Raja brachyura , Raja clavata , and Raja montagui

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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: No

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: In general, this is a very strong manuscript. The methods were very rigorous and thoroughly explained so that anyone may recreate this procedure. I have a few minor recommendations that I believe would contextualize the need for age validation in elasmobranchs, but the methods and results are the focus of this study.

Intro: I would like to see more detail in this section on the reasons why age reading success is species/diet/environmentally dependent and what aspects of life history makes it difficult to have a sweeping methodologically across the taxa. Moreover, why is ageing such an important skill to hone for elasmobranchs? Both of these points are addressed in the discussion, but I would like to see some hint of them in the introduction so that I am drawn into the importance of this study.

L44 I think this sentence can be rewritten to be stronger ("fish" is used twice)

L50 give indication of why those structures are used in elasmobranchs (calcified, records age similar to otoliths); move parentheses to end of sentence ("sharks, skates and rays (elasmobranchs)"

L57-8 This sentence feels out of place here wince we don't have context for this particular species, but the inclusion of a specific study and species is something that should be done in the introduction.

L59-61 More details on why the authors couldn't validate

L61 Unclear what the first discrepancy is, other than some papers invalidated vertebral growth

L61-4 This sentence and first half of the second sentence can be combined

L65 Provide examples of environmental factors (temperature? salinity?)

L77 More info on these "other studies" - species? underestimation? etc

Methods: Really strong section, very thorough and clearly explained

L96-7 This sentence should be start of results

L109-111 I think this sentence is missing some punctuation

L111-12 Also put this in results

L150 Eight readers at each institute or across all three?

Results: Thorough. Would benefit from in text reported test statistics and values/means.

L223 "on average" a statistical average? Is it significant?

Table 2 "Modal age range" is slightly confusing. I think it means the age range of the whole species sample as determined by the modal age of each individual across all reads, but it also could be the extent of the range in age read from the readers. Since it's already established that age was determined using mode read, "modal" can be removed from the term. Also, this needs units (years)

L234 "observed" implies the animal was monitored since birth and is thus validated to be 6. "estimated" would be more appropriate, since the true age is never known unless the animal was born and kept in captivity.

Sections 3.2-3.3 These sections are missing reported test statistics (especially p values) and values of what is being compared..

L282 "each both" is redundant

Discussion: Great analysis and inclusion of relevant studies/literature. Overall strong.

L346-7 Start discussion with your findings; are elasmobranchs understudied? That wasn't mentioned in the introduction; I recommend a broader opening paragraph where a recommendation of best method is made, like last sentence of abstract and in the conclusion.

L357-61 Great analysis!

L370 "observed" -> "estimated"

L371 The species doesn't live to 54, individuals in the species can live to 54

L372 "a minimum of 54 years" sounds like all tope sharks must live to 54. Change to "can live to at least"

L379 This sentence says the same thing... why would it say "although" if the age composition and growth curve are both similar in the two species?

L380 Give p value, not "no significant difference"

L391 Sentence end?

L394-5 This is a fairly large sample size for elasmobranchs, but not as much compared to teleosts

399 Start section 4.2 with this study's findings on vertebral structure. The beginning of every section should start with restating the relevant findings from the results (lines 404-408)

409 It's not expected, but it is common

L411 "APE and CV were lower, and in general PA was higher" This sentence also appears in line 222, and I'm confused by it again. It would help to give values with these types of statements such as APE and CV were x% lower and 75% of PA was higher than... In general, it's best to avoid saying "in general" (I couldn't resist)

415 There's an extra "vertebrae" in this sentence

412-29 Really great analysis and discussion here

L437 This is a really great and valuable section, but I would have liked to see it introduced in the introduction so that the reader has an understanding of why it is important to improve age studies in elasmobranchs

Conclusion: Good, but maybe not necessary as it's own section. If the last sentence is removed, it can be added to the discussion. Up to the authors, but I don't think this text needs to be on its own.

L456 Change "the authors suggest" to "the findings suggest" or "the findings point to" so that the suggestion is driven by data, not the authors' interpretation

L462-3 Make into one paragraph?

L465-7 Unnecessary sentence

Reviewer #2: General Comment to Authors

I like the concept of this study and I am always encouraged to read manuscripts seeking to improve the problematic field of aging. However, I have concerns some content, methodological aspects, and elements lacking in the discussion.

I make 5 over arching comments, which are unpacked in more detail in line comments below. I encourage the authors to think about the framing of this study, as there are some really good elements here. I am just not convinced that the method has resulted in a recommendation that is going to lead to the best estimates of true age in future work; only the most statistically constrained estimates in the context of reader agreement, and whether this same result would be achievable in a sample including longer lived species is questionable.

1. The introduction does not do a good job of situating the current ‘best practice’ of preparing and reading band pairs in vertebrae. The convention is to section anterior vertebrae as this generally results in higher age estimations; important for a field prone to systematic under aging through vertebral analysis, and convention since 2004 (Cailliet and Goldman 2004).

2. It is not explained how the authors identified the birth mark between methods.

3. The use of taking the ‘modal age’ in lui of consensus reads among agers is prone to introducing systematic under ageing bias – what if the oldest age estimates were most true of actual age? That consideration cannot be made within the current methodology. The variation on age estimates (fig 4 & 5) is quite large and only emphasizes my concern. The present method selects for comfortable age-bias/precision stats, but does not necessarily ensure that the most true/best/pre cautionary final age estimations have been made in reality.

4. The modeling approach of length at age data is quite antique. Its pretty firmly accepted to use a multi-model approach (not just a priori selection of the von bertalanffy function, Smart et al 2016)), and better yet a Bayesian model where priors of readily available information on size-at-birth/hatching and maximum size can be used to constrain the parameters of L0 and Linf (Smart and Grammar 2021).

5. My final comment extending from 429 argues that this study has selected the best preparation method for vertebrae to improve age bias/precision metrics, not necessarily the method that would produce the most accurate estimation of true age. The concept of counting whole vs section vertebrae is again quite antiquated and really, its the bias in younger age classes in this study which is most likely what has resulted in comparable age estimates between whole and sectioned vertebrae. It’s a dangerous assertion to encourage use of whole vertebrae for skate on the back of this work, as it will not be appropriate for individuals attaining older ages than the present studies sample size. This is known from over 40 years of age and growth research, and goes against best practice guidelines of research in the field that has led to the presently accepted conventions on vertebrae preparation for ageing.

Line Comments

Line 51

Add caudal thorns to this list.

Line 54

Reframe this paragraph; staining is not always used, and to my awareness its probably more common not to stain. There are studies that have discussed the relative null effect of staining, and these should be referred to here. See for example Burke et al. 2020, which subsequently cites some useful papers exploring the use of staining; along with others cited in the present MS. The other key point to note with staining (more for discussion) is that this technique was popular before photographic manipulation was possible. These days, its probably more effective to play around with brightness and contrast of images to achieve the same effect as staining – this is why staining is hardly used now; digital advancements have made it largely superfluous. Along with this is the development of techniques like XRF elemental mapping, where calcium and phosphate (the most abundant elements) can be discreetly viewed along the transect.

Burke, P. J., Raoult, V., Natanson, L. J., Murphy, T. D., Peddemors, V. & Williamson, J. E. (2020). Struggling with age: Common sawsharks (Pristiophorus cirratus) defy age determination using a range of traditional methods. Fisheries Research 231, 105706.

I also think it needs to be clear that vertebrae are most commonly sectioned, as previous work has identified under aging of whole centra;

Discussed a bit in following + other papers already cited in this MS. This introduction doesn’t really set the appropriate context for accepted methodological conventions in the field that are designed to improve readability and minimise under aging. This is important context to this present work – the convention is to section anterior vertebrae; this study largely seeks to validate or invalidate this convention for these species of skate.

Harry, A. V. (2018). Evidence for systemic age underestimation in shark and ray ageing studies. Fish and Fisheries 19, 185-200.

Cortés, E., Grant, M. I., Dureuil, M. & Ellis, J. R. (2024). Life history characteristics. In: R.W. Jabado, A.Z.A. Morata, R. Bennett, B. Finucci, J. Ellis, S. Fowler, M.I. Grant, A.P.B. Martins and S. Sinclair (Eds.), The Global Status of Sharks, Rays, and Chimaeras, pp. chapter 3, 33–38. IUCN/SSC Shark Specialist Group. IUCN, Gland, Switzerland and Cambridge, UK

Goldman, K. J., Cailliet, G. M., Andrews, A. H. & Natanson, L. J. (2012). Assessing the age and growth of chondrichthyan fishes. In Biology of Sharks and Their Relatives (Carrier JC, Musick JA & MR, H., eds.), p. 423. Boca Raton, FL: Taylor & Francis Group.

MacNeil, M. & Campana, S. (2002). Comparison of whole and sectioned vertebrae for determining the age of young blue shark (Prionace glauca). Journal of Northwest Atlantic Fishery Science 30, 77-82.

Line 64

The biochemical structure of vertebrae (phosphate hydroxyapatite [Ca10(PO4)6(OH)2]) is not variable between species. Its density, flexibility are influenced most likely by species morphology and stress forces needed in skeletal structure owing to swimming mode and ecological activity (along the spectrum of sedentary to highly mobile- if a species is swimming a lot, it needs a string skeleton to anchor large muscles to). Here, diet and other environmental factors are referred to, This releates to trace element incorporation in the Ca10(PO4)6(OH)2 matrix or elements entrapped in the protein matrix. These factors would only have minor relevance to structural properties (density, hardness, etc) of vertebrae in the context the authors are talking about here. These needs some revision to better describe structural properties of vertebrae, there composition, and what factors influence composition. Lisa Natanson explored these concepts late in her career;

James, K. C. & Natanson, L. J. (2020). Positional and ontogenetic variation in vertebral centra morphology in five batoid species. Marine and Freshwater Research 72, 887-898.

Natanson, L. J., Skomal, G. B., Hoffmann, S. L., Porter, M. E., Goldman, K. J. & Serra, D. (2018). Age and growth of sharks: do vertebral band pairs record age? Marine and Freshwater Research 69, 1440-1452.

Natanson, L., Andrews, A., Passerotti, M. & Wintner, S. (2018). History and Mystery of Age and Growth Studies in Elasmobranchs: Common Methods and Room for Improvement. In Carrier, J. C., Heithaus, M. R., & Simpfendorfer, C. A. (Eds.). Shark research : Emerging technologies and applications for the field and laboratory, pp. 177-194. Taylor and Francis Group. pp. 178-194.

However, I wonder how relevant this really is to the present paper? It hasn’t explored how varied vertebral structures hold up against vertebral preparation techniques. Better to set the context of conventional methods for vertebral preparation for ageing, and why it is we have arrived at the present convention of sectioning anterior vertebrae, where staining has limited benefit. That’s the state of knowledge this MS needs to build a hypothesis of that would improve the field.

Line 154

Please explain the process of identifying the birthmark for whole and sectioned vertebrae. Its typical to get a translucent band at the birthmark, so did counts begin from the second translucent band in whole vertebrae? Another advantage to sectioned vertebrae is that the birth mark is more easily identified, as in addition to a thin translucent band that is typically (but not always) laid, is that there will usually be an acute angle change in the intermedalia at the birth mark, and sometimes a visible ‘notch’. These characters cannot be identified in whole vertebrae, so how was the birth mark identified? Was the accuracy of its identification corroborated with sectioned vertebrae? One of the major error sources of age estimation is mis identification of the birth mark, and this study has not explained how it was identified, and what constituted a growth band there after. It just says growth bands were counted.

Line 187

It’s a bit of an old fashioned parametrization of the growth model used here. The parametrization that includes L0 is more useful, as L0 serves as a proxy for size at birth, with t0 has no biological meaning. The use of the parametrization with L0 also allows Bayesian A&G models to be used, and L0 and Linf can more easly have informed priors, based on knowledge of birth size and maximum size. Its probably not possible to accurate inform a prior of t0. Its not essential, but I would be highly encouraging of considering use of a more contemporary approach to modelling the length at age data

Please see

Smart, J. J., Chin, A., Tobin, A. J. & Simpfendorfer, C. A. (2016). Multimodel approaches in shark and ray growth studies: strengths, weaknesses and the future. Fish and Fisheries 17, 955-971.

Smart, J. J. & Grammer, G. L. (2021). Modernising fish and shark growth curves with Bayesian length-at-age models. PLoS One 16, e0246734. (includes very intuitive R package)

This section also needs to be clear that the a priori selected model is the von bertalanffy growth function. Again, see Smart et al 2016 above, as this strays from contemporary approaches to modelling length at age data whereby a multimodel approach is encouraged as best practice.

Line 429 and then general discussion remarks

Yes, but only for the young spectrum of age estimates encountered in this study. This discussion is crying out for explicit mention that a key constraint here is the young ages encountered <8. The key advantage of sectioning comes when highly compressed ‘older age bands’ need to be examined, when animals have 20, 30, 40 band pairs to count. It is not really all that surprising that whole vertebrae performed equal, or slightly better than section vertebrae I some instances for these young age classes where growth bands are inherently larger and more easy to count – the need for sectioning was not born out of difficulties counting bands in younger age classes, it was from difficulties encountered in counting bands in older age classes, which are not included in this study. Further, is there any insight to offer in where the larger age discrepancies in sectioned vertebrae arose? Was it birth mark, distal edge? That would be useful information; identifying vertebral zones leading to mis counts among readers.

The other factor here, is the assumption is made that band pairs equate to annual growth increments. There was more variability in the sectioned vertebrae, but how do you know that those age estimates were not better than whole vertebrae? By not sectioning, smaller more indiscreet bands are hard to identify, and may have been missed. Our built ‘best practice’ of ageing calls for sectioning for this reason (e.g., Cailliet and Goldmans catelouge of papers on this topic; built from over 30 years of examining ageing studies and trailing methods; along with prior mentioned better ability to identify the birth mark; Cailliet and Goldman 2004). What this study has done is identify the best preparation method of vertebrae from these species of skate, in these ‘age’ classes, that allows a wide range of readers to most commonly agree on their ‘age estimates’ and thereby produce the best age bias/precision stats. Whether underaging has occurred is an un answered question. An interesting thing here, and counter to most literature, is that higher age estimates were made for R. brachyura with whole vertebrae, but not for the other two species. Why do you think this is? Have whole vertebrae resulted in better age-bias stats because ambiguous bands, or smaller bands are less clear in whole vertebrae compared to section vertebrae so that there is effectively less source for error ? – have whole vertebrae potentially missed some smaller bands that could well represent annual depositions under the assumption band pairs are accreted annually for these species? If there are less banding structures to count, age-bias stats are inherently going to be lower

Line 237 states that sectioned vertebrae had higher ‘modal’ age estimates for R. clavate and R. montagui, but, these precision and bias stats were higher. Given that aging is about producing estimates that most reflect true age of the animal so that those ages can be fed into management initiatives, is it better to have higher more precautious ages with slightly worse age bias/precision stats, or lower more conservative ages with neater age bias/precision stats? Its an partly uncomfortable question that this study has raised, and it needs to be addressed. The goal of aging is not low APEs, its proving the best advice to management of growth of the species, and given we know underestimation occurs from vertebral ageing, it seems to make sense to use the precautionary principle and err toward older estimates, even if it means lower agreement among readers. Better to over age than under age.

Furthermore, the concept of selecting the ‘modal age’ is not something I entirely agree with – how to you know that the oldest age estimate wasn’t the more accurate to true age and that the modal age selection is not a form of methodological bias leading to systematic age underestimation? The variation on age estimates (fig 4 & 5) is quite large and only emphasizes my concern. Where any consensus reads done? (if not, again straying from conventional methodology), particularly with the ager who made the oldest age estimation for each individual so that they could make their case as to their decision to count the band structures they could identify? In a field where underage estimation is an issue, again, it’s a backward facing decision in respect to the history of this problematic field. The present method selects for comfortable age-bias/precision stats, but does not necessarily ensure that the most true/best/pre cautionary final age estimations have been made in reality. As such, the title is not entirely appropriate; it should be ‘Vertebral preparation to improve band pair identification agreement among readers’ or something to that effect.

These technicalities need some discussion. The field of ageing with vertebrae is plagued with inferences, and sometimes proven examples of systematic underestimation of age (e.g., the MRC study referred to for Tope, theres others for white sharks, where if we believed whole vertebrae estimates they’d have a max age of about 20, not 70+). My over arching point is that whole vertebrae may have resulted in clean APE’s and CV’s, but have they resulted in more accurate estimations of the age of these individuals – this is a separate point to the number of readers that agreed on an age, as that will err toward conservative counts. This discussion needs to consider these aspects – do whole vertebrae improve age estimates or just age precision metrics among readers? My view is that the latter is probably true and this discussion needs to be framed to answering this question as it’s the over arching unknown to come out of this study.

Some good well presented and discussed points are that anterior vertebrae resulted in higher age estimates generally, and were ‘more readable’ and that staining had very limited effect. This is corroborated in the literature, and this study adds to the evidence base of these factors. I have no issues with the PCA parts of this study, though I wonder if there is really enough latitudinal and longitudinal scope to provide robust insight. I’d also be interested to see the effects of reader experience on ages

Figure 2, please mark what was identified as the birth mark on this figure, and it would be useful to mark the identified band pairs counted also (if possible considering the modal age method). These images lack meaning otherwise for the reader

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Raja brachyura , Raja clavata , and Raja montagui

Dear Dr. Greenway,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

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

Reviewer #2: Yes

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

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: (No Response)

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Reviewer #2: I thank the authors for their response to my comments and improvements made to the manuscript. At heart, I do not agree with the modal age approach, but I am satisfied that the first paragraph of the discussion outlines the key constraint for the reader, and the purpose of its use herein. There is scope to justify the reasoning for this approach in the methods additionally, as it is a foundational decision made in the conduct of the methodology of this study, and an action that influences the results and their interpretation (see below comment for a suggestion, as you’ve actually provided a really good couple of sentences in a reply to a comment).

Considering this text now added to the start of the discussion, and reply to my comment 3., I wonder whether the title is entirely appropriate. The final sentence of the first discussion paragraph for example is correct, and also implies the title is misleading

“Therefore, our finding that anterior, whole, unstained vertebrae provided the highest reader precision and agreement should be interpreted within this context”

A more appropriate title as such would be a synonym of:

A comparison of vertebral preparation techniques to increase reader precision and agreement in vertebral band pair identification in northwest Atlantic skates.

This is what the study has done. I recommend saving the ‘age estimation’ for the follow up manuscript that you mention

A few responses to previous comment reply’s and just a few more line comments below. Well done to the authors, this was quite a large review and I appreciate the time in constructing considered responses.

Reply to comment 3.

The statement ‘modal age remains a well-established and widely accepted practice in age and growth validation studies’ is something I just do not consider to be true. Noting the authors list of referred to papers (Campana 2001; Goldman 2005; Vitale et al., 2021; ICES, 2022) in their response to comment 3., Campana 2001 for example speaks to ‘modal progression’ which is in the context of length class modes corresponding to ages, and as such, whether length modes in an annual breeding population can be used to estimate age from length; not taking the mode of age estimates across a range of readers who have examined banding patterns in calcified hard parts to determine age. I haven’t had much luck locating others cited, but ensure there is no confusion in ‘modal progression’ which is different to the present method. Regardless, better to justify the use of using modal ages to the readership more so than me. I like the justification provided in the first paragraph in the authors reply to the original comment 3., and would encourage its inclusion within the paragraph ending at line 194

‘…. modal age can potentially introduce a systematic underageing bias, particularly if the highest (oldest) age estimates more closely approximate true age. However, our primary objective was not to determine absolute accuracy but rather to assess how different preparation methods influence the readability and consistency of vertebral age estimates across multiple readers. The modal age approach enabled us to quantify reader consensus and exclude indeterminate cases (i.e., instances with no clear mode), thereby focusing the analysis on relative precision rather than absolute accuracy.’

Reply to comment 4.

This part of your response should be clearly stated in the methods speaking about which model was used

‘growth modelling in this paper was conducted exclusively to test whether the two types of

vertebrae provided the same results in terms of growth curves. For this reason, we decided to rely

only on the von Bertalanffy function, as it is more easily comparable with the existing literature.’

Its good to hear a robust age and growth study on the way. A point of caution is that the below part of the response is fundamentally incorrect; its all about fitting the best model to the length-at-age data at hand to produce the best fit for your data. Not exploring the best model for the data used in caution of setting a precedent does not make sense, and I don’t understand what is meant. Perhaps you are referring to the underlying sample size considering the follow up paper you mention. In anycase, the intention of a multimodel approach is not to one day revert back to a priori selection of a single model, just want to make sure that is clear.

“Moreover, considering the small sample size, we were concerned that applying a multi-model comparison could introduce a potential bias by indicating one model as “better” without a solid basis,”

This response has not addressed the use of t0 – a biologically meaningless parameter that can very easily be recalculated to L0 from Linf and K:

L0 = L∞(1− exp(k*t0))

Line 40

Would be good to add the age range here, as per previous review these results may differ for older animals “…were more precise for age classes … , offering..”

Line 117

This sentence is incomplete. State ‘draw conclusion on the most effective vertebrae preparation methods for the Raja genus’ or something similar

Line 240-44

I wonder if a measure of the number of published age and growth papers each reader has been an author on, an/or led or been senior author on, and/or been an ager in, and/or number of sp. aged would be better metrics? I for example in my first age and growth paper read about 1500 vertebrae of a single species in two populations before proceeding with age estimates for that paper (n>500). That means under the present definition I would have medium experience despite at that point, only having aged one species and not having had an accepted publication yet; I’d consider my experience low at that point. Something to consider. I understand the present system captures the spectrum of present experience, but it doesn’t confer much to actual robust contribution to the field as a measure of expertise.

Line 443

‘k’ in VBGF is not a growth rate but a coefficient of the curvature of the function that describes a proxy for the ‘growth completion rate’ determined from the curveature between L0 and Linf. Easiest to replace ‘growth rate’ with ‘k’ than to describe the nuances of this. It can be paired with statements like ‘higher k values indicates faster growth’. There are equations for calculating growth rate from VBGF parameters if wanting to pursue that, but I don’t see it as needed. It’s a common mistake in age and growth literature. In 4.1 discussion, and elsewhere please adjust use of 'growth rate' as a direct infernece from k, or substitute of using k.

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