A recurrent regulatory change underlying altered expression and Wnt response of the stickleback armor plates gene EDA

The work is significant because it lends new biological insight into the molecular mechanism of adaptive evolution in natural populations and also expands our knowledge of the regulatory landscape that controls EDA expression. This finding has both developmental and potential clinical significance. More generally, this work is remarkable in that it is also a rare instance in which a non-coding change appears to have repeatedly been the target of selection in multiple populations. It would have enhanced the study to know which factors bind to this region, or data on the phenotypic consequences of engineering a correction of this change, or of mutating the same base, but to a different substitution. Since such additional work would require a good deal more time and effort, on balance the reviewers decided it would be beyond the scope of this study to require such additional data.

However, we would like to ask you to provide more information on the status of the T to G change in a larger number of marine animals.

We have now carried out a PCR and sequencing screen to look for the presence of the T to G mutation in a population of migratory marine fish from Rabbit Slough AK. (This work was carried out in part by Shannon Brady, who has now been added a co-author of the manuscript). This survey showed that most completely plated marine animals are homozygous for the marine “T” allele, but a small number of fish are heterozygous carriers of the freshwater “G” allele (overall minor G allele frequency: 2.3%). We also typed these heterozygous fish with flanking markers, to distinguish between carriers of large, small, or recombinant freshwater EDA haplotypes. These data are summarized in a new Supplementary file 1, and a corresponding section has been added to both the Discussion (paragraph 5) and the Materials and methods (paragraph 3) to cover the new material.

Tone down your inference that there might be very few, perhaps only one mutation that is evolutionarily permissible, unless you can demonstrate with additional mutations in the enhancer that this is indeed the case.

We have examined the location of the marine and freshwater sequence change for predicted transcription factor binding sites, and computationally compared the predicted effects of all possible single base pair mutations at the positions surrounding the observed T->G change. While there are multiple mutations that can potentially disrupt the c-Jun site previously mentioned in the Discussion, the observed T->G change is the only base pair mutation that is predicted to both eliminate a c-Jun site in the marine enhancer, and to simultaneously create a new overlapping AP-2 alpha site in the freshwater sequence. We describe these predicted mutational effects in an enlarged paragraph 7 of the Discussion, and discuss how the dual effects of the T->G mutation could be an example of combined molecular constraints that limit the range of base pair mutations observed in low-plated populations.

We have also edited the Discussion to clearly state the possibility that the observed sharing of T->G changes is due to the relatively high frequency of the freshwater G allele in marine populations, rather than due to strong constraints on possible adaptive sequence changes among independently arising mutations (paragraph 5 and first sentence of paragraph 6).

Also, in your previous work, you did not distinguish between gene-flow from freshwater into migratory marine fish and ancestral presence in the marine population.

This distinction was not discussed in the original Colosimo et al. paper in 2005. However, the issue has subsequently been further analyzed using the geographic patterns found in the Colosimo et al. marine and freshwater EDA allele sequences. This analysis shows much greater geographic structuring among freshwater EDA alleles than among marine EDA alleles, which is consistent with repeated rounds of gene flow from freshwater fish into migratory marine populations, rather than selection from ancestral diversity already present in marine populations (Schulter and Conte, 2009). We now briefly refer to this point in the revised Discussion (see paragraph 5).

Nor did you explore potential reasons for the remarkable size of the common low-plated 16 kb haplotype.

Our new survey of migratory marine fish includes additional analysis of freshwater haplotype sizes in carrier animals, and identifies multiple examples of apparent recombinant haplotypes that are smaller than the previous observed 16 kb freshwater region (Supplementary file 1). We have also modified the Discussion to point out that the substantial size of the typical 16 kb freshwater haplotype may be due to coselection for multiple phenotypes arising from linked genes, and that NAKA may be an interesting example of a population that has fixed only the armor plate changes (see the end of paragraph 5 of the revised Discussion).