Investigating the Relationship between Growth Rate, Shell Morphology, and Trace Element Composition of the Pacific Littleneck Clam (Leukoma staminea): Implications for Paleoclimate Reconstructions
Round 1
Reviewer 1 Report
The work of Kempf et al represents an investigation of microstructural variability in relation to elemental composition in a cohort of Pacific littleneck clams (Leukoma staminea) cultured from shared broodstock. Despite being a very common species, it has only been sparsely been examined for sclerochronological utility. The authors used a microprobe approach to measure Sr, Mg and S variability in the shells of a selection of juvenile individuals and their parent clams. The use of microprobe analysis is as common as LA-ICPMS approaches these days, but provides an extremely detailed view of elemental variability in the shell. The authors also measured sulfur variability in the shell, which is under-investigated, with very few published measurements from any previous bivalve sclerochronological study.
For this reason, this work is of note and provides useful data and interpretation of elemental variability in future sclerochronological studies across Veneridae. However, improvements could be made to their quantitative analysis of the microprobe data, to provide more certain estimates of spatial variability of element counts. These would not involve new geochemical measurements, but would provide more precise statistical analysis of elemental results. I go into more detail about this suggestion and other suggested changes to the manuscript on a line by line basis below.
74: I would say that shells do not only record chemical properties of seawater in theory. This has been conclusively demonstrated, but as you mention at later, there are lots of exceptions to the general rule that require caution
80: There is a literature on juvenile TE values and their lower consistency within and between individuals which the authors could draw on. Juveniles of Glycymeridae (https://doi.org/10.1016/j.chemgeo.2019.01.008), Tridacnidae (http://dx.doi.org/10.1016/j.palaeo.2016.03.019) and other aragonitic taxa also show a reduced periodicity in the juvenile interval. These dynamics may be more common in bivalves than not, and could relate to the prioritization of accelerated growth above all other circadian rhythms during the juvenile stage.
176: Unclear what this sentence means, would be best to rephrase to have clearer diction. I think you are referring to the fact that the pore water geochemistry differs from the water column's?
227: In this sentence, are you saying you selected SEM sites to be the mirror image of the EMP analyses, e.g., the other side of the cut section?
Figure 3: in B and E, it appears that a polynomial or exponential fit would be better to the data rather than a linear fit. This would make more sense as the volume and associated tissue mass would be expected to increase nonlinearly as the length of the bivalves increased.
286: I am unclear on what this sentence means. Are you saying that adjacent growth lines often blur into each other? Or specifically the cessation lines and adjacent increments (the dark part and the light part)?
312: I understand the microprobe approach is different from LA-ICPMS in providing more qualitative results. But it would still be much more helpful to get more definitive descriptive statistics regarding the distributions of values in the different shell regions you describe. Are the data given back to you as rasters/TIFFs? If so, could you landmark them, set a scale, then import them into qGIS to use a spatial approach to draw outlines around the areas of interest, find the distribution of counts in the oOSL, iOSL, ISL, growth lines, etc? Those outlines could be saved as shapefiles. Then the shapefiles could be used to "cookie cutter" out the areas of interest from the data rasters and see the distribution of values present. This can be done in qGIS, but also R via the packages raster and sf. Those values could be presented as means/SDs or boxplots, and would be very useful for future studies attempting to compare their measurements to your results, since any El/Ca measurements would be expected to covary closely with the count data you present. At the very least, if your raw files don't appear as rasters, more formalized statistics would be much appreciated here rather than "~100 counts" or "~15 counts".
327: Does the medium juvenile have any differences in microstructure, growth patterns that could explain this lack of variability?
359: Could there be any sources of energetic stress to the clams during the culture interval, which would reduce their energy budget, reduce growth and reduce their ability to discriminate against substitutive ions during the calcification process?
570: please superscript all the 18s in δ¹⁸O
Author Response
Please see attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This is an interesting ms in that it would be very good to develop an understanding of a different bivalve to Arctica islandica (see aim expressed in Line 162)– though in fairness large amounts of research has also been done of Glycymeris and Panopea also. It is important to note that Leukoma is different to those taxa in respect to its life habit (shallow burrowing intertidal- which limits its use though assuredly interesting in terms of archaeology/human harvesting patterns) and lives for only a few years rather than centuries. The conclusion seems to be that this is not a taxon suitable for sclerochronology because of the very odd and inconsistent patters of bands in the juvenile animals – but perhaps this needs further investigation and more details of their husbandry revealed. It is certainly very useful to have trace element distributions in different parts of the shell and different microstructures as this has been a rather neglected issue which has only recently been highlighted.
I have some comments which might prove useful:
Taxonomy and phylogeny
It is important to stress how this clam relates to the others which are mentioned in various parts of the ms.
Line 46 – there seems to be some confusion of its name. It should read Leukoma staminea (Conrad, 1837). There is an extraneous pair of brackets. It would be sensible to say that it is a member of the Veneridae from the start.
Line 100 – closely related to what? In fact Saxidomus, Leukoma and Mercenaria are all venerids.
Line 102 – ‘clam’ is a very colloquial term. In fact because this ms specifically sets out to consider if there is any phylogenetic component to the patterns, it should be noted here that Pinna nobilis is very unrelated to the study taxon, being in the Pteriomorphia. Arctica gets mentioned a lot but the fact that it is not closely related only comes in very late in the ms
Collection details
How were the specimens actually? Are these intertidal? None of this is clear. Line 11 says that the individuals are all part of a ‘genetic cohort’ though there is no evidence presented that this is true given that the broodstock was a number of individuals and these were not screened genetically. It is rather important that we are told the tank conditions, what they were fed on etc, were tidal patterns simulated (if they are intertidal animals)?
Microstructural problems
Line 126 – ‘between 0.03 and 0.5 wt %’ is an odd statement. I don’t think that this is what the authors mean. It is usually noted that the maximum is 5% organics though this is not true, figures of up to 12% are known.
I have not had time to look at the literature of Prototharca staminea (as it used to be called) but I wonder if all previous studies really did say it had just two layers – it seems to have a very common venerid microstructure to me. Only one of the previous studies is referenced and this is clearly in error as it refers to nacre in the inner part of the shell and so is not a reliable source! It would be more sensible to make comparisons with other venerids than to Arctica , which as they note is in a different family (and indeed not very closely related). In fact apart from vague numbers of layers the microstructure of Leukoma is very different to Arctica.
Line 266 - ‘from the anterior margin to the ventral margin’ – I don’t really understand what that means in bivalve terminology.
Are you sure about the microstructure? Fig 4 is quite poor in terms of the quality of the SEM – also the caption suggests that there should be labels which appear to be missing. Are you sure that the orientations are right – the oOSL almost looks as though it is at right angles to what it should be? The prismatic layer in the ISL looks like myostracum to me. Similarly, Figure 6 are you absolutely sure that prismatic layer is not a myostracal sheet (which are common in venerids)? What is the strange layer above which looks to have etched back but is structureless? The possibility that some of the prismatic layers are myostracum rather than growth lines matters as the former are laid down by muscles and not the standard mantle processes – they might very well have very interesting geochemistries.
The growth lines in the juvenile clams studied here are rather perplexing– the variation in number 90 with a standard deviation of +/- 46 seems massive in an apparently closely related cohort held in identical conditions. Certainly I agree with the conclusion that this taxon looks inappropriate for sclerochronology – though presumably the annual bands may be better.
Other points
Line 120 – I wonder if ‘prepped’ is really the right term – ‘prepared’ would be more typical
Figure 1 – make the ‘red dots’ clearer for annual growth bands. I wonder if arrows might work better?
Line 175 – something wrong in this sentence – perhaps ‘water’ rather than ‘was’?
Line 221 – the need to view the sections in transmitted light is not clear (I suspect to check for the quality of the polish – in which case this might be omitted!)
Line 255 – extraneous symbol?
Line 266 – I am not at all sure what ‘terminal accretion’ means.
Line 294 – why just palaeontologists?
References – I do not know the specifics of the journal reference style. It is unusual if it has the use of upper cases in titles as it does at present, but regardless of that species names should start with a lower case (e.g. Saxidomus gigantea in Line 840, though they are all wrong).
Author Response
Please see attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
This is very well written paper on the relationship between growth rate, shell morphology and trace element composition in Leukoma staminae.
Authors have hypothesized that slower shell growth would correspond to reduced incorporation of trace/minor elements (Sr, Mg and S) in the aragonite lattice, as has been documented in other biomineralizing marine invertebrates. They found higher levels of S, Sr and Mg in the dark, slower-growing growth lines compared to the light, faster-growing increments, particularly in the inner shell layer, thus refuting their hypothesis. Authors also found that young L. staminea shells are likely not useful as archives of (paleo)environmental conditions, adult L. staminea shells are likely suitable if micrometer-scale variability in shell structure and chemistry is accounted for. This paper can be published in its current form.
Best wishes,
Reviewer
Correction needed:
(44) Schöne, B. R.; Dunca, E.; Fiebig, J.; Pfeiffer, M. Mutvei’s Solution: An Ideal Agent for Resolving Microgrowth Structures of Biogenic Carbonates. Look. Back Skelet. Diaries - High-Resolut. Environ. Reconstr. Accretionary Hard Parts Aquat. Org. 2005, 228 766 (1), 149–166. https://doi.org/10.1016/j.palaeo.2005.03.054.
The journal name is wrong, it is in "Palaeogeography, Palaeoclimatology; Palaeoecology"!
Author Response
(44) Schöne, B. R.; Dunca, E.; Fiebig, J.; Pfeiffer, M. Mutvei’s Solution: An Ideal Agent for Resolving Microgrowth Structures of Biogenic Carbonates. Look. Back Skelet. Diaries - High-Resolut. Environ. Reconstr. Accretionary Hard Parts Aquat. Org. 2005, 228 766 (1), 149–166. https://doi.org/10.1016/j.palaeo.2005.03.054.
The journal name is wrong, it is in "Palaeogeography, Palaeoclimatology; Palaeoecology"!
We have fixed this in the references.
