and provided with approximately 20% sucrose solution with access to at least 1 of Psychotria poeppigiana, Gurania eriantha, Psiguiria triphylla, and Psiguria warscewiczii as pollen sources.

Crossing for quantitative trait linkage mapping

To map the genetic basis of ocimene production, we crossed H. melpomene, which produces (E)-β-ocimene, to H. cydno, a closely related species which does not. We crossed these 2 species to produce F1 offspring and backcross hybrids in both directions. Female F1s are sterile, and so we mated male F1s to H. cydno and H. melpomene virgin stock females to create backcross families. Families created by backcrossing to H. melpomene had a phenotype similar to pure H. melpomene, suggesting that the H. melpomene phenotype is dominant. While we used 265 individuals to create the linkage map, we focused on backcross families in the direction of H. cydno, where the (E)-β-ocimene phenotype segregates for the QTL mapping (S1 Fig). We phenotyped and genotyped 114 individuals from 15 backcross families in the direction of H. cydno. Bodies were stored in dimethyl sulfoxide (DMSO) and stored at −20°C for later DNA extraction.

Genotyping and linkage map construction

DNA extraction was carried out using Qiagen DNeasy kits (Qiagen, Hilden, Germany). As previously described, individuals were genotyped either by Restriction site Associated DNA sequencing (RAD-seq) [64–66] or low-coverage whole genome sequencing using Nextera-based libraries [66,67]. A secondary purification using magnetic SpeedBeads (Sigma, Gallen, Switzerland) was performed prior to Nextera-based library preparation. Libraries were prepared following a method based on Nextera DNA Library Prep with purified Tn5 transposase [67]. PCR extension with an i7-index primer (N701–N783) and the N501 i5-index primer was performed to barcode the samples. Library purification and size selection were done using the same beads as above. Pooled libraries were sequenced by BGI (China) using HiSeq X Ten (Illumina). Parents were sequenced at an average depth of 30.23 (SD 12.67), grandparents at 12.58 (SD 11.47), and offspring at 9.77 (SD 4.98).

Linkage mapping was conducted following Byers and colleagues [66], using a standard Lep-MAP3 (LM3) pipeline [68]. Briefly, fastq files were mapped to the H. melpomene reference genome using BWA MEM [69]. Sorted bams were then created using SAMtools, and genotype likelihoods were constructed [70]. The pedigree of individuals was checked and corrected using IBD (identity by descent), and the sex was checked using coverage on the Z chromosomes by SAMtools depth. A random subset of 25% of markers were used for subsequent steps. Linkage groups and marker orders were constructed based on the H. melpomene genome and checked with grandparental data.

The map constructed contained 447,820 markers. We reduced markers by a factor of 5 evenly across the genome resulting in 89,564 markers with no missing data to facilitate computation. We log-transformed amounts of ocimene produced to conform more closely to normality. Statistical analysis was carried out using R/qtl [101]. We carried out standard interval mapping using the scanone function with a nonparametric model, an extension of the Kruskal–Wallis test statistic. The analysis method for this model is similar to Haley–Knott regression [102]. We used permutation testing with 1000 permutations to determine the genome-wide LOD significance threshold. To obtain confidence intervals for QTL peaks, we used the function bayesint. Phenotype data, pedigree, linkage map, and R script are available from OSF (https://osf.io/3z9tg/). Sequencing data used for the linkage maps are available from the European Nucleotide Archive (ENA) project PRJEB34160 [66].

Phenotyping of (E)-β-ocimene production

Chemical extractions were carried out on genital tissue of mature (7 to 14 days post-eclosion) male individuals of H. melpomene, H. cydno, and hybrids. Genitals were removed using forceps and soaked, immediately after dissection, in 200 μl of dichloromethane containing 200 ng of 2-tetradecyl acetate (internal standard) in 2-ml glass vials with polytetrafluoroethylene-coated caps (Agilent, Santa Clara, California, United States of America). After 1 h, the solvent was transferred to a new vial and stored at −20°C until analysis by GC/MS.

GC/MS analysis

DMAPP (90%), GPP (95%), and IPP (95%) were purchased from Sigma. FPP (98%) was purchased from VWR (Darmstadt, Germany). (R)-Linalool (95%) was purchased from Merck (Darmstadt, Germany). (S)-Linalool was isolated from coriander oil (Sigma) and purified through column chromatography.

Extracts from adult butterflies and samples from in vitro experiments were analysed by GC/MS using an Agilent (model 5977/5975) mass-selective detector connected to an Agilent GC (model 7890B/7890A) with electron impact ionisation (70eV). This instrument was equipped with an Agilent ALS 7693 autosampler and an HP-5MS fused silica capillary column (Agilent) (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm). Injection was performed in splitless mode (injector temperature 250°C) with helium as the carrier gas (constant flow of 1.2 ml/min). The temperature programme started at 50°C for 5 min and then rose at a rate of 5°C/min to 320°C, before being held at 320°C for 5 min. The compounds were identified through comparison with retention time and mass spectra of standard samples.

Chiral analysis of linalool was performed in an Agilent 7820A gas chromatograph equipped with flame ionisation detector (FID) using a chiral column Beta DEX 225 (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm, Supelco, Bellefonte, Pennsylvania, USA). The oven program started at 50°C for 1 min, followed by increasing the temperature at a rate of 3°C/min until 210°C, keeping this temperature for 5 min. A total of 1 μl of each sample was injected in splitless mode at a flow of 1.65 mL/min. The peak areas were used to calculate the percentage of each stereoisomer.

Heliconius cydno guided assembly and annotation transfer

H. cydno and H. melpomene had their most recent common ancestor 1.5 million years ago, and their absolute divergence is roughly 3% (dxy approximately 0.03) [41,71]. Due to this high degree of similarity, it is possible to map H. cydno RNA-seq reads to the H. melpomene genome. However, we wanted to accurately quantify gene expression in existing H. cydno samples (GenBank BioProject PRJNA283415 [34]) by reducing potential biases associated with RNA-seq reads carrying H. cydno specific alleles. RNA-seq reads from H. cydno with such variants have a lower probability to map correctly to the existing H. melpomene reference. This biases quantification and increases false-positive rates, documented extensively, specifically in the context of allele-specific expression studies [72].

A H. cydno trio (mother, father, and progeny) was previously Illumina sequenced (ENA study ERP009507) and assembled into maternal and paternal genomes with trio-sga [35]. The paternal genome had 34,566 scaffolds, a total size of 270,339,622 bp and a scaffold N50 of 25,716 bp, with 551 kb of gaps (paternal trio fasta file is available from OSF (https://osf.io/3z9tg/)). To improve gene contiguity, we used the progressiveCactus algorithm (v3) to align the H. cydno paternal haplotypic assembly to the chromosomal version of the H. melpomene genome (v2.5) [73–75]. The HAL database created by progressiveCactus was loaded to Ragout (v1.2) [76] to produce the final H. cydno reference-guided assembly (H. cydno reference fasta file; ordering information and unplaced scaffolds are available from OSF (https://osf.io/3z9tg/). The H. cydno guided assembly has 58 scaffolds, a total size of 261,056,210 bp, a scaffold N50 of 13,724,118 bp, and 8.3 Mb of gaps.

We then transferred the H. melpomene annotation (v2.5) to the H. cydno assembly. We used EMBOSS Seqret (v6.6.0.0) to convert the H. melpomene annotation file to the embl format [77], and we used RATT to transfer the H. melpomene annotation (reference) to the guided H. cydno genome (query). RATT is part of PAGIT, a post-assembly genome-improvement toolkit (v1.0) [78]. We searched for synteny between the reference and the query using MUMmer (v4.0) and detected possible errors such as start and stop codons or frameshift mutations [79]. After correcting such errors with the RATT pipeline, the annotation transfer to H. cydno was complete [80].

Annotation improvements

To ensure that our genes of interest from H. melpomene (those identified in S1 Table) were correctly annotated, we manually curated these genes in the H. cydno annotation. To find orthologs in H. cydno, we used the BLAT function in Apollo to search for H. melpomene exons [81,82]. We checked the gene models for splice sites and start and stop codons. The curated gene models were then exported from Apollo and manually included in the H. cydno annotation. We then subset the annotation to include only exons, because CDS sequences had not been properly annotated (Updatedannotation.gff). We then converted it to gtf file format using the gffread function of Cufflinks (Hcyd1.0_annotV2.gtf) [83] and filtered out exons longer than 30,000 bp (Hcyd1.0_annotV2.gtf; gtf_modify_Hcyd_annotV2.R). We finally used the gtf_modify_Hcyd_annotV3.R script to include unique H. cydno gene-ids (Hcyd1.0_annotV3.gtf).

To check that our genes of interest were correctly annotated in all 3 Heliconius species (H. melpomene, H. cydno, and H. erato), we used already published RNA-seq data of abdomens and heads to improve the annotations from GenBank BioProject PRJNA283415 [34]. We performed quality control and low-quality base and adapter trimming on the RNA-seq data using TrimGalore! [84]. We then mapped the reads to the H.melpomene genome v2.5 [75], our newly assembled H.cydno genome, and the H. erato genome [85] using STAR. In all cases, we ran 2pass and set a maximum intron length to 80,350 bp, which is the size of longest intron in the H. melpomene genome [86]. We used StringTie [87] to assemble transcripts genome wide and compared the new assembly of out candidate genes with the original annotation. H. melpomene genes HMEL022305g1 and HMEL037104g1 were merged into 1 new gene (now called HMEL037104g1 in Hmel2.5_edited.gtf) based on this evidence. Furthermore, genes HMEL007429g2 and HMEL007429g3 were also merged (now called HMEL007429g4 in Hmel2.5_edited.gtf), as the first 2 exons of HMEL007429g2 and the last exon of HMEL007429g3 are expressed as a single transcript. We also edited the H. erato annotation to split Herato0606.239 into Herato0606.239_a and Herato0606.239_b (Herato_edited.gff). We made no further changes to the annotation of H. cydno.

RNA-sequencing analysis

Gene expression analyses were performed using already published RNA-seq data from heads and abdomens of H. melpomene and H. cydno from GenBank BioProject PRJNA283415 [34], the same data used to improve the annotations. We used the reads after the trimming step done with TrimGalore! [84]. We then remapped the reads to the H.melpomene genome v2.5 [75] and our newly assembled H.cydno genome using STAR, this time including our manually edited annotations (Hmel2.5_edited.gtf and Hcyd1.0_annotV3.gtf). As before, we ran 2pass and set a maximum intron length to 80,350 bp. featureCounts [88] was used to produce read counts that were normalised by library size with trimmed mean of M values (TMM) normalisation [89] using the edgeR package in R [90]. To test for differences in expression of our candidate genes, we used the voom function from the limma package in R [91], which fits a linear model for each gene by modelling the mean–variance relationship with precision weights.

To test for male abdomen-biased expression within H. melpomene, we included 2 fixed effects, sex, and tissue, as well as including individual as a random effect (expression ~ sex + tissue + sex*tissue + (1|individual)). We were looking for genes with a significant interaction between sex and tissue, showing higher expression in male abdomens. To test for differences in expression between H. melpomene and H. cydno abdomens, we included 2 fixed effects, sex, and species, as well as an interaction term (expression ~ sex + species + species*sex). We were interested in finding differences in the extent of sex bias between species, again detected by a significant interaction term with higher expression in H. melpomene male abdomens.

p-Values were corrected for multiple testing using the Benjamini–Hochberg procedure for all genes in the genome-wide count matrix (17,902 for H. melpomene). For the interspecific comparison, we identified genome-wide orthologs from the annotation and produced a gene count matrix including both species. The ortholog list was limited to genes that had only 1 ortholog in each species (11,571 genes). Scripts are available from OSF (https://osf.io/3z9tg/).

In vitro expression and enzymatic assays

RNA extraction from male abdominal tissue of H. melpomene was carried out following a standard TRIzol protocol (Invitrogen, Glasgow, United Kingdom) and cDNA synthesised using 5x iScript Reaction Mix (Bio-Rad, Basel, Switzerland). DNA for HCYDOS was synthesised by Eurofins Genomics. For HCYDOS, we used a sequence obtained from whole genome resequencing data as the H. cydno reference genome had a gap in this region (sequence CJ565_B, S12 Table, Resequenced_mel_cyd.fa). Following the protocol from Champion pET101 Directional TOPO™ Expression Kit (Invitrogen), we amplified the full-length transcript of genes of interest from the cDNA by PCR using Q5 High-Fidelity 2x Master Mix (Biolabs, Ipswich, Massachusetts, USA), with gene-specific primers (S15 Table). The primers were designed for full-length transcript amplification for H. melpomene. For H. cydno, the primers amplified all but a 23-bp section at the 3′ end of the gene, the opposite end to the predicted active site. The plasmid contains an in-frame stop codon 26 bp from the end of our inserted H. cydno sequence. Therefore, transcription is terminated 23 bp from the end of our amplified sequence resulting in a protein which is expected to be of the same length as the true protein, the only difference being that the last 8 amino acids are different. We do not expect this change to affect protein folding.

The PCR products were purified using a MiniElute PCR purification kit (Qiagen) and then sequenced to confirm identity (S15 Table). Following sequencing, the PCR products were ligated into the expression vector pET101/D-TOPO and transformed into One Shot TOP10 Chemically Competent E. coli cells. Plasmids were extracted from cultures of successful colonies using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced again to confirm correct ligation in the vector using the T7 and T7-reverse primers (S15 Table).

Plasmids containing the genes of interest in the correct orientation were transformed into E. coli strain BL21 Star(DE3) for expression. Cell cultures were grown to an OD₆₀₀ of 0.5 and induced with 1 mM IPTG. After induction, the cells were cultivated for a further 2 h at 37°C and 250 revolutions per minute (rpm), before collection by centrifugation for 15 min at 6,000xg at 4°C. Expression of protein was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) (S5 Fig). Pellets were resuspended in chilled extraction buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.5, 1 mM MnCl2, 100 mM KCl, 3 mM dithiothreitol, 10% glycerol, protease inhibitor cocktail (Sigma)) and disrupted by sonication. Cell lysates were then centrifuged for 10 min at 9,000xg at 4°C and the supernatant (containing the soluble part of the cell lysate) retained.

TPS and IDS activity was assayed using the soluble fraction of the cell lysate. Protein concentration was estimated using a Qubit Protein Assay Kit (Invitrogen). A total of 80 to 100 ng of protein was added to each reaction in a total volume of 300 μl. We added different precursors from different steps in the pathway (Fig 1) to characterise enzymatic activity. Experiments were incubated at 30°C for 2 h at 200 rpm.

First, we added DMAPP and IPP (100μM each), the 2 building blocks at the beginning of the pathway. To form a terpene from these compounds, they first need to be combined to form GPP, which can then be converted to a terpene by TPS activity. If the enzyme is a multifunctional GPPS/TPS, as in I. pini, monoterpenes should be formed from DMAPP and IPP, via the production of GPP. Furthermore, if FPPS or GGPPS activity is present, FPP or GGPP could be formed from DMAPP and IPP, as well as sesquiterpenes or diterpenes if sesquiterpene or diterpene synthase activity is exhibited. We then carried out assays with GPP (100μM) and IPP (50μM). If the enzyme solely exhibits monoterpene synthase activity, the monoterpene could only be formed from GPP directly and not from DMAPP and IPP. Furthermore, the enzyme could be an FPPS or GGPPS and could therefore produce FPP or GGPP from GPP and IPP. FPP and GGPP could be converted to sesquiterpenes or diterpenes if sesquiterpene or diterpene synthase activity is exhibited. We also tested with GPP alone (100 μM) to test for monoterpene synthase activity directly. Finally, we carried out assays with FPP (100 μM) and IPP (50 μM). If the enzyme is a GGPPS as annotated, it should form GGPP from FPP and IPP, as well as potentially converting GGPP to diterpenes. This is also a test for sesquiterpene synthase activity, as sesquiterpenes should be formed from FPP if the enzyme is a sesquiterpene synthase. We also tested for enzymatic activity with (R)-linalool and (S)-linalool (100 μM).

To test for IDS activity, we repeated the above experiments with DMAPP and IPP, GPP, and IPP, and FPP and IPP, followed by treatment with alkaline phosphatase to hydrolyse the isoprenyl diphosphate products to their respective alcohols. These alcohols can then be detected by GC/MS analysis.

Dephosphorylation of GPP produces the monoterpene alcohol geraniol, while dephosphorylation of FPP produces the sesquiterpene alcohol farnesol. Our expectation for controls, without IDS or TPS enzymatic activity, is to find geraniol when GPP is provided and farnesol when FPP is provided. Linalool is a monoterpene alcohol which is an isomer of geraniol, and nerolidol is a sesquiterpene alcohol which is an isomer of farnesol. Furthermore, geranylgeraniol is a diterpene alcohol derived from the dephosphorylation of GGPP. If an enzyme is exhibiting IDS activity, we expect it to be able to catalyse the condensation of IPP and the other precursor provided, DMAPP, GPP, or FPP, to form larger molecules. Therefore, when provided with DMAPP and IPP, we expect to find either monoterpene or sesquiterpene alcohols, derived from GPP or FPP. When provided with GPP and IPP, we expect sesquiterpene alcohols derived from FPP. When provided with FPP and IPP, we expect larger diterpene alcohols, such as geranylgeraniol, to form via the formation of GGPP.

For TPS activity assays, reactions were stopped on ice and overlaid with 250-μl hexane and left at 25°C overnight. The hexane layer was then transferred to a new vial and stored at −80°C. For IDS activity assays, following incubation with the precursors, 20 units of alkaline phosphatase (Sigma) in alkaline phosphatase buffer was added to each reaction mixture and incubated at 30°C for 4 h at 200 rpm. Following this, 250-μl hexane was added and left at 25°C overnight. The hexane layer was then transferred to a new vial and stored at −80°C. Prior to analysis by GC/MS, 20 μL of a solution of 2-acetoxytetradecane in hexane (10 μg/mL) was added as an internal standard, and samples were concentrated to a volume of approximately 30 μL. Products were compared to control experiments where protein expression was not induced. GC/MS data are available from OSF (https://osf.io/3z9tg/).

Phylogenetic and selection analyses

To identify orthologs of the GGPPS in other Lepidoptera, we searched protein sequences from H. melpomene version 2.5 [64,75] against the genomes of H. erato demophoon (v1), B. anynana (v1x2), D. plexippus (v3), P. polytes (ppol1), P. napi (pnv1x1), M. sexta (msex1), B. mori (asm15162v1), and P. xylostella (pacbiov1) using the BLAST interface (tblastn) on LepBase [37,63]. We also included the previously identified orthologs from the H. cydno genome (S1 Data). To check that the predicted orthologs contained functional protein domains, we used the National Center for Biotechnology Information (NCBI) conserved domain search with default parameters [92]. We deleted any proteins found without complete functional domains, including a gene from H. erato, Herato0606.241, and the H. cydno ortholog of HMEL037104g1. We also did not include the H. cydno ortholog of HMEL22306g3, as despite showing transcription (S4 Fig), there were multiple stop codons within the coding region. For HCYDOS, we used a sequence obtained from whole genome resequencing data as the H. cydno reference genome had a gap in this region (sequence CJ565_B, S12 Table, Resequenced_mel_cyd.fa). We used this sequence for all functional assays, phylogenetic and selection analyses, and ancestral state reconstruction.

To focus on the Heliconius-specific duplications, we downloaded the transcript sequences for the H. melpomene and H. erato proteins from LepBase and exported transcripts for predicted genes in Apollo for H. cydno. (S1 Data). We used gene Herato0606.245 (GGPPS, shows high similarity to the GGPPS of the moth C. fumiferana) to root the tree.

To investigate the evolutionary relationship of the Heliconius GGPPS, we carried out a broader phylogenetic analysis with other known insect and plant IDS and TPS proteins. Protein sequences for these additional enzymes were downloaded from UniProt [93]. Heliconius protein sequences were obtained as described above. We used an ocimene synthase enzyme from Citrus unshiu to root the tree.

We aligned amino acid sequences using Clustal Omega on the EMBL-EBI interface [94]. Both sets of nucleotide sequences were aligned in MASCE using default parameters (gap creation: -7, gap extension: -1, frameshift cost: -30, stop codon not at the end of sequence: -100) [95]. Alignments were visualised using BoxShade (https://embnet.vital-it.ch/software/BOX_form.html). Phylogenetic trees based were constructed in PhyML using the model LG for amino acid sequences and GTR + G + I for nucleotide sequences [96]. These phylogenies were plotted using the package ape and evobiR in R version 3.5.2. [97–99]. Phylogenies and R code are available from OSF (https://osf.io/3z9tg/).

We performed selection analysis on the set of 34 Lepidoptera GGPPS sequences and the set of 17 Heliconius GGPPS sequences. To compare the selection pressure on Heliconius and Bicyclus with other lepidopteran GGPPS gene lineages which have not undergone extensive expansion, we used codon substitution models implemented in Phylogenetic Analysis by Maximum Likelihood (PAML) [100]. We constructed models under 2 assumptions: The first assumed 1 ω ratio (the ratio of nonsynonymous substitutions (dN) to synonymous substitutions (dS)) for the whole tree, and the second assumed 2 ω ratios, 1 for Heliconius and Bicyclus and the second for the rest of the tree. The models were compared using a likelihood ratio test (LRT).

Similarly, to analyse the selection pressures on HMELOS and HCYDOS, we implemented codon substitution models in PAML using the Heliconius gene tree. We constructed models under 5 assumptions: The first assumed 1 ratio for the whole tree, the remaining models assumed 2 ω ratios, the first ω ratio for either the HMELOS branch, the HCYDOS branch, the branch leading up to HMELOS and HCYDOS or the HCYDOS and HMELOS branch varying together under a single ratio and the second ω ratio for the rest of the tree. To test whether the HMELOS, HCYDOS, or the branch leading up to these genes differs from the background ratio, each 2-ratio model was compared to the 1-ratio model using LRTs. To test whether the ω ratio differs between the HCYDOS and HMELOS branches, the 2-ratio models where the respective branches were allowed to vary were compared using LRT, and both models were compared to a third model where both the HCYDOS and HMELOS were allowed to vary under the same ratio.

Ancestral sequence reconstruction

The amino acid sequence of the LCA of HcydOS and HmelOS was calculated using the aaml program in PAML. The set of 17 Heliconius sequences used in the phylogenetic analysis was manually refined to minimise gaps in the multiple sequence alignment (MSA) (Heliconius_ASR_edited_aligned). This is because there are no models for insertions and deletions in PAML programs. The manually refined Heliconius sequences were aligned using Clustal Omega on the EMBL-EBI interface with default parameters [94]. A phylogenetic tree for these Heliconius sequences was generated in PhyML with 1,000 bootstraps; the best model was JTT + G + I + F. Marginal ancestral sequence reconstruction was performed using the aaml program in PAML using an empirical model and wag.DAT substitution rate matrix. To investigate intraspecific variation in the presence of these amino acid differences, we aligned sequences from whole genome sequencing of 10 individuals of H. melpomene and 10 H. cydno from Panama (S12 Table, Resequenced_mel_cyd.fa). Two sequences were generated per individual, not representing true haplotypes. We aligned amino acid sequences using Clustal Omega on the EMBL-EBI interface [94]. Alignments were visualised using BoxShade (https://embnet.vital-it.ch/software/BOX_form.html). Manually curated sequences and gene sequences from resequenced individuals are available from OSF (https://osf.io/3z9tg/).