Combining stable isotope analysis with DNA metabarcoding improves inferences of trophic ecology

Abstract

Knowing what animals eat is fundamental to our ability to understand and manage biodiversity and ecosystems, but researchers often must rely on indirect methods to infer trophic position and food intake. Using an approach that combines evidence from stable isotope analysis and DNA metabarcoding, we assessed the diet and trophic position of Anthene usamba butterflies, for which there are no known direct observations of larval feeding. An earlier study that analyzed adults rather than caterpillars of A. usamba inferred that this butterfly was aphytophagous, but we found that the larval guts of A. usamba and two known herbivorous lycaenid species contain chloroplast 16S sequences. Moreover, chloroplast barcoding revealed high sequence similarity between chloroplasts found in A. usamba guts and the chloroplasts of the Vachellia drepanolobium trees on which the caterpillars live. Stable isotope analysis provided further evidence that A. usamba caterpillars feed on V. drepanolobium, and the possibilities of strict herbivory versus limited omnivory in this species are discussed. These results highlight the importance of combining multiple approaches and considering ontogeny when using stable isotopes to infer trophic ecology where direct observations are difficult or impossible.

Fig 1. The sequence libraries of Anthene usamba caterpillars contained similar ratios of chloroplast to non-chloroplast sequences as those of phytophagous lycaenid species, whereas aphytophagous lycaenid species had no chloroplast sequences in their libraries.

Fig 2. Stable isotope values are consistent with C. mimosae ants and A. usamba being functionally herbivorous on V. drepanolobium, while predatory Salticidae sp. and Clubiona sp. spiders are elevated by one trophic level.

Error bars show standard errors.

Fig 3. Estimated relationships between fraction of A. usamba’s nitrogen and carbon derived from V. drepanolobium (p and q, respectively), and A. usamba’s nitrogen and carbon isotopic fractionation (Δ¹⁵N and Δ¹³C, respectively) in a simple two-source mixing model.