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Ken Reels
Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-β-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-β-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-β-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.
Copyright: 2021 Darragh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The H. cydno assembly is available from OSF ( ) and was assembled using previously published sequencing data available from ENA study ERP009507. Sequencing data used to make linkage maps is available from ENA study PRJEB34160. RNA sequencing data of H. cydno and H. melpomene heads and abdomens was obtained from GenBank BioProject PRJNA283415. Raw data and scripts, as well as GC/MS data, are available from OSF ( ).
Funding: KD and AO were supported by the Natural Research Council Doctoral Training Partnership (grant NE/L002507/1) and KD additionally by a Smithsonian Tropical Research Institute Short Term Fellowship -programs/fellowships. KJRPB, IAW, RMM and CDJ were supported by the European Research Council (grant 339873 SpeciationGenetics). RMM was also supported by a Deutsche Forschungsgemeinschaft Emmy Noether fellowship _funding/programmes/individual/emmy_noether/index.html (grant GZ:ME4845/1-1). PR was supported by the Jane and Aatos Erkko Foundation AP was supported by a Natural Research Council studentship (PFZE/063) and a Smithsonian Tropical Research Institute Short Term Fellowship -programs/fellowships. JWD was funded by a Herchel Smith Postdoctoral Research Fellowship -fellowships and a Smithsonian Tropical Research Institute Fellowship -programs/fellowships. WOM was supported by the Smithsonian Tropical Research Institute -programs/fellowships and National Science Foundation (grant DEB 1257689). SS thanks the Deutsche Forschungsgemeinschaft (grant Schu984/12-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Anti-aphrodisiac pheromones vary both qualitatively and quantitatively between Heliconius species [18]. Some compounds are only found in particular clades or species, while others, such as (E)-β-ocimene, are found in distantly related Heliconius species. This phylogenetic pattern suggests that these pheromones evolve rapidly, with gains and losses common throughout the evolutionary history of Heliconius [14]. Heliconius cydno, a species closely related to H. melpomene, does not produce (E)-β-ocimene [14,18], most likely representing a loss of (E)-β-ocimene production as this compound is present in other Heliconius species. This provides us with the opportunity to study the genetic basis of this rapidly evolving trait between species.
(A) IPP and DMAPP are first formed from the mevalonate pathway. IPP and DMAPP are the substrates for isoprenyl disphosphate synthases (GPPS, FPPS, and GGPPS). IDSs produce isoprenyl diphosphates of varying lengths, depending on the number of IPP units added. Isoprenyl diphosphates (GPP, FPP, and GGPP) are themselves the substrates used by TPSs to make terpenes of various sizes. For example, monoterpene synthases produce monoterpenes, such as ocimene, from GPP. For illustration, (E,E)-α-farnesene is used as a representative sesquiterpene, and phytol as a diterpene. (B) Proposed biosynthetic pathway in H. melpomene. Reciprocal best BLAST hits are highlighted in bold. IDSs are in red and their products, isoprenyl diphosphates, in blue. BLAST, basic local alignment search tool; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; GPPS, geranyl diphosphate synthase; IDS, isoprenyl diphosphate synthase; IPP, isopentenyl diphosphate; TPS, terpene synthase.
Here, we identify the genes involved in the biosynthesis of (E)-β-ocimene in the butterfly H. melpomene and analyse the evolution of terpene synthesis in Heliconius and other insects. To determine candidate TPS genes, we identified pathway orthologs in H. melpomene and carried out a genetic mapping study between H. melpomene and H. cydno. We identified a genomic region associated with the production of (E)-β-ocimene and searched for candidates within this region. We then identified genes with up-regulated expression in the genitals of male H. melpomene, where (E)-β-ocimene is produced. We tested the TPS function of our candidate genes, as well as an ortholog in H. cydno, by expression in Escherichia coli followed by enzymatic assays. We carried out phylogenetic analyses, selection models, and ancestral state reconstruction, to place our discoveries relative to previously identified plant and insect TPS genes.
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.
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).
(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 ( ). 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.
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.
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