Extant cartilaginous fishes share trabecular and areolar mineralization patterns, but not tesserae, and evidence for a paedomorphic chimaera skeleton

Reviewer #1 (Public Review):

Summary:

It seems as if the main point of the paper is about the new data related to rat fish although your title is describing it as extant cartilaginous fishes and you bounce around between the little skate and ratfish. So here's an opportunity for you to adjust the title to emphasize ratfish is given the fact that leader you describe how this is your significant new data contribution. Either way, the organization of the paper can be adjusted so that the reader can follow along the same order for all sections so that it's very clear for comparative purposes of new data and what they mean. My opinion is that I want to read, for each subheading in the results, about the the ratfish first because this is your most interesting novel data. Then I want to know any confirmation about morphology in little skate. And then I want to know about any gaps you fill with the cat shark. (It is ok if you keep the order of "skate, ratfish, then shark, but I think it undersells the new data).

Strengths:

The imagery and new data availability for ratfish are valuable and may help to determine new phylogenetically informative characters for understanding the evolution of cartilaginous fishes. You also allude to the fossil record.

Opportunities:

I am concerned about the statement of ratfish paedomorphism because stage 32 and 33 were not statistically significantly different from one another (figure and prior sentences). So, these ratfish TMDs overlap the range of both 32 and 33. I think you need more specimens and stages to state this definitely based on TMD. What else leads you to think these are paedomorphic? Right now they are different, but it's unclear why. You need more outgroups.

Your headings for the results subsection and figures are nice snapshots of your interpretations of the results and I think they would be better repurposed in your abstract, which needs more depth.

Historical literature is more abundant than what you've listed. Your first sentence describes a long fascination and only goes back to 1990. But there are authors that have had this fascination for centuries and so I think you'll benefit from looking back. Especially because several of them have looked into histology and development of these fishes.

I agree that in the past 15 years or so a lot more work has been done because it can be done using newer technologies and I don't think your list is exhaustive. You need to expand this list and history which will help with your ultimate comparative analysis without you needed to sample too many new data yourself.

I'd like to see modifications to figure 7 so that you can add more continuity between the characters, illustrated in figure 7 and the body of the text. Generally Holocephalans are the outgroup to elasmobranchs - right now they are presented as sister taxa with no ability to indicate derivation. Why isn't the catshark included in this diagram?

In the last paragraph of the introduction, you say that "the data argue" and I admit, I am confused. Whose data? Is this a prediction or results or summary of other people's work? Either way, could be clarified to emphasize the contribution you are about to present.

Reviewer #2 (Public Review):

General comment:

This is a very valuable and unique comparative study. An excellent combination of scanning and histological data from three different species is presented. Obtaining the material for such a comparative study is never trivial. The study presents new data and thus provides the basis for an in-depth discussion about chondrichthyan mineralised skeletal tissues. I have, however, some comments. Some information is lacking and should be added to the manuscript text. I also suggest changes in the result and the discussion section of the manuscript.

Introduction:

The reader gets the impression almost no research on chondrichthyan skeletal tissues was done before the 2010 ("last 15 years", L45). I suggest to correct that and to cite also previous studies on chondrichthyan skeletal tissues, this includes studies from before 1900.

Material and Methods:

Please complete L473-492: Three different Micro-CT scanners were used for three different species? ScyScan 117 for the skate samples. Catshark different scanner, please provide full details. Chimera Scncrotron Scan? Please provide full details for all scanning protocols.

TMD is established in the same way in all three scanners? Actually not possible. Or, all specimens were scanned with the same scanner to establish TMD? If so please provide the protocol.

Please complete L494 ff: Tissue embedding medium and embedding protocol is missing. Specimens have been decalcified, if yes how? Have specimens been sectioned non-decalcified or decalcified?

Please complete L506 ff: Tissue embedding medium and embedding protocol is missing. Description of controls are missing.

Results:

L147: It is valuable and interesting to compare the degree of mineralisation in individuals from the three different species. It appears, however, not possible to provide numerical data for Tissue Mineral Density (TMD). First requirement, all specimens must be scanned with the same scanner and the same calibration values. This in not stated in the M&M section. But even if this was the case, all specimens derive from different sample locations and have, been preserved differently. Type of fixation, extension of fixation time in formalin, frozen, unfrozen, conditions of sample storage, age of the samples, and many more parameters, all influence TMD values. Likewise the relative age of the animals (adult is not the same as adult) influences TMD. One must assume different sampling and storage conditions and different types of progression into adulthood. Thus, the observation of different degrees of mineralisation is very interesting but I suggest not to link this observation to numerical values.

Parts of the results are mixed with discussion. Sometimes, a result chapter also needs a few references but this result chapter is full of references.

Based on different protocols, the staining characteristics of the tissue are analysed. This is very good and provides valuable additional data. The authors should inform the not only about the staining (positive of negative) abut also about the histochemical characters of the staining. L218: "fast green positive" means what? L234: "marked by Trichrome acid fuchsin" means what? And so on, see also L237, L289, L291
Discussion

Please completely remove figure 7, please adjust and severely downsize the discussion related to figure 7. It is very interesting and valuable to compare three species from three different groups of elasmobranchs. Results of this comparison also validate an interesting discussion about possible phylogenetic aspects. This is, however, not the basis for claims about the skeletal tissue organisation of all extinct and extant members of the groups to which the three species belong. The discussion refers to "selected representatives" (L364), but how representative are the selected species? Can there be a extant species that represents the entire large group, all sharks, rays or chimeras? Are the three selected species basal representatives with a generalist life style?

Please completely remove the discussion about paedomorphosis in chimeras (already in the result section). This discussion is based on a wrong idea about the definition of paedomorphosis. Paedomorphosis can occur in members of the same group. Humans have paedormorphic characters within the primates, Ambystoma mexicanum is paedormorphic within the urodeals. Paedomorphosis does not extend to members of different vertebrate branches. That elasmobranchs have a developmental stage that resembles chimera vertebra mineralisation does not define chimera vertebra centra as paedomorphic. Teleost have a herocercal caudal fin anlage during development, that does not mean the heterocercal fins in sturgeons or elasmobranchs are paedomorphic characters.

L432-435: In times of Gadow & Abott (1895) science had completely wrong ideas bout the phylogenic position of chondrichthyans within the gnathostomes. It is curious that Gadow & Abott (1895) are being cited in support of the paedomorphosis claim.

The SCPP part of the discussion is unrelated to the data obtained by this study. Kawaki & WEISS (2003) describe a gene family (called SCPP) that control Ca-binding extracellular phosphoproteins in enamel, in bone and dentine, in saliva and in milk. It evolved by gene duplication and differentiation. They date it back to a first enamel matrix protein in conodonts (Reif 2006). Conodonts, a group of enigmatic invertebrates have mineralised structures but these structure are neither bone nor mineralised cartilage. Cat fish (6 % of all vertebrate species) on the other hand, have bone but do not have SCPP genes (Lui et al. 206). Other calcium binding proteins, such as osteocalcin, were initially believed to be required for mineralisation. It turned out that osteocalcin is rather a mineralisation inhibitor, at best it regulates the arrangement collagen fiber bundles. The osteocalcin -/- mouse has fully mineralised bone. As the function of the SCPP gene product for bone formation is unknown, there is no need to discuss SCPP genes. It would perhaps be better to finish the manuscript with summery that focuses on the subject and the methodology of this nice study.