DOI: 10.5061/dryad.c2fqz61jz
title: NMR spectra of a cardenolide isolated in Heliopsis helianthoides (Asteraceae)

description: Factors mediating coexistence of closely related species have long been of
 interest in the origin and maintenance of biodiversity.  Niche
 differentiation, for example, facilitates stable coexistence by reducing
 interspecific competition. Here we study adaptive divergence in congeneric
 seed bugs that often co-occur at small spatial scales: the small milkweed
 bug (Lygaeus kalmii) and the false milkweed bug (L. turcicus). L. kalmii
 primarily feeds on milkweeds (Apocynaceae) and sequesters toxic
 cardenolides as an anti-predator defense; little was previously known
 about L. turcicus. We report the primary host plant of L. turcicus is
 false sunflower Heliopsis helianthoides (Asteraceae) and that its seeds
 produce cardenolides, the second report of this toxin among Asteraceae. We
 find divergent trends in performance and defense of the bugs on the two
 hosts: L. kalmii had &gt;30% higher growth rate on milkweeds than on
 false sunflower, while L. turcicus performed equivalently on each species.
 Conversely, L. turcicus sequestered more effectively from false sunflower,
 while L. kalmii sequestered equivalently on the two seeds. The thick shell
 of false sunflower seeds contributes to the disparity in performance, and
 differences in seed chemistry are hypothesized to drive differences in
 sequestration. In particular, false sunflower cardenolides are converted
 to a diversity of breakdown products by its specialist (L. turcicus),
 while L. kalmii sequesters these compounds intact. Thus, adaptation to
 host-plants is asymmetrical for growth and defense between these two seed
 bugs. Shared plant chemistry apparently bridged host-shifts in this group
 of insects, with adaptive divergence to their food niches contributing to
 diversification.
description: <em>H. helianthoides</em> seeds were purchased from
 commercial vendors. Freeze-dried seeds (50 mg for seeds) were ground to a
 powder (Retsch Mixer Mill 300). 1.6 mL meOH with 20 μg digitoxin
 (Sigma-Aldrich, MO, USA) was added to each sample, along with ~30 FastPrep
 beads. Samples were agitated twice for 45s at 6.5 m/s on a FastPrep-24
 homogenizer and centrifugated for 15 min at 12,000 rpm. Solids were
 discarded, and samples were dried in a centrifugal concentrator at
 35 <sup>o</sup>C. Samples were resuspended in 250 μL of meOH,
 and each resuspended sample was vortexed 3 times with 1.5 mL of hexanes
 and centrifugated for 15 min at 12,000 rpm. The hexane layer was discarded
 to de-fat each sample. Samples were once again dried down, resuspended in
 250 μL of MeOH, and filtered with a MultiScreen Solvinert 0.45 μm
 low-binding hydrophobic PTFE 96-well plate (Millipore, Burlington, Mass).
 We analyzed samples using HPLC following the method used by Züst et al.
 (2019). Fifteen μL of the samples were injected into an Agilent 1100
 series HPLC (Agilent, Waldbronn, Germany) equipped with a diode-array
 detector. Cardenolides were eluted at a flow rate of 0.7 mL/min across an
 acetonitrile gradient (0–2 min 16% acetonitrile, 24 min 70% acetonitrile,
 30-40 min 95% acetonitrile, and a 10 min post-run with 16% acetonitrile).
 Peaks with symmetrical absorption maxima between 216 and 222 nm were
 considered to be cardenolides. Cardenolide concentrations were calculated
 based on peak areas at 218 nm and were standardized by the concentration
 of digitoxin in the sample and the sample’s dry mass. Peak areas were
 calculated from HPLC chromatograms using the chromatographR
 package. Nuclear magnetic resonance (NMR) spectra were
 recorded on a Bruker 600 Avance III HD console and
 BBFO<sup>+</sup> cryoprobe.  All 1D data
 (<sup>1</sup>H, <sup>13</sup>C) and 2D data
 (DQCOSY, gCOSY, HSQCAD, gHMBCAD, HSQCTOCSY, ROESYAD) were obtained with
 Bruker standard sequence. Sample was measured in
 CD<sub>3</sub>OD (99.5%) and the residual solvent signals were
 δ<sub>H</sub> 3.31/δ<sub>C</sub> 49.15. NMR data
 were processed with MestReNova version 15.0.0-34764 Mestrelab Research
 S.L.
description: # NMR spectra of a cardenolide isolated in Heliopsis helianthoides
 (Asteraceae)
 [https://doi.org/10.5061/dryad.c2fqz61jz](https://doi.org/10.5061/dryad.c2fqz61jz) ## Description of the data and file structure The data set is organized in main directories that correspond to different NMR sequence and a file of all processed NMR data. Within each of these directories, there is one directory for each spectrum acquired. These directories have extension .fid and their name specifies the NMR sequence. Each of these directories includes files that contain the raw NMR data (fid), the acquisition parameters (procpar), a log file (log), a text file (text). Below is a summary of the main directories. To view the files, please download the .zip file and uncompress it. Processed files with extension .fid are ready to be viewed in NMRViewJ or MestReNova. Sunflower cardenolide.mnova can be viewed in MestReNova In case of any questions, please do not hesitate to contact the authors. ### Files and variables #### File: NMR\_Cardenolide\_Heliopsis.zip **Description:** 1H.fid DQCOSY.fid gCOSY.fid gHMBCAD.fid HSQCAD-full.fid HSQCTOXY-20ms.fid HSQCTOXY-80ms.fid ROESYAD.fid Sunflower cardenolide.mnova