Patricia J Wittkopp Senior and Reviewing Editor; University of Michigan, United States Artyom Kopp Reviewer; UC Davis, United States In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Ancient mechanism for the evolution of Bicoid function in fly development" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Artyom Kopp (Reviewer #3). The other two reviewers remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

All three reviewers agree that the methodology is a significant strength of the paper. After discussion, however, the reviewers and editors felt that the results presented here are very much in line with what is already known about the functional changes brought about by evolutionary amino acid substitutions in the BCD homeodomain. Furthermore, the conclusions were based on testing variants in Drosophila melanogaster, which does not allow for an analysis of the contributions of "trans" evolutionary changes in target sequences, and in interacting proteins. In the end, it was the lack of a significantly new advance in understanding BCD protein evolution/function that led to the decision.

Reviewer #1:

This study sought to characterize the amino acids changes that have evolved to confer functionality on Bicoid since the ancestral duplication that produced this gene in the lineage leading to cyclorrhaphan flies. The study focused on the homeodomain and used a transgenic rescue assay to test the effect of variants on target gene expression and overall ability to rescue in vivoin vivo. These experiments were complimented by in vitroin vitro analysis of binding site preferences of variants.

The authors found that the Bcd homeodomain in an otherwise Zen protein could perform much like Bcd. They then used ancestral sequence reconstruction (ASR) to generate and test ancestral sequences. This allowed them to identify an ancestral-like sequence that could function like Bcd although it was unable to fully function like this transcription factor, in for example, repressing translation of caudal. The authors then sought to determine which Bcd specific amino acids in the homeodomain were responsible. They found that the K at position 50 conferred much of the characteristics of Bcd in vitroin vitro and in vivoin vivo and that the R at position 54 also contributed to Bcd function in vivoin vivo. None of the other amino acid differences tested made any detectable contribution, although since rescue was not complete they and other changes outside the homeodomain presumably also have minor roles.

Overall the experiments are described clearly and the work confirms that the amino acids at positions 50 and 54 have made a major contribution to the evolution of Bcd functionality as inferred from several previous studies cited in the manuscript.

A case is made in the Discussion sectionDiscussion section for how these amino acid changes may have facilitated the usurpation of the ancestral gene regulatory network for anterior patterning. However this study only assesses changes in the homeodomain of Bcd and therefore there appears to be an assumption that the binding sites in the target genes have not changed in sequence and nor has there been any other changes in trans. Therefore this speculation should be toned down or at least the caveats should be explained in more detail.

It would also have been interesting to determine how Otd with a Bcd homeodomain and other variants with particular amino acid substitutions performed in these assays to further test what ancestral system Bcd replaced and how this was achieved.

The Introduction sets out the case for the merits of the ASR approach to identify mutations underlying evolutionary differences. However, while ASR can be a powerful approach, I think the Introduction is too biased and there is an implication that horizontal comparisons do not generally allow the genetic changes underlying phenotypic differences to be identified (e.g. from wording such as 'differences that correlate with phenotypic differences' and 'identified allelic differences that reproduce diversity in gene expression and developmental outcomes'. On the contrary mapping approaches and functional analyses of variants between species have identified many mutations underlying population and species differences (e.g. see Martin and Orgogozo, 2013) and continue to do so. Therefore a more balanced Introduction is needed.

Reviewer #2:

The article by Liu et al.et al. aims to understand the evolution of the very interesting fly protein Bicoid. They use a cutting edge tool for reconstructing the most likely ancestor of it and its paralog Zen, and testing the functions of variations on the modern and ancestral proteins in establishing and patterning the anterior posterior axis. I think the work is very well done, but in the end, does not advance our knowledge of the properties and evolution of Bicoid significantly. The importance of the transition from Q50 to K50 is well known and has been for around 30 years, and it is no surprise that it is far and away the most significant change in the protein. Position 54 is also well known to have important functions.

These results should be the starting point of the paper, not the main conclusions. The really interesting thing to me is why these are not sufficient to give complete Bicoid rescue? Is it still a question of intramolecular epistasis that prevents the combination of k50 and r54 from activating all bicoid targets? If so, which sites are involved? Or is something else going on? Perhaps the cis-regulatory elements in D., melanogaster have coevolved with the Drosophila Bcd homeodomain? I realize testing the latter hypothesis is outside the scope of the approach of this paper, but a more systematic probing of the potential of interactions among residues within the homeodomain with the two large effect changes could support or eliminate the intramolecular epistasis hypothesis, and could give more support to searching for other explanations.

As it stands I feel that the field would not be advanced significantly by this work, since we are still at the point of knowing that q50 to k50 led to a big change in the sequences bound by Bicoid, but we have no further understanding of how this led to Bicoid becoming the anterior determinant of the cyclorrhaphans.

Reviewer #3:

I think this is an exemplary paper. As the authors explain in their very eloquent Introduction, incorporating explicit phylogenetic structure and ancestral character reconstruction into comparative studies produces much more rigorous results than simple pairwise comparisons between species. This is as true for development as it is for morphology and DNA sequences, but unfortunately the application of phylogenetic approaches is much rarer in evo-devo studies than in other sub-fields of evolutionary biology – – due at least in part to the difficulty of collecting appropriate data, but also perhaps to cultural factors. This paper raises the bar in both respects. The authors have performed a very nicely structured comparison of reconstructed ancestral proteins to investigate how their function evolved through time on a defined evolutionary lineage. At the same time, they have integrated a wealth of in vivoin vivo, in vitro, and biophysical data to characterize the functions of both extant and ancestral proteins. The result is a very thorough and rigorous paper. In some respects, the results are not entirely surprising – – the single mutation that was found to play by far the largest role in the evolution of protein function is the one that has been hypothesized to do just that for decades. However, in addition to confirming the role of this mutation, the authors were able to quantify its contribution to phenotypic change at different levels of biological organization (from binding affinity/occupancy rate to target gene expression to morphological phenotype), identify a second AA substitution that acts in concert with it, and to rule out the role of multiple other fixed AA changes (within the limits of their assays, of course). So the exceptionally laborious approach taken by the authors has paid off.

[The decision letter after re-review follows.]

As you know, the initial reviews and discussion identified this as interesting and unusually rigorous work demonstrating the phenotypic effects of lineage specific amino acid substitutions in flies. However, concerns were also expressed about the novelty of this work and impact on the field given that the amino acids tested had previously been suggested to be involved in the trait studied. After much debate, I decided initially to reject this work based on these grounds. You have done a great job highlighting the differences between this prior work and your new findings in the comprehensive response to reviewers and appeal letter. After consulting with all three of the original reviewers, we agree that the changes to the manuscript address our concerns. I am thus happy to now accept this work for publication.