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Poison frogs sequester chemical defenses from arthropod prey, although the details of how arthropod diversity contributes to variation in poison frog toxins remains unclear. We characterized skin alkaloid profiles in the Little Devil frog, Oophaga sylvatica (Dendrobatidae), across three populations in northwestern Ecuador. Using gas chromatography mass spectrometry, we identified histrionicotoxins, 3,5- and 5,8-disubstituted indolizidines, decahydroquinolines, and lehmizidines as the primary alkaloid toxins in these O. sylvatica populations. Frog skin alkaloid composition varied along a latitudinal gradient across populations in a principal component analysis. We also characterized diversity in arthropods isolated from frog stomach contents and confirmed O. sylvatica specialize on ants and mites. To test the hypothesis that poison frog toxin diversity reflects species and chemical diversity in arthropod prey, we (1) used liquid chromatography mass spectrometry to chemically profile consumed ants and mites, and (2) used sequencing of cytochrome oxidase 1 to identify individual prey specimens. We show that chemical profiles of consumed ants and mites cluster by frog population, suggesting different frog populations have access to chemically distinct prey. We identified 45 ants and 9 mites isolated from frog stomachs, finding several undescribed species. Finally, by comparing chemical profiles of frog skin and isolated prey items, we were able to trace the arthropod source of four poison frog alkaloids, including 3,5- and 5,8-disubstituted indolizidines and a lehmizidine alkaloid. Together, our data shows the diversity of alkaloid toxins found in O. sylvatica can be traced to chemical diversity in arthropod prey.

Our recent publication titled "Ant and Mite Diversity Drives Toxin Variation in the Little Devil Poison Frog" aimed to describe how variation in diet contributes to population differences in toxin profiles of poison frogs. Some poison frogs (Family Dendrobatidae) sequester alkaloid toxins from their arthropod diet, which is composed mainly of ants and mites. Our publication demonstrated that arthropods from the stomach contents of three different frog populations were diverse in both chemistry and species composition. To make progress towards understanding this trophic relationship, our main goal was to identify alkaloids that are found in either ants or mites. With the remaining samples that were not used for chemical analysis, we attempted to identify the arthropods using DNA barcoding of cytochrome oxidase 1 (CO1). The critique of Heethoff, Norton, and Raspotnig refers to the genetic analysis of a small number of mites. Here, we respond to the general argument of the critique as well as other minor issues detailed by Heethoff, Norton, and Raspotnig Kluwer Academic/Plenum Publishers