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The manuscript entitled “Genomic data suggest parallel dental vestigialization within the xenarthran radiation” by C.A. Emerling et al. presents evidence for the parallel evolution and gradual decay in dental regression in xenarthran lineages. It is a very consistent and robust manuscript, based on a well-designed analytical methodology. 

The authors ground their hypotheses on two major lines of evidence: sequence analysis of gene pseudogenization and dN:dS analysis of nucleotide sequences, for eleven core genes involved in enamel and tooth formation. Both provide compelling support to the main hypotheses of independent and stepwise loss for enamel/teeth. 

The scientific background and the specific aims of the manuscript are well-developed while remaining clear to the non-specialist. The manuscript is well-written and the authors provide a wealth of details about how the dataset was constructed and analyzed. The in-text figures are designed clearly while conveying a large amount of information. All necessary material is presented or made available to support the authors’ claims; relevant supplementary tables (26 six of them, all meaningful to me) and figures also provide extra details to the keen reader. I appreciated that the discussion addresses how the molecular findings integrate with the (scarce) paleontological evidence available from the relevant Eocene/Oligocene Xenarthra fossils, and the changes in the ecology/diet of the sloths.

I only regret that some sentences in the main text sometimes lean towards a non-necessary finalist presentation of the evolutionary processes. For example l.34 reads “sloths halted their dental regression; l.468-9 reads “stem dasypodid armadillos independently inactivated AMTN”. My remark might only reflect the fact that English is not my mother tongue, and that this phrasing sounds finalist to the French reader that I am. 

I am fully convinced by the various claims made in the manuscript concerning the independence, parallelism, and gradual characteristics of the dental regression processes identified in the various subclades within Xenarthra. I agree with the authors that the lack of SIM signal observed in the DMP1 and MEPE genes only reinforces the observations made on the 9 other genes more specifically involved in enamel/tooth development.

I am a bit surprised that not a single SIM was recovered for any of the genes under scrutiny for the ancestral branch of the entire Xenarthra super-order, considering that dental regression is a feature observed even in the earliest-known xenarthran fossils (as indeed the authors acknowledge in the discussion). I think that this observation might deserve a little more consideration in the discussion: it could be emphasized that this is a clear limitation of such a functional study based solely on the analysis of coding sequences and that the processes identified here might just reflect one side of the coin. Adding to this, I wonder why only the coding sequences of the focal genes were targeted. It is obvious from the results (AMTN, ODAM) that key mutations leading to pseudogenization might have occurred in non-coding sequences of these genes or promoter regions also. Were the intron sequences excluded for any specific reason (like strong divergence among xenarthrans, or else)? I would appreciate the authors briefly justify this choice.

Gathering the gene (exonic) sequences dataset analyzed in the paper for such an extensive sample within modern Xenarthrans (31 species represented) is quite impressive. The sources of the data analyzed are extremely diverse: whole-genome sequencing, shotgun and target capture sequencing, targeted amplicon sequencing. Yet, this data collection might also be one of the few limitations of the paper: even with such a broad effort, a lot of genetic information is missing from the final dataset. Except for the few fully sequenced analyzed, it is unclear whether the missing exons of various genes are the reflection of a biological reality or rather of the incomplete molecular sampling due to imperfect amplification or capture and/or lack of depth in the sequencing. This difficulty is exemplified by the DMP1 and MEPE datasets (see below). Since these missing pieces of genes might themselves be involved in the identification of inactivated genes, it slightly blurs the general message.

For the ACPT gene, specifically, the authors state that its CDSs were not included in the baits designed for target sequence. Why? Due to this choice, a majority (19 out of 31) of xenarthran taxa are not directly represented in the ACPT alignment. Thus direct evidence of pseudogenization for this gene is scarce. However, figure 1 indicates that for 11 non-sequenced armadillo species, the dN/dS ratio estimates suggest a possible gene inactivation. Could the authors be more specific as to how this result is obtained since only a single direct sequence is available for all 12 most basal armadillos? 

When compared with the information conveyed in figure 1, it seems that some of the identifications for the genes between ‘missing-or-pseudogene_phylogeny-based/delta’, ‘pseudogenized/psi’, ‘unknown/?’ and ‘pseudogene_dN:dS-based/omega’ lack consistency among the various markers and taxa. For example, some ‘delta’ identifications are made for some taxa for which not a single exon sequence of the marker of interest was retrieved (as ‘missing’), but sometimes it is given for taxa that show only a few of the marker exons (as pseudogene based on the phylogeny’). I would rather have these two situations split into two different summary letters, for they do not convey the same information at all.

I’ll provide a few examples, below, of situations that put the emphasis on the not-so-obvious link between the supplementary tables S5 to S15 and the interpretation that is made of them within figure 1.

Table S6 – AMBN gene
The sequence for Cabassous tatouay is represented in the dataset by a single exon (exon6) which is deemed putatively functional, but the other 9 expected exons are missing. This sequence shows a “?” in figure 1. However, nine other taxa lack one or several exons (up to 5 for Euphractus sexcinctus) for this gene and yet are all deemed functional for AMBN, because no obvious signature of pseudogenization is found in the remaining exons. 
 Isn’t there a risk, in such a situation – which corresponds to the majority of the markers analyzed – to underestimate the actual inactivation of the markers? In other words, how can we be confident that, with one or several missing exons, the protein produced is still efficient and not sub-functional? And from how many absent exons should one deem that the protein is not functional anymore? It seems fairly arbitrary to me at this stage (and if so, which is fine, it should at least be specified).

Table S7 – AMELX gene
 For Cabassous tatouay, again, only one exon out of 4 is present in the dataset (and not mutated). Yet this time the gene is deemed inactivated via ‘delta’ in figure 1. Why is the interpretation different for this marker from the AMBN case? 

Table S8 – AMTN gene
 Priodontes maximus shows only one (non-mutated) exon out of the 7 expected and is identified with a delta (pseudogenized based on phylogeny I guess) like Totypeutes tricinctus for which not a single exon was found (thus recorded as a missing gene for this one). Again the use of a delta for both categories seems misleading to me.

Table S14 – ODAM mutations
 Among Vermilingua, Myrmecophaga tridactyla show the 10 expected exons of the marker, with 6 of them showing inactivating mutations, and yet it is identified as a “delta” - inactivated based on the phylogenetic bracketing – instead of the “psi” like Tamandua tetradactyla. Why?

Table S9 – DMP1
 All 31 taxa are presented as having a functional DMP1 in figure 1. Yet, the 5 expected exons are only documented for the 11 complete genomes analyzed, whereas the other 20 taxa showed the lack of at least 1 and up to 4 of these exons.  Based on the observation that the present exons never show a SIM signature, it sounds reasonable to extrapolate that these sequences are functional as the authors do. Yet, the limited completeness of the dataset for this marker (and the others too) should be directly accessible from the main text to be obvious to help contextualize the interpretation made. 

Dear Christopher Emerling and colleagues,

I read with interest your preprint entitled « Genomic data suggest parallel dental vestigialization within the xenarthran radiation ». In this research article, you report your findings on the patterns of sequence evolution related to the vestigialization of teeth development in xenarthran, a group of mammals known to have experienced changes in their teeth ranging from regressive modifications to a complete absence.  

You collected data and explored the evolution of sequences corresponding to eleven known genes involved in tooth development in anteaters, armadillos and slugs. You present a phylogenomic analysis that revealed different patterns of mutations across Xenarthra. You conclude that the regressive evolution linked to tooth development in these mammals could not be linked to a single mutation, and instead followed parallel trajectories in the different clades and occurred relatively rapidly.

I find that the title reflects the main message of the study. 

I find that the abstract is relatively concise and presents the main results and conclusions of the study.

In the introduction, the theme of “regressive” evolution is presented as the underlying theme of the paper. The specific domain of teeth loss in jawed vertebrates is also explained, along with background information on specific and well-known genes involved in tooth development and differentiation. The Xenarthra taxon and its teeth traits are presented in enough detail so that the reader can understand the research questions and hypothesis. The references provided in the introduction are relevant and cover not only the most recent research but also covers older literature. 

In the Materials and methods, the information related to the gene set being analysed is presented in sufficient details. I just checked the gene symbols and became aware that the approved gene symbol for ACPT is now ACP4 (and at the same time I learned that its whole name is testicular acid phosphatase ! https://www.genenames.org/data/gene-symbol-report/#!/hgnc_id/HGNC:14376). The taxonomic sampling is presented with details on each sample. Protocols for the experiments of molecular genetics are given in a manner enabling reproducibility. The sequences OP966064-OP966335 were not unavailable at the time of review. The procedure for sequence analysis are missing some minor details (e.g. BLAST version). The Bioproject PRJNA907496 was unavailable at the time of review. I have to say that I releasing the southern naked-tailed armadillo genome assembly as a Zenodo dataset is not acceptable, the international nucleotide sequence database collection (INSDC www.insdc.org) lists the sole repositories for such data. 

In the paragraph Dataset Assembly, line 225 Supplementary dataset S1 does not correspond to sequence alignments, I guess you meant dataset S2-S23. In this paragraph, I was expecting to find the rationale to consider an exon as missing (ie yellow in supp tables S5-S15).

Line 219 you mention that “sequences were assembled and occasionally combined”, how does this link to the supp table 2 ? Did you use a cut-off for base quality before proceeding to mutation analysis and did you look for hetero or homozygosity in the case of inactivating mutations?

Overall there is no specific code or script provided. This latter point could be improved as the analysis of the branch model dN/dS ratio with the various models and their statistical analysis (lines 245-266) is of methodological interest.

In the results, you reported the findings on gene mutation patterns across xenarthrans in a condensed format in Figure 1. While this figure is extremely informative, I think that it holds too many pieces of information. I have a suggestion to help the reader follow the figure along with the text: you could position the matrix of shared inactivation on the left as it is the starting point in terms of data, and then have the species tree (with the root oriented toward the right). In my opinion, the mix of colors and symbols is extremely difficult to analyze. A possibility would be to split the figure in two, with one panel showing the data matrix, and another panel with the tree and traits. Also, the use of serif characters in figures is usually avoided (you used it for sub-order names and common names). 

Line 410-414: I suggest that you follow the scientific convention on significant figures for omega values.

In Figure 2, which provides key data, I notice that the alignments of the different genes do not always include the translation, you could maybe insert a consensus translation over the alignments in these cases. The presentation of the different genes does not follow a simple rule (such as alphabetic ordering), so different mutations of the same gene are not presented side by side. Since the objective here is to show shared inactive mutations, It may be better to include all taxa for a given gene with different SIMs : e.g. AMTN exon1 next to exon4, with all taxa. And to highlight mutations, the use of a dot for identical residues and of lowercase for silent mutations may help.

On figure 3, the color scale is too small to be readable. In the text, you mention lines 423-427 that patterns of relaxed selection are strikingly similar among genes having the same or similar role during tooth development. This statement lacks some explicit description, as a matter of fact, I found that DSPP and MMP20 share a similar pattern, and that EDMA and ODAM also share a similar pattern. You could expand this section to draw conclusions that are more in line with the results.

Discussion: 

Line 442 :I was wondering about the odds to identify shared inactivation mutations across the xenarthrans radiation estimated at 68 million years ago? Intuitively and maybe naively, I would expect that a single ancestral inactivating mutation could be followed by subsequent mutations of different scopes, including larger deletions. The finding that exon 11 of ACPT is deleted in most xenarthran would fit such a scenario.

Line 458 a typo at coeval ? 

In this discussion, I was expecting you to provide some more elements on the limitations of your work and your approach to finding mutations.

Overall your interpretation of the data takes into account the older and newer studies in the field, and they are reasonable given the provided results. I especially appreciated the broader implications regarding gene loss and evolution, their tempo, and the relationship with life traits.

Supplementary data:

I was surprised to find the legends for the supplementary datasets and figures in the supplementary_Tables_S1-S26. I suggest you to add a text file for all supplementary datasets and figures caption, but since it is on the Zenodo page this may be unessential. Dataset S1 does not contain all FASTA alignments, I guess you omitted the range: Dataset S1-S24 !

The main strength of your study is that I find it extremely well performed, and associated with relevant conclusions.

The main weakness is that I could not find in the manuscript the details to ensure that all necessary permits or approvals were obtained for collecting the animals. I acknowledge that this can be difficult for Museum samples, but such details should be available for samples from the Animal Tissue Collection of ISEM, for which you gave voucher information. For these vouchers, I could no find any information on the permits that enabled animal collection, even from the cited references. The reference Gibb et al 2016 mention only two permits for Bradypus torquatus and Tolypeutes tricinctus. Moreover, some samples presented here are not described in Gibb et al 2016 (e.g T-1476, T-1722, T-1631, T-2977, T-JL556). Delsuc et al 2018 refers only to Mylodon darwinii. Whether we like it or not, this is an ethical requirement for a scientific publication. In my quality of reviewer, I should also point out that a Benefit-sharing statement is a condition for publication in numerous journals, such as Molecular Ecology (https://onlinelibrary.wiley.com/page/journal/1365294x/homepage/forauthors.html).