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Aleksandar Mitrovic headed home a late winner in Lisbon to leave the Selecao with only the play-offs if they are to reach Qatar. Three European teams will prevail from the spring’s showdowns with the likes of Austria, Scotland and Russia also competing.
After showing his frustration at the final whistle, Manchester United star Ronaldo provided an optimistic outlook on Monday.
"Football has shown us time and time again that sometimes the most difficult paths are the ones that lead us to the most desired outcomes," Ronaldo said on Instagram.
The 36-year-old veteran could be facing his last World Cup after making four straight appearances in the tournament since 2006.
"Yesterday's result was tough, but not tough enough to get us down," he said. "The goal of being in the 2022 World Cup is still very much alive and we know what we have to do to get there.
"No excuses," he said. "Portugal is on its way to Qatar."
Portugal looked good at the start of the game against Serbia, scoring after two minutes through Renato Sanches. But they struggled afterwards, allowing the visitors to equalise through Dusan Tadic in the 33rd minute and substitute Mitrovic scored the winner.
Ronaldo was upset after the late goal, complaining and yelling at his teammates from midfield as he waited for play to resume. After the final whistle, he sat on the field by himself, shaking his head and looking desolate. Some teammates and opponents attempted to console him.
Veteran goalkeeper Rui Patricio was blamed by some for the first goal, as he couldn't hold onto the ball after Tadic's shot deflected off a Portugal defender.
Coach Fernando Santos is also under pressure, amid calls for his resignation in the Portuguese press. Headlines such as “Miserable” and “World Shame” featured on newsstands on Monday.
"I'm sad and frustrated but I still have confidence in myself and in my players," Santos said. "The last results were negative but there is no reason we shouldn't have faith in ourselves."
Portugal were coming off a draw at already-eliminated Ireland. The team entered the final round level on points with Serbia after seven matches but had the advantage of being able to qualify with a draw because of a better goal difference.
Instead, Portugal must face the play-offs with the other nine group runners-up and two group winners from the UEFA Nations League. The 12 countries will play next March in three four-team brackets, with the seeded teams getting a home game in the single-legged semi-finals.
Additional reporting by AP.