Ms Austin declared her intentions to step down from the assembly and its works towards treaty in Victoria via a public Facebook live stream on Tuesday night. She followed this up with a post on Wednesday morning to thank her supporters and reiterate her stance as a "fighting from the ground up kind of woman".

The Ballarat-based Austin was one of three south-west representatives elected by Indigenous Peoples to join the assembly, which also features representatives from traditional owner groups.

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Ms Austin has maintained since first standing for election that the culturally significant trees on country between Buangor and Ararat would be a sticking point for her in the last stage of highway duplication works.

About 50 protesters were arrested on Tuesday at Dobie where the directions tree was removed, however, the tree's cultural significance is refuted by other Aboriginal traditional owners.

In an emotional live stream, Ms Austin said she had made a "culturally informed decision" on how to best position herself and her voice - and the assembly was no longer the right place.

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"I believe in a treaty but I don't believe in this treaty," Ms Austin said on social media.

"The conflicting agendas are bold. You cannot say you're all for elements of treaty - self-determination, community control, speaking of grassroots voices - you can't do that then make irreversible decisions in destroying sacred country at the same time, especially during the pandemic."

Major Roads Projects Victoria confirmed a Fiddleback Tree was removed from near Hillside Road on Monday but had not been identified as culturally significant by the Registered Aboriginal Party and was unlikely to pre-date European settlement.

Eastern Maar Aboriginal Corporation chairman Jason Mifsud confirmed the tree had undergone extensive surveying by cultural heritage experts and "wasn't in a list of trees that we were able to negotiate through the environment process."

"Eastern Maar Aboriginal Corporation, which does represent 12 family groups - including numerous people from the Djab Wurrung who sit on our board including senior women - have found that the cultural surveying that came back to us all passed our cultural thresholds," Mr Mifsud said.

"The trees that need to be saved, are being saved."

Major Roads Projects Victoria stated the Directions Tree identified in the Federal Court proceeding is at the northern end of the alignment and has not and would not be removed.

Premier Daniel Andrews said on Tuesday direct consultation with the 12 families that comprise the traditional ownership group of the land was ongoing and significant changes to the design of the road had been made.

He said it was a "dangerous road" where many people had lost their lives and he was "determined to get it upgraded".

Mr Andrews said there had been legal action, consultation and an agreement reached, but acknowledged that not everybody would back that.

He said that at some time "we have to be faithful to that agreement".

Mr Andrews said if they waited for "100 per cent consensus" the deadly stretch of road would go unchanged.

Victoria's first Aboriginal Lidia Thorpe said: "Dan Andrews gets everyone excited with his COVID updates and then they evict the land protectors of Djab Wurrung country, who have looked after this country for thousands of generations.

"They're destroying our cultural heritage, particularly that ancestor tree, and it should not have been desecrated the way that it was."

The Courier has contacted Ms Austin for further comment and the First People's Assembly of Victoria.