Animals have evolved a wide range of anti-predator adaptions, with prey investing in multiple anti-predator defences that offer a ‘defence portfolio’ to protect against predators. Yet why specific defences occur together or in isolation remains an evolutionary puzzle. Here we examine the evolution of eyespots, a conspicuous visual anti-predator marking, and other anti-predator defences in skates and rays, a diverse group of cartilaginous fishes. We demonstrate that eyespots are more likely to evolve in a step-wise fashion from other conspicuous markings, evolutionary gains of multiple conspicuous markings are followed by evolutionary reductions in the number of conspicuous markings, and conspicuous markings are lost more than they are gained. Moreover, skates and rays exhibit alternative evolutionary trajectories in their anti-predator defences that are shaped by key ecological factors. Species with robust anti-predator defences rarely evolved eyespots, while eyespots were more common in smaller bodied species that lack robust defences and live in brighter visual environments. These results demonstrate that accounting for multiple anti-predator defences can resolve why iconic anti-predator defences, like eyespots, appear in some taxonomic groups but are conspicuously absent in others.   

The diverse range of animal body colorations and patterns exemplifies evolutionary adaptations driven by predation to enhance prey survival. Eyespots, characterized by their distinctive concentric rings of contrasting colors, are a widely known example due to their potential role in misleading predators and thereby reducing predation risk. The functional role of eyespots has long been presumed to be a multifaceted antipredator mechanism, potentially by creating the illusion of larger or more threatening organisms, startling predators, or diverting attacks away from vital body parts. The survival benefits of bearing eyespots have been repeatedly demonstrated in terrestrial environments, and research increasingly examines the perceptual basis of their deceptive effects on predators. However, despite eyespots being widespread among fish and cephalopods, their effect on marine predator behavior remains largely speculative. In this study, we compared behavioral responses to eyespots using artificial prey in terrestrial (Gallus gallus, red junglefowl) and marine (Chiloscyllium punctatum, brown-banded bamboo shark; Negaprion brevirostris, lemon shark) model predators and found that eyespots elicited divergent reactions. While red junglefowl delayed their attacks when presented with eyespots, both shark species attacked eyespot-bearing prey faster, suggesting that eyespots may serve different functions across different predators. Although we observed opposing responses in terrestrial and marine predators, we discuss how both may reflect context-dependent interactions with an antipredator signal. Our study highlights the complex role of eyespots in predator-prey dynamics and the importance of ecological context in evaluating the effectiveness of these markings.

Eyespots are taxonomically widespread color patterns consisting of large concentric rings that are commonly assumed to protect prey by influencing the behaviors of predators. Although there is ample experimental evidence supporting an anti-predator function of eyespots in terrestrial animals, whether eyespots have a similar deterring function in aquatic animals remains unclear. Furthermore, studies in terrestrial systems suggest that the protective function of eyespots depends on ambient light conditions where predators encounter them, but this effect has never been tested in aquatic environments. Here, we examine how eyespots influence behavioral responses in an aquatic environment under different visual environments, using laboratory-reared three-spined sticklebacks (Gasterosteus aculeatus) as model predators. Specifically, we experimentally examined behavioral responses of sticklebacks toward artificial prey patterns (control vs. eyespots) under two different light environment treatments (low vs. high). We found that eyespots did not postpone attacks from sticklebacks. However, sticklebacks approaching eyespots stopped more frequently than sticklebacks approaching prey items with a control pattern. Sticklebacks were (marginally) slower to attack prey in the low-light treatment, but the light level did not influence stickleback behavioral responses toward eyespots. We conclude that eyespots can modulate some behaviors of an aquatic predator, albeit with a different functional role from that previously demonstrated in terrestrial species.

Many animals possess eyespots, conspicuous eye-like markings thought to deter predators. However, experimental evidence for their antipredator function comes largely from terrestrial systems, especially butterfly–bird interactions, and it remains unclear whether eyespots provide similar protection in aquatic environments. Here, we experimentally test whether eyespots reduce predation risk by deploying artificial prey with and without eyespots in shallow coastal waters of Bimini, The Bahamas, and monitoring survival using video recordings. We found that fish showed no bias in initial strikes between prey types, and that eyespots did not reduce predation risk. These results provide no support for the hypothesis that continuously visible eyespots confer survival benefits in this marine system, in contrast to many terrestrial studies reporting antipredator effects. By providing a marine comparison point, our experiment adds rare predation data to a field dominated by terrestrial studies, broadening the empirical basis for understanding how eyespots operate in nature.