This work describes an interesting phenomenon in the plant Zostera marina: populations with higher rates of asexual reproduction present a higher proportion of genetic mosaic individuals. However, I think the description and discussion of the phenomenon needs some reconsideration and the text will benefit from further work to improve its clarity. In the following list of comments the first points two require special attention and the rest are minor suggestions for the improvement of the manuscript:
1) Throughout the text, the observed phenomenon is described as an “accumulation of somatic mutations”. However, I think that it will be more accurate just to state that there is an increase of individuals that are genetic mosaics in asexual populations. The expression “accumulation of somatic mutations” seems to suggest there is some kind of mutation load associated to those mutations. What does really mean “accumulation of somatic mutations”? I think the authors should keep in mind that they are working with molecular markers that are assumed to be neutral. Thus, the phenomenon described is independent of the “accumulation of (somatic) deleterious mutations” (i.e. increase of mutation load) that will led to a mutational meltdown described by Lynch and Gabriel (1990). The observed difference between the sexual and asexual populations is in the incidence of (neutral) genetic mosaicism and this should be the focus of the work. I also recommend to denote genotypes as genetic mosaic and non-mosaic (or something equivalent) rather that “genotypes with somatic mutations” and “genotypes free of somatic mutations”. Mutation of somatic origin might be present in the non-mosaic offspring of a mosaic individual (either originated by sexual reproduction or originated by asexual reproduction due to genetic drift among cell-line within the meristem of a mosaic individual).
2) The text gives a considerable importance to the potential fitness effects of the described phenomenon with the discussion focused on some potential mutational meltdown and the different consequences of sex and clonality. The discussion of the fitness effects of the observed pattern are focused on an increase of mutation load due to asexuality (something that is theoretically sound but was not measured in this work) but fitness effects of mosaicism are practically ignored. Pineda-Krch and Lehtilä (2004, http://www3.interscience.wiley.com/journal/118796683/abstract) review the different fitness effects of genetically heterogeneous organisms which include some beneficial effects of mosaicism. Authors should rethink the focus of this part of the discussion. In any case, the title of the article should not refer to fitness effects that were not studied in the present work.
3) The introduction ends by stating the two hypothesis to be tested in the article. The introduction does not offer a clear explanation why mosaic individuals are expected to be more frequent in asexual populations (hypothesis (ii)). Instead, a much clear explanation is offered in the discussion. I think that the introduction will improve if the mechanics of asexual and sexual reproduction regarding the number of cell-lines involved were discussed instead of the “accumulation of somatic mutations”. The higher proportion of mosaic individuals at marginal populations of Zostera marina (hypothesis (i)) is a consequence of a previous observation (Reusch et al. 1999) and the confirmation of hypothesis (ii) rather than an independent hypothesis in itself. While I agree with the importance of the geographic distribution of the phenomenon I wonder if it would better be discussed only in the discussion section, leaving more space in the introduction to develop hypothesis (ii).
4) I have the impression that the words chimera (http://en.wikipedia.org/wiki/Chimera_(genetics)) and mosaic (http://en.wikipedia.org/wiki/Mosaic_(genetics)) are used in the text as synonymous (with the meaning of mosaic). Please, clarify this (if necessary).
5) Write in words “at least 20” (instead of “>20”), “20 or more” (instead of “20+”) (or its intended meaning if different)
6) Numbers between 1 and 9 are sometimes written in letters and sometimes in figures (e.g. “they were divided into 2 classes, those with and without additional 3rd and fourth fragments at the two hypervariable loci”, from page 9). It is easier to read if they are homogeneously written (some journals require this to be in letter).
7) In page 7, the sentence “Here, two hypervariable loci that display complex genotypes were excluded and used for somatic mutation analysis (see below).” seems a bit confusing. The reader might have the impression that these two loci are among the seven loci discussed in the previous sentence. I suggest to remove the sentence altogether, since the use of these two loci is described in another subsection. If the sentence is maintain, I think it would be better if it is reworded into something like: “At this point of the work, two additional loci were also considered but excluded because of their hypervariability and complex gentypes (they were used for somatic mutation analysis as explained below)”
8) The text regarding Psex needs some clarification (page 8). Is Psex a likelihood (of a model) or a probability (of an event)? If it is a likelihood, what is the significance of being lower than 0.05 (likelihoods usually take very small values)? Should be interpreted as a p-value? Is it a computer program (“Psex claculates [...]”) or a statistic (“[...] to calculate Psex.”)?
9) The acronym MLG (multilocus genotype?) is used without being defined. Also it should be specified if it refers to the SLGs or to the nine-locus genotypes.
10) Page 9. Change “AIC criteria” for “AIC” (the 'C' in AIC stands for criterion).
11) The mosaic nature of some individuals with composite genotypes was checked by genotyping different section along a leaf. Some detail of this procedure are missing on the materials and methods. In particular, it will be interesting to specify for how many individuals this was checked.
12) Page 16. In the sentence: “It will be worthwhile to extend mutation studies to functional genes underlying sexual reproduction”, what is it meant? “Underlying function genes”? “functional genes involved in sexual reproduction”?