The Eye-Closing Mechanism in Pufferfish has Evolved From Puffing-Related Musculature: Phylogenetic Evidence

Eye closure is a fundamental protective mechanism for the vertebrate visual system, yet it is rare among fully aquatic vertebrates. Pufferfishes (Order Tetraodontiformes: Family Tetraodontidae) exhibit a unique form of eye closure, termed "iris-like eye closure," in which the cornea is covered by radially-directed movements of the surrounding skin rather than by eyelids. A previous study has documented this behavior and its associated musculature in a single species, but its phylogenetic distribution and evolutionary origin are unresolved. Dissection for comparison of 51 specimens representing all tetraodontiform families revealed that the cutaneous muscle (CT), a sheet-like subcutaneous muscle, is present only in Tetraodontidae and Diodontidae, whereas a specialized concentric subdivision, the cutaneous muscle orbicularis (CTO), is restricted to Tetraodontidae. Mapping the presence or absence of CT and CTO onto different previously published phylogenetic trees, followed by ancestral state reconstruction, indicates that CT occurred only once, in the common ancestor of Tetraodontidae and Diodontidae, presumably in association with the evolution of body inflation. CTO subsequently evolved only within Tetraodontidae through differentiation of the orbital portion of CT, enabling iris-like eye closure. These results suggest a stepwise evolutionary scenario in which a preexisting muscular system was co-opted for a novel protective function. Iris-like eye closure in tetraodontids therefore represents an independent evolutionary solution to corneal protection, functionally convergent with eye-closing mechanisms in other vertebrate lineages. This study highlights the diversity of morphological strategies for eye protection in aquatic vertebrates and underscores the role of muscle repurposing in the evolution of novel functional traits.