Evolutionary convergence of colour pattern in mimetic species is tightly linked with the evolution of chemical defences. Yet, the evolutionary forces involved in natural variations of chemical defences in aposematic species are still understudied. Herein, we focus on the evolution chemical defences in the butterfly tribe Heliconiini. These neo-tropical butterflies contain large concentrations of cyanogenic glucosides, cyanide-releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host-plant, so that their concentrations may depend on host-plant specialization and host-plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different cyanogenic glucosides using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defences both within and among species. Based on the most recent and well-studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host-plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defence variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.

Butterfly collection

We sampled butterflies throughout Heliconiini distribution to collect the maximum number of species. Wild butterflies were caught from 2016 to 2018 across Peru (n = 286), Panama (n = 45), Ecuador (n = 24) and Brazil (n = 20), using a hand net. We used 375 individuals from 33 species, covering 43% of the Heliconiini tribe (Appendix. 1), and 55 subspecies (Tab. 1). Individuals were killed by freezing on the day of capture (approximately –18°C). Wings were cut at their attachment point to the body and preserved dried in an envelope and placed in a silica gel containing box to absorb humidity. In order to preserve the integrity of CG molecules, bodies were conserved in a plastic vial containing 100% methanol and kept in freezer (approximately –18°C).

Cyanogenic glucoside extraction in methanol

For each butterfly specimen, the butterfly body and the methanol medium were transferred in a glass tube. Methanol was evaporated at room temperature until the tissue was fully dried using Savant Automatic Environmental SpeedVac System AES1010 with VaporNet. For each specimen, body and wings were weighed before being crushed together into a fine powder in a glass mortar and pestle using liquid nitrogen. Two mL of 100% methanol were added to the powder before stirring for 1 hour at room temperature. Extracts were centrifugated for 20 minutes at 1600 rotations per minute, filtered using 7 mm diameter glass pipettes and cotton, filtered again with a MultiScreen 0.45 µm hydrophilic, low protein binding plate, and centrifuged five minutes at 3500 rotations per minute. Raw filtrates were diluted 50 times in milliQ water, vortexed and stored in fridge until liquid chromatography and tandem mass spectrometry (LC-MS/MS) injections.

Liquid chromatography and tandem mass spectrometry

The protocol used in this study has been previously optimized to identify and quantify CGs in butterfly methanol filtrates (Briolat et al., 2019; de Castro et al., 2019a). Analytical LC-MS/MS was performed using an Agilent 1100 Series LC (Agilent Technologies, Germany) coupled to a High Capacity Trap-Ultra ion trap mass spectrometer (Bruker Daltonics, Germany). Chromatographic separation was carried out on a Zorbax SB-C18 column (Agilent; 1.8 μM, 2.1x50 mm). Mobile phase A was composed by deionized water containing 0.1% (v/v) formic acid. Mobile phase B was acetonitrile supplemented with 50 μM NaCl and 0.1% (v/v) formic acid. The gradient was: 0 - 0.5 min, isocratic 2% B; 0.5 - 7.5 min, linear gradient 2% - 40% B; 7.5 - 8.5 min, linear gradient 40% - 90% B; 8.5 - 11.5 isocratic 90% B; 11.6 - 17 min, isocratic 2% B. Flow rate was set to 0.2 mL/min and increased to 0.3 mL/min between 11.2 to 13.5 min. During the liquid chromatography step, initially neutral CGs were associated with Na⁺ cations and analysed with mass spectrometer in the positive electrospray mode.  The oven temperature was fixed at 35°C.

In addition to the 375 butterfly samples, we ran blank control sample and a reference sample. Blank was methanol gone through the whole protocol extraction, and the reference sample was a mix of every butterfly filtrates. CGs were identified by comparison to standard solutions (aliphatic were chemically synthesized at PLEN, Møller et al., 2016, cyclopentenoid were donated by Lawrence Gilbert and Helene Engler, Engler et al., 2000). We made three calibration curves based on three commercial standards: linamarin, lotaustralin/epilotaustralin and amygdalin (commercial, Sigma Aldrich), from 0.1 to 20 ng/µL each. Blanks, standards, calibration curve and reference sample were run first. The reference sample was injected every ten butterfly samples.

Chemical data analyses

Mass spectra were analysed using the software Bruker Compass DataAnalysis 4.3 (x64). We targeted sodium adducts [M+Na⁺] of linamarin [retention time (RT) 2.4 min at m/z 270], lotaustralin [RT 5.4 min at m/z 284], epilotaustralin [RT 5.5 min at m/z 284], tetraphyllin B [RT 1.3 min at m/z 310], epivolkenin [RT 2.3 min at m/z 310], tetraphyllin A [RT 4.9 min at m/z 294], gynocardin [RT 1.4 min at m/z 326], dihydrogynocardin [RT 1.4 min at m/z 328] and amygdalin [RT 6.4min at m/z 480] (Briolat et al., 2019; de Castro et al., 2019a). For every targeted CG compound, the total concentration was estimated based on the Extracted Ion Chromatogram (EIC) peak areas, and on a regression calculated from the standard curve (in ng of CG/mL of butterfly extract). We reported the concentration of each CG in every butterfly in µg of CG/mg of dried butterfly weight.