PREMISE OF THE STUDY: Pachypodium (Apocynaceae) is a genus of iconic 
stem-succulent and poisonous plants endemic to Madagascar and southern Africa. 
We tested hypotheses about the mode of action and macroevolution of toxicity in 
this group. We further hypothesized that while monarch butterflies are highly 
resistant to cardenolide toxins (a type of cardiac glycoside) from American 
Asclepias, they may be negatively affected by Pachypodium defenses, which 
evolved independently.
METHODS: We grew 16 of 21 known Pachypodium spp. and quantified putative 
cardenolides by HPLC and also by inhibition of animal Na+ /K+ -ATPase (the 
physiological target of cardiac glycosides) using an in vitro assay. Pachypodium 
extracts were tested against monarch caterpillars in a feeding bioassay. We also 
tested four Asclepias spp. and five Pachypodium spp. extracts, contrasting 
inhibition of the cardenolide-sensitive porcine Na+ /K+ -ATPase to the monarch's 
resistant form.
KEY RESULTS: We found evidence for low cardenolides by HPLC, but substantial 
toxicity when extracts were assayed on Na+ /K+ -ATPases. Toxicity showed 
phylogenetic signal, and taller species showed greater toxicity (this was 
marginal after phylogenetic correction). Application of Pachypodium extracts to 
milkweed leaves reduced monarch growth, and this was predicted by inhibition of 
the sensitive Na+ /K+ -ATPase in phylogenetic analyses. Asclepias extracts were 
100-fold less potent against the monarch compared to the porcine Na+ /K+ 
-ATPase, but this difference was absent for Pachypodium extracts.
CONCLUSIONS: Pachypodium contains potent toxicity capable of inhibiting 
sensitive and cardenolide-adapted Na+ /K+ -ATPases. Given the monarch's 
sensitivity to Pachypodium, we suggest that these plants contain novel cardiac 
glycosides or other compounds that facilitate toxicity by binding to Na+ /K+ 
-ATPases.