In this paper, the authors compare DNA methylation of native and introduced house sparrows before and after (eight hours) an immune challenge (LPS). The paper is largely well-written and justified, representing an important contribution to the avian and species invasion literature. My line edits and comments are provided below, prefaced by some general comments.

1) For the readership of this journal, some brief but general background on DNA methylation could be helpful.

2) More information on the selection of the dose and time period of the LPS challenge is needed.

3) The statistical analyses could be clarified. In particular, it was unclear if/how the authors account for sex and other relevant information (e.g., multiple populations sampled within both the native and introduced range), and GLMMs may be more informative than the t/F-tests (especially as information about variance can be extracted from the random effects).

L10: I found the mention of longitudinal epigenetic studies here to be a bit misleading, as the authors are only comparing DNA methylation before and 8 hours after immune challenge (longitudinal suggests, to me, monitoring over longer time period).

L11: Going off the above, the text here somewhat implies DNA methylation were monitored over an 8-hour period. I would suggest rephrasing to “investigating changes in DNA methylation within-individual house sparrows (Passer domesticus) prior to and eight hours after simulated infection”

L27-28: For a general audience (as at JAV), it would be helpful to briefly define DNA methylation.

L47-48: A citation or two here would be helpful.

L63: I don’t think “over time” is necessary here, as it is implied by “response to simulated infection”. Going off some earlier comments, I think the emphasis on temporal change implies a more continuous/longer assessment of DNA methylation than what was performed here (T0 and T8 comparison).

L76: Did the authors only focus on a single sex? If not, could sex play a role in the results (or potentially confound geographic / invasive vs native differences)?

L82: Should be “DNA/RNA Shield”

L83-85: Can the authors provide reference for why this particular dose of LPS was used and why the eight hour timepoint was used for comparison?

L142 and elsewhere: The “test” in t-test and F-test does not need italics.

L142-143: If the comparison is between the T8-T0 difference between native and introduced populations, it is unclear why the F-test (if for test of variance, be explicit) and Pearson’s correlation are used? Pearson’s correlations do not appear in the Results. It is also unclear if/how the date of first introduction plays into the analysis.

L144: Within each of the native and introduced categories, the authors have 3-4 populations. An alternative approach to data analysis would be to use GLMMs to model the T8-T0 count difference, keeping native/introduced as the fixed effect and accounting for population as a random effect. This would allow the authors to quantify heterogeneity within the native and introduced range as well as to include any potential control variables (e.g., sex if applicable). Would DNA methylation be sensitive to any variation in time between capture and blood collection?

L146: Across the Results, please spell out any integers less than 10 (e.g., “only one differentially methylated CpG site; L163).

L157 and elsewhere: For any statistical results, please provide the test statistics, not just the p values.

L160: It isn’t entirely clear what this analysis is doing differently than the direct comparison among native and introduced individuals (and should be described in the Methods).

L184: More background in the Methods on why the eight-hour timepoints was chosen could help with interpretation here.

Figure 1: Overlaying the T0 and T8 means and confidence intervals (derived from either the t-test or more explicit GLMM) would help emphasize overall results here.

Figure 2: The figure may be slightly easier to read as a lollipop plot, with lines connecting the points with the zero-difference X axis.