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Revise your submission according to the comments received from two distinguished referees.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors start from the hypothesis that Globodera might edit the DNA sequence of HYP1 in some somatic cells. They test this by attempting to distinguish rare edited somatic alleles from inherited germline variation, using Cas9-enriched long-read ONT sequencing of native DNA. If somatic editing were occurring, they would expect rare alleles carrying large, complex rearrangements of sequence motifs relative to the common germline alleles. Contrary to the hypothesis, they find only 2-4 GrHYP1 alleles among 11,378 sequences from an inbred G. rostochiensis population, and conclude that GrHYP1 is not diversified by somatic editing. They then ask the same question of G. pallida, where the updated dataset contains far fewer rare alleles than previously reported, and trace the earlier signal to nanopore basecalling error within the repetitive domain and to PCR template switching in single-worm amplicon data. Having excluded editing, they propose that the remaining germline diversity arises through error-prone homologous repair of double-strand breaks, and that genome-wide nucleotide diversity is sustained by migration between subpopulations despite small local effective population sizes.

I find the central conclusion convincing. The demonstration that two independent technical artefacts can each reproduce the exact pattern predicted by the hypothesis is valuable well beyond this system. I recommend this article for publication and I have some minor revision/questions:

In lines 129-139, the authors ask what mutations produce the complex sequence motif that defines HYP1, and they use the transgenic yeast system to conclude that erroneous homologous DNA repair of double-strand breaks are the dominant mechanism. The yeast experiment establishes that these mechanisms can generate the observed motif architecture, but not that it is the mechanism operating in Globodera. In Fig. 3., the authors establish the original construct sequence as the baseline, not the natural allelic+ diversity in G. pallida populations. Could the authors comment on whether the mutations recovered in yeast share diagnostic features with the natural GpHYP1 alleles, for instance, microhomology at rearrangement junctions or comparable size and motif-boundary distributions? Some support of this kind, or an explicit statement of the limitation would strengthen this inference.

Reviewer #2: The HYP family in PCN (potato cyst nematodes) refers to hyper-variable apoplastic effector proteins that play an essential role in how sedentary plant-parasitic nematodes establish compatible interactions with their host plants. Among HYP effectors, the HYP1 gene displays an extraordinary, unprecedented level of sequence and structural diversity, even among individual nematodes. In the submitted article, the authors tested the hypothesis that a developmentally programmed editing process exists in both species of PCN, G. rostochiensis and G. pallida. Although the results did not confirm the existence of such an editing process in plant-parasitic nematodes, they also explore other mechanisms that might explain how such a complex sequence motif structure could have arisen in the HYP1 gene.

The manuscript is well written, quite interesting (even if the main hypothesis was not validated) and I fully support its publication after the implementation of some minor comments below:

- Whilst I acknowledge that the authors present and discuss their results concisely, it seems to me that reading this article would benefit from both better structuring (subheadings) in the ‘Results/Discussion’ section and a review of what should be presented as supplementary data or within the main text. There is a great deal of supplementary data, and almost every main result or figure has a corresponding entry in the supplementary data, which readers are invited to consult. I suggest : i) providing clearer guidance to the reader regarding the supplementary information presented in the supplementary documents in relation to the figures ; ii) and also reorganizing some parts like adding the supplementary document entitled ‘HYP1 mutations observed in a transgenic yeast system’ – which contains important information – to the main text, and instead placing the paragraph in the results section (lines 116 to 128) relating to potential confounding effect in the supplementary materials (as this will not alter the main conclusion set out just before).

- Line 42-43 : Can you support by a reference the statement that free living nematodes have large census population sizes and efficient dispersal ? This was not studied in Cole & Viney, 2018 and I have the feeling that is may be an overinterpretation of the fact that free living nematodes are more aboundant in soils. I am not sure that in arable field a PPN species will not have a census population size as large of that of a free living nematode species.

- Line 79 : as the M&M is at the end, please indicate in the Results the names of the nematode populations (ie line 19 and Newton) used in this study. Qualify the Newton population as a field population maintained in laboratory rather than a « non inbred » population (there is natural inbreeding reported in many G. pallida populations, don’t think that Newton is an exception).

- Investigating mutations using the yeast system was quite interesting but if I am correct yeast is a good model with great functional conservation accross animals for nucleotide excision or mismatch repair but I don’t know if this holds true also for recombinational mutations. May be a point of discussion or a paragraph to add in the introduction in relation to this part of the results.

- Figure 4D : I have not understood the interest of Figure 4D and have not seen any corresponding text in lines 152-160.

- Lines 161 - 179: I am not convinced of the value of this paragraph and I think it should be deleted. Indeed, it adds nothing new: even if the estimate of Ne differed from that already published for G. pallida, it would be impossible to compare them, as the value obtained by the authors is closely linked to the population’s rearing conditions in the laboratory. Furthermore, as the authors themselves mention, their estimate of Ne could be overestimated, as many parameters of the ideal population, as defined in the Wright–Fisher model, are / can be biased (an ideal population is characterised by the absence of migration, mutation, selection and generational overlap, by equal sex ratios, a constant size from one generation to the next, and random mating).

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Reviewer #2: No

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Diversity at the HYP1 locus in potato cyst nematodes does not result from developmentally-programmed somatic mutations

PONE-D-26-37792R1

Dear Dr. Hanlon,

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Kind regards,

Tushar Kanti Dutta, Ph.D.

Academic Editor

PLOS One

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