the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 23 Apr 2026
Ground-truthing the application of compound-specific stable isotopes of amino acids to planktic foraminifera tests from Santa Barbara Basin
Abstract. Planktic foraminifera form shells that are preserved in ocean sediments and are used in a variety of paleoproxy and biostratigraphic applications. However, species-specific ecology can complicate the interpretation of planktic foraminifera-based proxies, and limited options exist for examining the ecology of extinct species. Here we apply test-bound compound-specific stable isotopes of amino acids (CSI-AA) to examine the trophic ecology of extant planktic foraminifera. We measure CSI-AA in planktic foraminifera shells collected in sediment traps from the Santa Barbara Basin, CA, specifically the three most abundant species in this region: Globigerina bulloides, Neogloboquadrina incompta, and Turborotalita quinqueloba. The nitrogen CSI-AA of all three species suggest that planktic foraminifera have metazoan-like metabolisms, and that trophic position estimates using CSI-AA are appropriate for planktic foraminifera. All three species had trophic positions near 2 (primary consumer), with no evidence for mixotrophy or photosymbionts. Carbon CSI-AA, in combination with a Bayesian stable isotope mixing model, indicates that the three species occupied separate niches based on diet. Globigerina bulloides fed opportunistically on all groups of phytoplankton available in Santa Barbara Basin, adjusting its diet with seasonal changes in phytoplankton assemblage. Turborotalita quinqueloba specialized in diatoms and heterotrophic bacteria. Neogloboquadrina incompta consumed heterotrophic bacteria and some phytoplankton. Our results align with the current understanding of each species’ ecology while further defining their niches. Our findings suggest that CSI-AA is a promising tool for understanding the trophic ecology of planktic foraminifera, and we make recommendations for future applications of CSI-AA to fossil specimens.
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Status: final response (author comments only)
- RC1: 'Comment on egusphere-2026-2228', Anonymous Referee #1, 24 May 2026
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RC2: 'Comment on egusphere-2026-2228', Anonymous Referee #2, 22 Jun 2026
General comments:
The manuscript entitled “Ground-truthing the application of compound-specific stable isotopes of amino acids to planktic foraminifera tests from Santa Barbara Basin” by Doherty et al. presents a novel and potentially important application of compound-specific isotope analysis of amino acids (CSI-AA) to investigate the trophic ecology of planktonic foraminifera. The study addresses a long-standing question regarding the feeding ecology and trophic position of these organisms, and the dataset is both valuable and difficult to obtain. Overall, the manuscript is clearly written, the analyses are generally well executed, and the discussion is thoughtful and comprehensive. I believe this study has the potential to make a significant contribution to our understanding of planktonic foraminiferal ecology.
However, I have several major concerns regarding the assumptions and implementation of the Bayesian stable isotope mixing model, particularly the selection and treatment of endmembers. Because several key conclusions of the manuscript depend on these analyses, I believe that additional justification, clarification, and in some cases further sensitivity analyses are necessary before the results can be fully evaluated. In particular, the exclusion of zooplankton as potential endmembers requires stronger justification, and I encourage the authors to assess whether the main conclusions remain robust when zooplankton are incorporated into the model framework.
In addition, several methodological details are currently insufficiently explained, making it difficult for readers to assess the robustness of the analyses. Clarifying these points and expanding the discussion of alternative interpretations would substantially strengthen the manuscript. I therefore recommend revisions, and encourage the authors to address the points outlined below.
I am generally supportive of publication after these issues have been satisfactorily addressed.
Major points
1. Structure of the Introduction
The introduction briefly introduces the CSI-AA approach (Lines 55–64), but the explanation remains rather general. Since this method is central to the study, it would be helpful to provide more specific background information, such as the range of trophic position (TP) values that have previously been reported for planktonic foraminifera. This would help readers better understand the significance of the present results.
In contrast, the section from Line 66 onward contains a relatively detailed description of the Santa Barbara Basin (SBB). At this stage, it may be sufficient to provide only the essential rationale for selecting this study site. The more detailed description of the study area could instead be moved to the Materials and Methods section, for example under a subsection such as “Sampling Location” or “Study Area,” where the environmental and oceanographic characteristics can be described more thoroughly.
2. Definition of seasonal groups for G. bulloides
The manuscript defines October–January as “fall/winter” and February–July as “spring/summer.” However, the rationale for this classification is not clear. In oceanographic studies, January–March are often considered winter months, so it seems somewhat unusual that February and March are included in the spring/summer category. If there is a site-specific oceanographic or ecological basis for this seasonal classification in the study area, I encourage the authors to provide an explanation and supporting justification.
3. Bayesian stable isotope mixing model
The Bayesian stable isotope mixing model uses five endmembers: cyanobacteria, diatoms, dinoflagellates, heterotrophic bacteria, and nanoeukaryotes. However, it is unclear why zooplankton groups such as copepods and ciliates were not included as potential endmembers.
At least for G. bulloides, laboratory studies have demonstrated active feeding on Artemia, and N. incompta is also capable of capturing and consuming Artemia under culture conditions. Given these observations, excluding zooplankton a priori appears somewhat arbitrary. If zooplankton were included in the model and subsequently found to contribute negligibly, that would provide support for their exclusion. However, the rationale for omitting them from the outset should be explained.
I therefore encourage the authors to evaluate whether the conclusions remain robust when zooplankton endmembers are incorporated into the model and to present the results of such an analysis.
In addition, Table A1 indicates that multiple taxa are grouped within each endmember category. It is not entirely clear how these taxa are treated in the mixing model. Are contributions first estimated for individual taxa and then aggregated into the five categories presented in the pie charts? Alternatively, Figure A1 appears to suggest that the model is parameterized directly using the five grouped categories.
If the latter is the case, please clarify how variation among taxa within each category is incorporated into the model. Because endmember selection and definition can strongly influence the inferred source contributions, a more detailed explanation of the modeling framework would be valuable.
4. Treatment of Phe in the Δ13C analysis
Related to the previous point, the authors state in the description of Figure 3 that Phe was excluded because the foraminiferal values fell outside the range represented by the endmembers. This seems reasonable.
However, the manuscript should clarify how the Δ13C values were subsequently calculated. Since Δ13C is defined relative to the average value of the essential amino acids, was this average recalculated after excluding Phe? If not, an amino acid that was deemed inappropriate for the analysis would still influence the calculated Δ13C values of the remaining amino acids. Please clarify the procedure used.
More fundamentally, if the Phe values of the foraminifera fall outside the range of all selected endmembers, does this suggest that the current set of endmembers may be incomplete or inappropriate? Please discuss this possibility.
In particular, I would again encourage the authors to present analyses that include zooplankton endmembers and evaluate whether doing so improves the consistency of the model and the placement of Phe within the expected isotopic space.
5. Selection of endmembers and baseline δ13C values
When interpreting trophic relationships using stable isotopes, baseline isotopic values can vary substantially among regions. Therefore, it would be helpful to clarify whether the endmembers used in this study were from environments with comparable isotopic characteristics to the studied region.
The justification for endmember selection should explicitly address this issue. Demonstrating that the chosen endmembers are representative of the isotopic baseline conditions relevant to the study area would strengthen confidence in the interpretation of the mixing-model results.
6. Interpretation of the Bayesian mixing model results
Assuming that the estimated source composition is correct, are the observed δ15N values quantitatively consistent with the inferred diet composition?
Such a validation would provide an important independent test of the model results. If the observed trophic isotope signatures can be predicted from the inferred dietary composition, this would constitute strong support for the overall validity of the analytical framework. I therefore encourage the authors to perform and present such a consistency check.
7. Feeding ecology of G. bulloides
The interpretation of G. bulloides as primarily herbivorous warrants further consideration. For example, Takagi et al. (2025) used metabarcoding to characterize eukaryotic communities associated with planktonic foraminifera. Their results showed that although diverse algal taxa were detected in G. bulloides, algal sequences represented only a very small fraction of the total eukaryotic community, which was dominated by heterotrophic eukaryotes. These organisms are likely to include zooplankton prey. Furthermore, the long spines characteristic of G. bulloides have often been interpreted as structures that facilitate the capture of motile prey. Consistent with this view, Spero and Lea (1996) reported direct observations of feeding in freshly collected specimens:
“Approximately 400 G. bulloides were maintained in the laboratory during the course of this study. Freshly collected specimens were often observed feeding on small zoo- and phytoplankton (e.g. copepods, nauplii or dinoflagellates) that were trapped in the spine-rhizopodial matrix.”
These observations indicate that predation on small zooplankton occurs not only under laboratory conditions but also in natural populations. Given these lines of evidence, I encourage the authors to discuss the possibility that zooplankton may represent an important component of the diet of G. bulloides. This consideration is particularly relevant in light of the mixing-model assumptions discussed above, and it would be valuable to reassess the results with zooplankton included as potential endmembers.
Minor points
Figure 2
The purpose of panel A is not entirely clear. If its inclusion is intended to convey specific information, please clarify in the text and/or caption what readers are expected to learn from this image. In addition, if the photograph is retained, I recommend replacing the mechanical pencil used for scale with a calibrated scale bar, which would provide a more accurate and standard reference for size.
Table 1
The last two rows (the weight-related information) appear to represent components of the total dry weight. Therefore, the table could be improved by formatting these entries in a way that more clearly indicates their nested relationship to the total dry weight.
Figures 5 and 9
If I understand the figures correctly, Figures 5 and 9 appear to present comparable information using three trophic-position estimators (TP-Ala, TP-Glx, and TP-Ala-multi). It may therefore be possible to combine these figures into a single multi-panel figure with the three estimators displayed side by side. Such a presentation could facilitate comparison among the different approaches and improve the overall organization of the results.
Figure A1
According to the figure caption, the dotted line represents the mode of the histogram. However, the indicated position does not appear to coincide with the histogram peak. Please verify that the dotted line is plotted correctly and revise either the figure or caption if necessary.
Citation: https://doi.org/10.5194/egusphere-2026-2228-RC2
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This manuscript demonstrates the compound-specific isotope analysis (CSIA) is a new tool for evaluating the trophic position and trophic niche of planktonic foraminifera species. I expect that the Authors worked hard on the present study, and I recognize that the data reported here are novel and valuable. Although I have no major concern in this manuscript, I feel that its quality could be improved before publication, because several parts of manuscript are insufficiently explained (which are described below).
Concerns:
1. The Authors measured the isotope ratios of amino acids preserved in foraminifera tests, however, this manuscript does not describe the procedure how the tests were purified. I think that the purification procedure should be included in this manuscript. Is there any fractionation of C and N isotopes of amino acids during the purification process?
2. The Authors compared the isotope ratios of Glx and Ala (and TP-Glx and TP-Ala), based on the cap-delta Glu and Ala reported in Décima et al., 2017; Chikaraishi et al., 2009. Although those cap-delta are likely common for major metazoans with trophic levels lower than 3 (because there are many literatures), I doubt whether these cap-delta values can be used generally for major protist species. The generality should be discussed in this manuscript, because it is critical to applying the findings of the present study to future studies.
3. On line 43, the Authors wrote ‘The foraminifera themselves can also assimilate inorganic nitrogen’. Does this mean that the foraminifera can biosynthesize many amino acids including essential amino acids, de novo? If so, how does this biosynthetic ability impact the isotope results of the present study? Please explain what this sentence means.