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Douglas Ross, who was one of four Scottish Tory MPs who said Mr Johnson should no longer be in Number 10, said the number of Conservatives who voted against the Prime Minister was “far higher than many people were expecting”.
On Monday, a crunch vote secured Mr Johnson’s future, for now, after 148 told the 1922 Committee they had no confidence in him and 211 serving MPs backed the Prime Minister.
But Mr Ross told STV News: “I think the Prime Minister does have to reflect on such a significant number voting no confidence in him.”
And when asked by the broadcaster if Mr Johnson should resign, Mr Ross, who serves as an MP as well as an MSP, said: “Well, I think he has to look at that.”
He added: “That’s a decision for the Prime Minister but we know when Theresa May had a greater percentage number of MPs supporting her in 2018 when she faced a confidence vote, people were immediately saying it wasn’t good enough.
“Well, last night 41% of Conservative MPs felt that they couldn’t support the Prime Minister and that is a very high number.
“A hundred and forty eight is far higher than many people were expecting.”
And the leader told BBC’s Reporting Scotland he voted for the Prime Minister to go because Mr Johnson’s actions were “unacceptable”.
“For those who set the rules to then break the rules, I think, is very difficult to come back from,” he said.
“So, as I say, it’s not the timing I would have chosen but, ultimately, there was a vote last night and I had to make a decision, and that’s why I went with my original thought that the Prime Minister’s actions were unacceptable and I couldn’t support him.”
Mr Ross has changed his mind on the Prime Minister’s future before, and when asked if it was now his final position, he said: “Yes.”
He added: “I understand how political opponents and some journalists want to frame it like that.
“The one thing that changed throughout this several months where I’ve criticised the Prime Minister’s actions was war in Europe.”
Of Scotland’s six Tory MPs, only Scottish secretary Alister Jack and David Duguid backed him.
Earlier in the day, the chief whip for the Scottish Conservatives at Holyrood said the result of Monday’s vote of confidence in the Prime Minister has left a “dark shadow” looming over the party.
Stephen Kerr told BBC’s Good Morning Scotland that “politically, undoubtedly, he (Mr Johnson) is damaged” by the outcome of the vote, but said he could not predict how long the Prime Minister will remain in his position.
He added that the results are “not good for the Conservative Party in general”.
“The Government’s trying to do some very good and worthy things, and this whole issue of the Prime Minister’s leadership is like a dark shadow over all of the good things that the Government is doing,” Mr Kerr said.
But he downplayed suggestions that a snap general election could be called in the aftermath of the ballot, pointing to recent opinion polls placing the Tories in second place behind Labour.
And Mr Murray, Labour MP for Edinburgh South, said it was “now a matter of when he [Mr Johnson] will go, rather than if”.
“If Boris Johnson thinks after last night’s vote he can command the respect and support of the country, then why doesn’t he take the short trip to Buckingham Palace, request that the Parliament and Government is dissolved, and go to the country and let the people decide?” Mr Murray said.
“That would be the best way to resolve this situation.”
The Labour MP said tabling a motion of no confidence on behalf of opposition parties could risk “undermining” the impact of 148 Tory MPs voting against their leader.
First Minister Nicola Sturgeon said the result was the “worst of all worlds for the Tories”, calling the Prime Minister a “lame duck” after it emerged just two MPs north of the border backed his leadership.
“At a time of huge challenge, it saddles the UK with an utterly lame duck PM,” Ms Sturgeon tweeted on Monday night.
“And for Scotland, it just underlines the democratic deficit – only two of 59 (Scottish) MPs have confidence in the PM.”