Tranmere's Cole Stockton netted in the last minute to end Morecambe's perfect start with a 2-1 victory.
Stockton fired home either side of Hughes seeing red in the 63rd minute for nudging his head towards sub Kayode Odejayi, his second the winner in the dying seconds of normal time.
It had all started so well for the Shrimps, previously the only side in the entire Football League with a 100 per cent record, as Hughes scored just before half-time.
Morecambe, who topped League Two before kick off, opened the scoring from a 39th-minute corner, Jamie Devitt's delivery headed into the danger zone by Alan Goodall with Hughes rising unmarked and directing home.
Andrew Fleming should have doubled the Shrimps' advantage but blazed over at the start of the second-half with Stockton firing Tranmere level after a defensive mistake in the 58th minute.
Then came Hughes' moment of madness and it was left to Stockton to have the final say as he guided home his second from Matt Hill's free-kick late on.