The scandalous pest
Population genomics supports clonal reproduction and multiple gains and losses of parasitic abilities in the most devastating nematode plant pest
Koutsovoulos et al.  have generated and analysed the first population genomic dataset in root-knot nematode Meloidogyne incognita. Why is this interesting? For two major reasons. First, M. incognita has been documented to be apomictic, i.e., to lack any form of sex. This is a trait of major evolutionary importance, with implications on species adaptive potential. The study of genome evolution in asexuals is fascinating and has the potential to inform on the forces governing the evolution of sex and recombination. Even small amounts of sex, however, are sufficient to restore most of the population genetic properties of true sexuals . Because rare events of sex can remain undetected in the field, to confirm asexuality in M. incognita using genomic data is an important step. The second reason why M. incognita is of interest is that this nematode is one of the most harmful pests currently living on earth. M. incognita feeds on the roots of many cultivated plants, including tomato, bean, and cotton, and has been of major agricultural importance for decades. A number of races were defined based on host specificity. These have played a key role in attempts to control the dynamic of M. incognita populations via crop rotations. Races and management strategies so far lack any genetic basis, hence the second major interest of this study.
The authors newly sequenced the full genome of eleven strains from Brazil and added nine already available samples from Africa and North-America. They report that, in all likelihood, M. incognita is indeed a purely asexual species. This is supported by (i) the confirmation that the genome is in its major part haploid, and (ii) a spectacularly high level of linkage disequilibrium, which does not decline with genetic distance between loci at a 100kb scale. The absence of sex and recombination is associated in M. incognita with a remarkably low amount of genetic diversity - one order of magnitude less than in typical sexual nematodes - and an heavy load of deleterious mutations, as measured by the ratio of non-synonymous (=amino-acid changing) to synonymous (=amino-acid conservative) diversity in coding sequences. The other important result of this study is that the population substructure in M. incognita is in no way related to host races or geography. The tree genetic clusters that are identified include strains from several continents and feeding on a diversity of host plants.
The implications of this work are numerous. First, the results suggest that M. incognita is an ancient asexual. Asexuality, which was here demonstrated via linkage disequilibrium analysis, must be ancient enough for diploidy (or, in this case, maybe triploidy) to have been lost - i.e., formerly homologous chromosomes have accumulated enough mutations to be assembled as distinct entities. So we are not talking about a highly successful clone having recently spread the world - rather a long-term obligate parthenogen. Asexual organisms are deprived of the source of genetic variation offered by recombination, which is why asexuality is thought to be an evolutionary dead-end. Long-term asexuals are uncommon and even the most famous ones, bdelloid rotifers, are suspected to experience between-individual genetic transfers . M. incognita is apparently a true 'evolutionary scandal', and as such deserves particular attention from molecular evolutionary geneticists.
The lack of any host race effect on the genetic diversity of M. incognita is another important finding. So-called 'races' have largely contributed to shape researchers' view of the structure of the species so far. This study demonstrates that a mental effort is now needed to forget about races, and consider host-specificity for what it is - a phenotypic trait. This result implies that many host shifts must have independently occurred in the three M. incognita genetic lineages, suggesting an arms race between plants and nematodes, which in the absence of sex and recombination must be entirely mutation-driven on the nematode side. Genes functionally involved in the arms race might therefore be expected to have experienced convergent evolution, if distinct M. incognita lineages have adopted the same solutions to overcome plant defenses. The present study paves the way for such a genome scan. The authors rightly discuss that the strong adaptive potential of M. incognita, at least in terms of host shift, despite no sex and tiny amounts of genetic diversity, is a paradox that would deserve to be further investigated.
 Koutsovoulos, G. D., Marques, E., Arguel, M. J., Duret, L., Machado, A. C. Z., Carneiro, R. M. D. G., Kozlowski, D. K., Bailly-Bechet, M., Castagnone-Sereno, P., Albuquerque, E. V., & Danchin, E. G. J. (2019). Population genomics supports clonal reproduction and multiple gains and losses of parasitic abilities in the most devastating nematode plant pest. bioRxiv, 362129, ver. 5, peer-reviewed and recommended by Peer Community in Evolutionary Biology. doi: 10.1101/362129
 Hartfield, M. (2016). Evolutionary genetic consequences of facultative sex and outcrossing. Journal of evolutionary biology, 29(1), 5-22. doi: 10.1111/jeb.12770
 Debortoli, N., Li, X., Eyres, I., Fontaneto, D., Hespeels, B., Tang, C. Q., Flot, J. F. & Van Doninck, K. (2016). Genetic exchange among bdelloid rotifers is more likely due to horizontal gene transfer than to meiotic sex. Current Biology, 26(6), 723-732. doi: 10.1016/j.cub.2016.01.031
Nicolas Galtier (2019) The scandalous pest. Peer Community in Evolutionary Biology, 100077. 10.24072/pci.evolbiol.100077
Revision round #22019-06-25
Decision round #2
I concur with the reviewer that the manuscript has been substantially improved. The scope of the study has broadened, and I find the overall message clear and compelling. The analysis of coverage, the distinction between so-called "heterozygous" and "homozygous" variation and the linkage disequilibrium analysis are important, informative additions. The title was appropriately amended and reflects, I think, the more ambitious nature of the study.
The reviewer has a couple of comments, which deserve to be considered.
First, the reviewer suggests analysing the variation between homeologous regions within a sample (major comments 1 to 4), when the text currently focuses on the between-samples ("haploid") variation. This would be a really nice addition, if possible. I am not sure, however, that separating true variants (between homeologs) from spurious variants (due to assembly/duplication issues) based on coverage is easy to do in this case - figure S1 suggests that the coverage distributions of the two categories of variants overlap quite a bit. Please let us know what you think is doable here.
The other important comment made by the reviewer (major comments 5-6) is that the population genetic analyses have been done in an unusual way, i.e., by comparing each sample to the reference. This has an unclear meaning, which depends on how the reference was generated (single individual? pool of individuals? from which origin?). The reviewer rather suggests analysing multiple alignments across the newly sequenced strains, which indeed should provide more reliable estimates of, particularly, piN, piS and their ratio. This is clearly a sensible recommendation, which I think should be followed.
I have a related, minor comment: for the same reason, I find the "homozygous SNP" vs "heterozygous SNP" terminology quite misleading. A SNP is normally a position in a genome at which between-individual variation has been detected - i.e., a variable colon in a within-species alignment, or a vector of genotypes. Such a vector should not be qualified as homozygous or heterozygous. Furthermore, because the M. incognita genome is haploid, one would not expect to find any heterozygous genotype at all. Yet, because the authors have applied a variant calling method that assumes diploidy, and because of assembly/duplication errors or partial di/triploidy, a large number of apparently heterozygous variants were called. I would suggest refraining from calling a SNP "homozygous" or "heterozygous", and using "variant" rather than "SNP" when referring to a genotype predicted by the variant caller. The word "SNP" should be restricted to the new analysis suggested by the reviewer, when sequences from the distinct strains have been multiply aligned.
I would suggest following these very last suggestions, which I think should help improve this excellent manuscript even further.
Reviewed by anonymous reviewer, 2019-06-13 21:59
Revision round #12018-09-19
Decision round #1
The two reviewers have expressed relevant and important comments on various aspects of the study. I concur that the current manuscript should be extensively revised in order to reach, and convince, a wider evolutionary biology audience, as expected for a manuscript recommended by PCI.
Reviewer 2 recapitulates the main results of the study and identifies a number of issues requiring clarification, rewriting, and/or re-analysis. This reviewer also suggests that the current title does not optimally reflect the content of the study - I agree.
In addition, Reviewer 1 mentions a number of analyses that could be made in order to better characterize the population genomics and molecular evolution of M. incognita, with a focus on its supposed asexuality. I agree that this is a missed opportunity, especially knowing that previous publications on the subject, some by authors of this manuscript, have opened very interesting questions (eg Castagnone-Sereno & Danchin 2014 JEB).
The two reviews are highly complementary and provide a number of clearly expressed recommendations, which I think should greatly help improve the manuscript.
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