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Mario is Nintendo's mascot, originating from the original Donkey Kong arcade game. Later on, Nintendo decided to add Mario in Mike Tyson's Punch-Out!! as the referee. Mario is mentioned during Little Mac's discussion with Don Flamenco when he makes a comment about Mario's hair. Instead of his usual overalls, Mario is seen wearing a white, long sleeved shirt with black pants. He kept his signature red cap.
- In Super Smash Bros. for Wii U, an Event Match references to Mario's appearance as the referee: The match is over but a wireframe Little Mac won't stop attacking a defenseless Little Mac, and you play as Mario (Fire Mario Alternate to resemble the referee color scheme) to KO Wireframe Mac.
- In the Super Mario games, Mario is voiced by Charles Martinet, who also provided most of the voice acting in Super Punch-Out!! on the SNES.