DOI: 10.5061/dryad.ghx3ffbjt
title: Variation of chemical compounds in wild Heliconiini reveals ecological and historical contributions to the evolution of chemical defences in mimetic butterflies

description: 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.
description: <b>Butterfly
 collection</b> We sampled butterflies throughout
 Heliconiini distribution to collect the maximum number of species. Wild
 butterflies were caught from 2016 to 2018 across Peru
 (<i>n</i> = 286), Panama (<i>n</i> = 45), Ecuador
 (<i>n</i> = 24) and Brazil (<i>n</i> = 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).   <b>Cyanogenic glucoside
 extraction in methanol</b> 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.   <b>Liquid chromatography
 and tandem mass spectrometry</b> 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<sup>+</sup> 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 <i>et al.</i>, 2016, cyclopentenoid were donated
 by Lawrence Gilbert and Helene Engler, Engler <i>et al.</i>,
 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.   <b>Chemical data analyses
 </b> Mass
 spectra were analysed using the software Bruker Compass DataAnalysis 4.3
 (x64). We targeted sodium adducts [M+Na<sup>+</sup>] of
 linamarin [retention time (RT) 2.4 min at <i>m/z</i> 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
 <i>m/z</i> 310], tetraphyllin A [RT 4.9 min at
 <i>m/z</i> 294], gynocardin [RT 1.4 min at
 <i>m/z</i> 326], dihydrogynocardin [RT 1.4 min at
 <i>m/z</i> 328] and amygdalin [RT 6.4min at
 <i>m/z</i> 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.