General comments – thank you to the authors for considering the suggested modifications to the manuscript. The additional analyses provide more insight into the genetic structuring of spadefoot toad populations across Europe, particularly showing how strong the spatial signal is (consistent with the idea of limited connectivity amongst populations). I agree with the authors that sample sizes limit the population-level analyses that can be performed (e.g. Ne estimates, FIS). The discussion is more readable having removed repetitive text.

The additional information about the local context of the Italian populations was useful. I flagged this population because a result of Dest ~ 1 implies that there is no overlap in alleles with most other populations, suggesting something very unusual about the population, especially as there was some discussion about the previous recognition of Italian populations as a distinct subspecies.

Some additional minor comments:
L64 “that conform with national or regional law”
L67-68 Some clarification of the DNA extraction method is needed. Two DNA extraction kits are mentioned. Do you mean that samples were extracted either with the DNeasy Tissue kit OR the 96 DNA Plant Kit, not “with and without” as currently stated. Does “with and without” refer to the use of the robotic workstation?
L139-140 I haven’t used Bottleneck for a very long time, but my memory is that there are several settings that could be modified for each mutation model. Can you include information here on any modifications you made to the default settings? If none, then state that the default settings were used.
L171 and elsewhere – mantel should be capitalised. I.e. Mantel test
L204-205 The last sentence is more like discussion than results. It could be moved to the Methods section to describe the statistical test.
L237-238 This first sentence reads as if it is the outcome of the current study. Could you rephrase slightly by saying “Previous studies have indicated that the common spadefoot toad….”
L283-285 I think you could provide a little more explanation of the BAPS result. The Mantel test showed highly significant isolation-by-distance. Taking this spatial signal into account, the BAPS analysis emphasised the highly localised genetic structure of toad populations. This is consistent with the overall lack of connectivity between populations that was mentioned in the Introduction.
L318-320 Check that this sentence is stated correctly. I would have thought that the clustering of central French populations with BE, NL and DE would have been evidence that the populations have been introduced. Given the small sample size, it’s questionable whether you can say anything about this issue at all. If keeping it, make it clearer what the actual sample size is (N = 4?) in an earlier sentence (perhaps in line 312) so that readers are primed to understand that inference will be limited.

I have some additional editorial suggestions (line numbers refer to the larger, continuous line numbers in the ‘clean’ manuscript version):

L37 “Recognizing the impact of genetic diversity on populations” – Suggest re-write “importance of genetic diversity for population health”
L72 Change to “conform with”
L107 Many locations had sample sizes that are too low for these analyses. These should have only been applied to locations with sample sizes >10, for example.
L118 Allelic richness – given that many of your populations have sample sizes less than 5, you need to indicate that you are extrapolating allelic richness for these. This analysis is used to subsample larger populations with uneven population sizes to the minimum population size (in your case this would have been 1 or 2, not 5). Therefore, caution is needed when extrapolating allelic richness for populations that don’t meet the minimum population size threshold used.
L122 and elsewhere: RStudio is a separate piece of software that provides a user interface for the R statistical software. You need to cite the version of R that you are using, not RStudio. Replace “RStudio” in the methods with “R” to indicate the statistical software.
L138-139 Check whether the QGIS citation is appropriate. It looks like the full citation has been put here rather than the shorter format that should be used for in-text citations.
L139-141 “After determination of the optimal number of clusters” - It might be better to describe this analysis as a hierarchical analysis of population structure. Also indicate whether this analysis was done for both Structure and BAPS analyses.
L147 “Finaly, to investigate the possibility of recent population declines” – It is more accurate to say “investigate a genetic signature of recent population declines”. If this text is used, then you can delete the following sentence (starting “Given nearly all populations”) as it becomes redundant.
L170-214 Now that I haven’t seen the manuscript for a while, I’m finding this results section quite dense on re-reading and hard to pull out the key findings. I would suggest reconfiguring this section around your key take-home messages rather than describing each analysis separately (better to use multiple lines of evidence to support a key observation) or in too much detail. The section would also benefit from breaking the text into more paragraphs, rather than as a single block. Perhaps it can be split into the broad-scale genetic structure vs finer-scale genetic structure results? This is my interpretation of the main messages and brief suggestions for structuring the text:
1. Differentiation metrics ranged from X to Y and you found a strong spatial signal in the observed differentiation patterns (significant IBD test & as visualised in PCoA). I think you can remove the detailed description of the PCoA as readers can interpret this easily from the figure.
2. State that at the highest level of structure, you identified an east-west genetic clustering of populations in Structure (confirm this was also seen in BAPS) analyses. Perhaps note the location of a potential geographic barrier (e.g. mountain ranges) to explain the divide between the clusters.
3. State that further structuring was observed within these major clusters using a hierarchical analysis, and this was most evident in the eastern cluster. Deal with the eastern cluster first, noting the strong differentiation of Italian populations evidenced in differentiation statistics, on the DAPC and distinct clustering in the Structure/BAPS analyses. State remaining populations were all found in the XY drainage basin/s.
4. For the western cluster, briefly state there were two main sub-clusters in the Structure analyses and what features these aligned with (by drainage basin?) and confirm further regional structure was observed in the BAPS analysis as this method explicitly incorporates spatial information.
The text already follows some of this structure, but I think the key is to make a general statement for each conclusion at the start of the sentence or paragraph, then group the lines of evidence to support the particular statement, rather than describing each analysis separately as done currently.
Also, be aware that Structure analyses can be biased in the presence of significant IBD (See https://onlinelibrary.wiley.com/doi/full/10.1111/ddi.12816). Check that the BAPS results support any of the conclusions you are making from the Structure analyses.
L192-195 Please include the deltaK figure or other output for BAPS and Structure analyses in the Supp Material so that we can see the evidence for the selection of the optimal K value for each analysis. The BIC figure is provided for the DAPC analysis so the equivalent is also needed for the other two analysis methods.
L218-219 “The observed trends…” – This sentence needs some prior context. I suggest modifying the previous sentence slightly: “We present a comprehensive overview of nuclear genetic diversity and population genetic structure across a significant portion of the western range of the common spadefoot toad, Pelobates fuscus, using samples collated over a 34 year timeframe (1986 – 2020) and across XX locations from adult and juvenile life-stages where available”. You could then delete the years from Line 223.
L227-277 I’m wondering if the discussion might flow better by swapping these two paragraphs around. It’s hard to disentangle the influence of contemporary population declines vs. the signal of post-glacial recolonisation on the genetic diversity & structure results. To make it easier for the reader I think it would be best to describe the historical pattern first (current L252-260), then the contemporary conservation context second (current L227 – 250).
L282 “Notably, it is only when we impose a requirement for eleven or more clusters in STRUCTURE” – but the distinction of the Italian pop was quite clear in DAPC and BAPS analyses. Could be worth restating that the Italian populations were distinct in DAPC and BAPS analyses, but less so in Structure analyses (if you agree this is the case).
L369 “restauration” – should be “restoration”
Figure 1 caption: Add cross-reference to the appropriate K=2 Structure figure in the supp material. Otherwise I think it might be more accurate to include the K=2 Structure assignments in panel A, rather than plotting both the eastern and western sub-clustering results on the same figure when these analyses were done separately. The sub-clusters are visualised in panels b and c anyway. For panels c and d, it’s not clear why different locations were included in each – can you add some explanation to the caption?
Figure 2 Add panel labels ‘a’ and ‘b’ rather than referring to ‘above’ and ‘below’
Supplementary Materials:
Figure S4 caption – “Meaning of the symbols in Figure S4.” I think the figure number needs to be updated?
Figure S10 – is this figure necessary? i.e. isn’t it the same as K=2 in Fig S9?
Figure S11 – how is the mean calculated? Across multiple runs? If so, can you include the number of replicate runs in the caption.
Figure S12 I suggest it would be better to describe this as the ‘eastern cluster’ rather than the ‘first cluster’. Would be good to include the delta K plot for this analysis.
Figure S13 Same comments as for S11 and S12
Figure S14 Better to describe this as the ‘western cluster’? Also include delta K plot.
Figure S15 Include number of runs
Figure S20 – Move this to the Materials and Methods section above. I think it should be the first figure in the Supp Material.

Thanks to the authors for making the previously suggested changes, especially attending to streamlining the genetic structure results section which is much clearer now. The glm results modelling genetic diversity against time and longitude were an interesting addition to the manuscript.

I noticed two small errors:
L182 There seems to be an incomplete sentence "PC2-4 further"
L274 "rare alleles become more frequent in the population" - normally a bottleneck is associated with a loss (extinction) of rare alleles through genetic drift, which contributes to the flattening of the allele frequency distribution. It would be unusual for rare alleles to become more frequent as a result of a bottleneck, unless there was some selection process operating.