We made artificial models of Heliconius doris butterflies, 100 of each of 4 morphs for a total of 400 per transect. These models were placed on leaves, trunks or twigs in visible, well-lit areas at 10m intervals along a 4 kilometer transect in each site. The placement of each model was carried out so as to mimic the natural perching behavior of Heliconius butterflies and provide a visible target for potential avian predators. The distinct model morphs were placed along the transect in a regular order. From 376 to 416 models were placed per site and left for 72 hours, after which, models were collected. Damage was clearly visible in the malleable wax bodies and paper wings of several models. Damages were catalogued as either (i) "invertebrate attack" when bearing the visible fine marks of arthropod mandibles, often on the wax bodies, (ii) "Avian Attack" when bearing the characteristic U or V shape marks on the wax or (iii) "Unknown Predator" when a severe attack was evident but a specific mark was not found, such as when wings were torn or wax bodies broken in pieces. Models that bore attack marks characteristic of invertebrates were not included in the data analysis (n= 97 out of 2,271), as there is currently no literature regarding invertebrates carrying the cognitive capacity necessary to make the associations between unpalatability and warning color patterns central to Müllerian mimicry. Furthermore, missing models were also excluded from the analyses as we are unable to determine if they were displaced by falling forest debris, human action or attacked by natural predators.

The evolution of mimicry in similarly defended prey is well described by Müllerian mimicry theory, which predicts the convergence of warning patterns in order to gain the most protection from predators. However, despite this prediction, we can find great diversity of color patterns amongst Müllerian mimics such as Heliconius butterflies in the neotropics. Furthermore, some species have evolved the ability to maintain multiple distinct warning patterns in single populations, a phenomenon known as polymorphic mimicry. The adaptive benefit of these polymorphisms is questionable since variation from the most common warning patterns is expected to be disadvantageous as novel signals are punished by predators naive to them. In this study we use artificial butterfly models throughout Central and South America to characterize the selective pressures maintaining polymorphic mimicry in Heliconius doris. Our results highlight the complexity of positive frequency-dependent selection, the principal selective pressure driving convergence amongst Müllerian mimics, and its impacts on interspecific variation of mimetic warning coloration. We further show how this selection regime can both limit and facilitate the diversification of mimetic traits.

Funding provided by: Agence Nationale de la RechercheCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001665Award Number: ANR-10-LABX-25-01Funding provided by: National Science FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000001Award Number: 1736026