the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 22 Jan 2026
Experimental assessment of benthic foraminifera as salinity bioindicators: Integrating morphological and eDNA approaches
Abstract. Benthic foraminifera are extensively used as bioindicators for paleoenvironmental reconstruction, yet environmental DNA (eDNA) analysis provides a powerful lens to uncover their community diversity and environmental responses. Currently, quantitative experimental studies on their responses to salinity gradients are scarce. Here, sediments from the intertidal zone of Qingdao Bay were subjected to a 10-week controlled culture across 13 salinity levels (0–60 psu), and community dynamics were analysed using both morphological and eDNA approaches. Foraminifera exhibited high tolerance to extreme low (0 psu) and high (60 psu) salinities, and community diversity (Margalef index) increased significantly with salinity (p < 0.05). The relative abundance of calcified Rotaliida declined with increasing salinity (p < 0.01), allowing for the establishment of a robust linear regression model. The eDNA analysis revealed that soft-bodied Monothalamiids – often overlooked morphologically – reached up to 76.2 % relative abundance (average 56.1 %). In contrast, the salinity-driven increase in high-Mg calcite-shelled Milioliida was observed only through morphological analysis. These results demonstrate the distinct but complementary nature of morphological and molecular methods. This study addresses the scarcity of experimental constraints on salinity responses, offering a calibrated reference for applying foraminifera in both ecological assessment and paleo-reconstruction.
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Status: open (until 05 Mar 2026)
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RC1: 'Comment on egusphere-2026-196', Anonymous Referee #1, 31 Jan 2026
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AC1: 'Reply on RC1', Yanli Lei, 09 Feb 2026
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We appreciate the reviewer’s time and their insightful assessment of this study. Their professional suggestions have been invaluable in improving the quality of our manuscript. Our responses to the specific points are provided below.
- We sincerely thank the reviewer for raising this critical point regarding taxonomic assignment and validation.
1.1 Taxonomic Classification Methodology:
In the original manuscript, we omitted the detailed parameters for taxonomic annotation. We have now clarified this in the revised Methods section. Specifically, after quality filtering, OTUs with fewer than 10 reads or present in fewer than 3 samples were removed to minimize artifacts. The representative sequences of the remaining OTUs were taxonomically annotated using BLASTn (version 2.7.1) against the Protist Ribosomal Reference database (PR2) (Guillou et al., 2012). OTUs identified as Foraminifera but unable to be assigned to specific major orders were categorized as "Others".
1.2. Phylogenetic Analysis and Micro-diversity:
We fully agree that a phylogenetic analysis provides essential proof of the taxonomic affiliations.
Since our dataset contains nearly 2,000 OTUs, constructing a single tree with all sequences would result in visual clutter and make interpretation difficult. Therefore, to verify the classification and visualize micro-diversity as requested, we selected the top 50 most abundant OTUs to represent the community structure (comprising 20 Rotaliida, 20 Monothalamids, 5 Textulariida, and 5 Milioliida). We constructed a Maximum Likelihood tree (Supplementary Fig. S1) using these representative OTUs alongside corresponding reference sequences extracted from the PR2 database, rooted with Gromia oviformis as the outgroup.
The resulting tree confirms that our OTUs cluster consistently with known foraminiferal lineages, validating our taxonomic assignments. Furthermore, it reveals high micro-diversity within dominant groups, particularly in the Ammonia (Rotaliida) and Ovammia (Monothalamids) clades.
- Thank you for your suggestions. We will add detailed morphological and eDNA data to the supplementary materials of the manuscript and include the numerical values for total abundance at the top of the bar chart in Figure 3A.
The responses to the specific comments are as follows:
line 58 (and line 75-76): Yes, it should be phrased as "13 different salinity levels." Thank you for pointing this out.
line 84: We used the Guillard f/2 medium for algal culture, and this will be added to the manuscript.
lines 108-110: In this study, the amplified target fragment was 380 bp in length. Paired-end sequencing with 250 bp reads was employed, resulting in a potential overlapping region of approximately 120 bp between forward and reverse reads. Thank you for this thoughtful suggestion. These technical details will be incorporated into the main text.
lines 111-114: OTUs with fewer than 10 reads or appearing in fewer than 3 samples were removed to eliminate potential contaminants. Representative sequences were extracted from the OTU table and taxonomically annotated against the PR2 database (Protist Ribosomal Reference Database, Guillou et al., 2012) using BLAST (version 2.7.1). OTUs not classified under Foraminifera (rank 3 in the PR2 database) were subsequently discarded. We will also include the detailed workflow in the corresponding section of the manuscript.
lines 115-116: We appreciate your feedback. The original description was indeed too brief, and we have revised the relevant section in the manuscript to provide a more detailed explanation. “The OTU table was used to quantify the species number and relative abundance of each foraminifera taxon under different salinity conditions, as well as the alpha diversity indices of the community. Spearman correlation analysis between each taxon (as well as community diversity) and salinity was performed using SPSS software. For data showing significant correlations, figures were generated in Origin with linear fitting equations, plotted using a 95% confidence interval.”
Figure 2: Yes, the samples were fixed and stained with Rose Bengal dye to differentiate living organisms. As mentioned in Section 2.2 (Materials and Methods), we have incorporated this point into the figure caption based on your suggestion to enhance reader comprehension.
Figure 5: Thank you for the opportunity to clarify Figure 5. We have revised the figure legend to address your questions directly.
The chord diagram visually compares the foraminiferal community composition revealed by eDNA (Molecular) versus morphological methods. Each outer semicircle represents a method’s dataset, independently normalized to 100% to show its internal taxonomic proportions. The inner colored arcs represent taxa; their width equals the sum of that taxon’s relative abundance from both methods, indicating its overall detection strength across the study. The connecting ribbons show how each taxon’s abundance is partitioned between the two methods—ribbon width at the connection point equals its relative abundance within that method.
You correctly interpreted the key patterns: eDNA was dominated by Monothalamiids, Rotaliida, and “Others,” with minimal signal from Textulariida and Milioliida, which were primarily detected morphologically. A wide ribbon connecting to a large outer segment (e.g., Rotaliida to Morphology) confirms that taxon as a major component of that method’s community.
line 171: In this study, OTUs annotated as Foraminifera yet unassigned at the Order level or lower were collectively categorized as "Others." As for the detailed explanation regarding the phylogenetic tree, it has been addressed in the response to the first point; please refer to that section.
line 204: Thank you for pointing out the missing reference. We have now added the appropriate citation to the manuscript.
In environments with low salt concentrations, there is a demonstrable decline in the feeding and metabolic efficiency of foraminifera (Nigam et al., 2006; Sujata et al., 2011). Conversely, the collective of foraminifera exhibits a higher degree of tolerance to physiological stress induced by elevated levels of salinity (Debenay, 1990; Gull et al., 2025).
Debenay, J.-P.: Recent foraminiferal assemblages and their distribution relative to environmental stress in the paralic environments of West Africa (Cape Timiris to Ebrie Lagoon), J. Foraminifer. Res., 20(3):267–282, https://doi.org/10.2113/gsjfr.20.3.267, 1990.
Gull, H. M., Tawabini, B. S., Amao, A. O., Prayudi, S. D., Ayranci, K., and Kaminski, M. A.: Benthic Foraminiferal Response to a Salinity Gradient in the Uqair Hypersaline Lagoonal System, Gulf Coast of Saudi Arabia, Micropaleontology, 71(3):261–279, https://doi.org/10.47894/mpal.71.3.04, 2025.
Nigam, R., Saraswat, R., and Kurtarkar Raikar, S.: Laboratory experiment to study the effect of salinity variations on benthic foraminiferal species – Pararotalia nipponica (Asano), J. Geol. Soc. India, 67:41–46, https://doi.org/10.1007/BF02709367, 2006.
Sujata, K. R., Nigam, R., Saraswat, L., and Rajeev, N.: Regeneration and abnormality in benthic foraminifera Rosalina leei: Implications in reconstructing past salinity changes, Riv. Ital. Paleontol. Stratigr., 117(2):309–318, https://doi.org/10.13130/2039-4942/5970, 2011.
line 228: Thank you for your suggestion. We have implemented it by annotating the abundance values directly above the corresponding bars in Figure 3A, which enhances clarity for readers. As shown, the data confirm that foraminiferal abundance is lower under low-salinity conditions compared to moderate- and high-salinity conditions.
line 230: We thank you for their careful observation regarding Figure 3B. You are absolutely correct that Figure 3B shows that the Milioliida group consists of only one species (Quinqueloculina seminula) and this species richness remains constant across salinities. However, the discussion in Line 230 refers specifically to the "relative abundance" (proportion of the total community) rather than species richness. As illustrated in Figure 3A, the relative abundance of Milioliida clearly increases with salinity (from ~0% at 0 psu to ~45% at 55 psu), which aligns with our discussion on biomineralization mechanisms favoring high-Mg calcite in hypersaline waters.
Changes in Manuscript:
To prevent future confusion, we have modified the sentence to explicitly cite Figure 3A and clarified the distinction between abundance and richness:
"At higher salinities, the formation of shells was found to be more favorable for Milioliida, thereby explaining the higher relative abundance observed at elevated salinities in classical morphological analyses (Fig. 3A)."
line 249: Thank you for this important clarification regarding terminology. We agree that eDNA sequencing primarily identifies Operational Taxonomic Units (OTUs) rather than biological species directly.
Changes in Manuscript: "Firstly, while morphological analysis in this experiment identified only 13 species, the eDNA method demonstrated considerable sensitivity, identifying 1,977 OTUs which were annotated to 103 species, thereby better reflecting the diversity within the foraminiferan group."
line 262: We agree with you that the diversity of Milioliida in the morphological dataset is very low (only one species). In the original text, we used the term "relatively monotypic" to describe this. To be more precise and address your comment, we have revised the sentence to explicitly acknowledge that this group consists of a single species, while emphasizing that its high relative abundance (which eDNA failed to capture) is the key insight provided by morphology.
Revised text:" In the eDNA dataset, the porcelaneous Milioliida group constituted a negligible proportion of the total reads; consequently, the community response to salinity gradients within this group could not be detected. In contrast, a statistical analysis of classical morphology reveals that (Fig. 3A/B), although the Milioliida assemblage is monospecific (represented only by Quinqueloculina seminula), its abundance is substantial, constituting a high proportion of the total biological count."
lines 263-265: Thank you for your suggestion. This statistical description is based on the results presented in Figures 3A/B. Detailed quantitative data on foraminiferal abundance will be provided in the Supplementary Materials. Please refer to the specific language revisions in the previous response (Line 262).
lines 277-280: Thank you for providing the references; they are greatly helpful for our manuscript and future research. We have incorporated the suggested content and made the following revisions:
To address this challenge, environmental RNA (eRNA) has emerged as a promising tool for identifying metabolically active communities, due to its rapid degradation outside living cells which better reflects contemporary biological activity (Chen et al., 2025; Qiao et al., 2025). Although eRNA methodology in foraminiferal research is still evolving and not yet standardized, recent studies—such as those revealing anaerobic metabolic adaptations in subseafloor foraminifera and the role of primary production in shaping benthic eukaryotic interactions—highlight its potential to uncover physiological and ecological responses inaccessible to traditional approaches. The continued development and integration of eRNA with controlled laboratory culture systems will help directly resolve how salinity fluctuations influence active foraminiferal community structure and function, thereby extending the findings of this study toward more mechanistic, ecophysiologically meaningful insights.
General comments: Thank you for your suggestion. We will carefully review the manuscript. The term "in situ" has been revised as advised.
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AC1: 'Reply on RC1', Yanli Lei, 09 Feb 2026
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RC2: 'Comment on egusphere-2026-196', Christopher Lowery, 02 Feb 2026
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This paper by Cao and others describes a culturing experiment of benthic foraminifera collected from the inter-tidal zone of Qingdao Bay and exposed to a range of salinities. The cultured populations were then examined by traditional morphometric techniques and via eDNA sequencing. These analyses agreed in the broad results but differed in interesting and revealing ways, particularly the dominance of organic-walled monothalamids that don’t show up in morphological datasets, and in the abundance of miliolids, which represent a small sliver of the genetic diversity but a large portion of the tests in some high salinity samples. This is a very cool study! I definitely think it deserves to be published in Biogeosciences, but I think some changes need to be made first.
First, and most importantly, I’m sorry if I missed this but I did not see any Data Availability Statement, nor any data tables associated with this paper. I was specifically looking for foraminiferal count data, but I also don’t see DNA data included anywhere in whatever format that is usually provided. I am not familiar with this journal’s data policies but in my opinion raw data MUST be provided as a supplement or linked to in a recognized online repository. Additionally, the total abundance of foraminifera (and of each species) for each culture should be included somewhere, perhaps as a 5th panel in Figure 3.
Second, the sampling methodology needs to be explained further. How was the mud collected? How much material? Was it split 13 ways for the individual cultures, or were 13 samples taken? Was it sieved prior to culturing? What was the volume per culture? Etc.
Finally, I think there could be more discussion of the strength of this morphology + DNA approach. What are we missing when we only use one or the other? What does this mean for our understanding of benthic foraminiferal assemblages?
I have some other comments and suggestions below, by line number. I am not a DNA specialist and so unfortunately was not able to evaluate those methods and results. I would be happy to review a revised version of this manuscript.
Sincerely,
Chris Lowery
University of Texas at Austin
Line 10 – not sure “yet” is the right word here. Maybe “and”
Line 11 – what do you mean “their” responses? Foraminifera? eDNA? It would be good to clarify. If you mean foraminifera broadly, you might also consider adding a clause at the start of this sentence that says something like “Foraminifera are commonly used to assess salinity changes in estuary settings, but quantitative experimental studies of their responses to salinity gradients are scarce.” I think it highlighting the widespread recognition of the importance of salinity in estuarine benthic forams will help emphasize the importance of your study.
Line 13 (and throughout) – PSU is an acronym should be capitalized
Line 15 – diversity “increased significantly” but by how much? Would be good to state the value here (ditto with the relative abundance of Rotaliida in the next line).
Line 22 – check this figure for grammatical errors (“were found to be survived”)
Line 30-32 – need some citations for these field observations
Line 33 – Streblus has been synonymized with Ammonia (which you use later on through the text). Please be consistent.
Line 49 – might be good here to mention the group of foraminifera that are widespread but fail to fossilize, as foreshadowing of your results
Line 67 – how many samples were collected? How far apart were they? How much material was collected? What was the sampling technique? How reflective is the intertidal zone of conditions in the rest of the bay? If the cultures weren’t all done on the same sample split 13 ways that should be stated (and would be a potential source of error). Also, was the sample sieved before culturing? Was this an already mature adult population that you subjected to a range of salinities or were these gametes that grew to adulthood during the experiment?
Line 69 – if the precision of the refractometer is 1 per mil., why are you reporting a salinity to a precision of 0.1 per mil?
Figure 1 – do you have a photo of the sampling location? You’ve got a bit of room here (and a lot of unused space in the map box) to include a photo of the sampling area. It would also be helpful to show a much closer map view of the sampling location. Finally, the scale bar is barely readable, and should be much larger.
Line 76 – what do you mean salinity “gradients?” Aren’t these just salinity *values* for the individual dishes?
Line 86 – how was pH maintained? (e.g., was a buffer added?)
Line 92 – I don’t know what ISO 23040:2021 is. Can you briefly explain the steps from Rose Bengal staining to species identification here? Also, did you use a consistent volume of sediment across all replicates? Did you count the dead assemblage too? Also, did you examine the living foraminifera in original intertidal sample to obtain a baseline with which to compare the population at these different salinities? I’m curious how similar/different the cultured communities are from the natural one.
Line 125-127 – did the total abundance of foraminifera show any trend across salinity gradients? It would be interesting to see the total numbers of foraminifera reported here too.
Line 129 – “the total” number of species? Or number of individuals?
Line 161 – cool! What are the names of those 103 species?
Figure 5 – what do the colors in (A) represent?
Line 170-176 – I’m not a DNA person so forgive me if this is obvious but the eDNA analysis is on bulk sediments from the sample, right? Wouldn’t this include a significant number of sequences from specimens that were alive prior to sample collection? Obviously there are trends here with salinity but this relict DNA would represent some error, right? Can you talk here about how that may affect your results, and how you deal with it? Especially given how, e.g., miliolids show different abundance trends compared to the morphological data.
Line 197-201 – I feel like this discussion of salinity response in Ammonia is missing the context of the range of interpreted ecophenotypes often associated with this genus (Ammonia tepida, e.g.) and the extensive cryptic diversity within this genus (see, for example, Goetz et al., 2025, JFR https://doi.org/10.61551/gsjfr.55.1.45). Given the taxonomic complexity of this genus I think it would also be very interesting to compare your DNA results with existing genetic data and species designations.
Line 205-207 – is this positive relationship between salinity and diversity also true in the wild? Has anyone done a thorough study of living populations in Qingdao Bay? Just based on marginal marine habitats I’m familiar with in the Gulf of Mexico region, there are a lot of low-diversity, high-salinity assemblages (for example see Poag’s book: https://books.google.com/books?id=HsUbBgAAQBAJ&lpg=PP8&ots=I0zTbcLg9O&dq=poag%20gulf%20of%20mexico%20foraminifera&lr&pg=PP1#v=onepage&q=poag%20gulf%20of%20mexico%20foraminifera&f=false). I think it would really strengthen your paper
Line 214 – “primitive” seems needlessly normative here.
Line 227 – what do you mean “it can be deduced?” Presumably you have count data that show this directly? (again, I think it would be helpful to show these count data, and also they need to be included as a supplemental table or uploaded to an online repository).
Line 228 – what’s the evidence that the tests dissolved?
Line 230-231 – If the tests dissolved (or didn’t dissolve, in the high salinity sample) wouldn’t the DNA results show the same relative abundance in both, since presumably the starting stocks were the same?
Line 238-239 – benthic foraminiferal assemblages have been used as a paleosalinity indicator before, and it would be good to cite some of those studies here.
Line 240-241 – Impact of salinity of Mg/Ca derived temperature has been known since at least Lea et al., 1999; see review in Katz et al. (2010 JFR https://doi.org/10.2113/gsjfr.40.2.165)
Line 247 – “it has been demonstrated” – by whom?
Line 250 – I think the large proportion on monothalamids here is a really cool result and something to highlight in this paper, and something you can tease out a bit more in the discussion: what does it mean for traditional foraminiferal analyses if we’re consistently missing this apparently large group? What are we missing about benthic foram ecosystems?
Line 286 – what functional relationships were identified?
Line 289-291 – do you mean reconstruction of environments in Qingdao Bay or generally? I think I would be cautious of assuming these linear relationships hold true across all estuary environments, since there are a lot of different local controls on foram populations. At the very least, it would be good to have further discussion in the Discussion about how applicable these results might be more broadly.
Citation: https://doi.org/10.5194/egusphere-2026-196-RC2
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The manuscript of Cao et al reports a study on foraminifera diversity in coastal sediments as assessed by morphological and molecular techniques, across a gradient of 13 different salinities. They collected more than 9000 foraminifera specimens and produced a large dataset which could be subjected to statistical analysis. They found correlations between foraminifera biodiversity and salinity which could be useful for interpreting paleo-salinities in sediment core records. The application of barcoded high throughput illumina sequencing to foraminifera was impressive and I think the conclusions are supported by the results, but there is key of information missing in the methods regarding taxonomic annotation of the DNA data. The are two major issues for the authors to consider when revising their manuscript:
1. Phylogenetic analysis : Currently, there is no explanation in the methods how the authors determined the taxonomic affiliation of the sequences. What database was used ? How were they compared (BLASTn?). This is essential information that is lacking and needs to be added. The authors should subject their foraminifera OTUs to a phylogenetic analysis, aligning them against known Foraminifera species sequences and then present the tree as a proof that the sequences indeed are related to those groups. It will also show any patterns in micro-diversity within the groups that could be interesting.
2. Quantitative results of the abundance of foraminifera groups should be presented. It is unclear whether this is displayed in Fig 3A or not. If Fig 3A is showing the relative abundance across 1000s of counted foraminifera this should be stated. The number of counted tests should be written as numbers at the top of each bar chart so the reader can see how many were counted at each salinity.
Specific comments on the text:
line 58 (and line 75-76): Do you mean 13 salinity gradients or 13 different salinities (and the use of the 13 different salinities represents a gradient?)
line 84: What was the medium used to grow the algae?
lines 108-110: For readers unfamiliar with the amplicon size, please state the size of the amplified product and the length of the paired end sequencing reads and how much overlap between forward and reverse pairs was possible (important for determining quality). It is stated on lines 159-160 (380 base pairs) but would be good to put it here in the methods as well.
lines 111-114: There is key information missing here, how were the foraminifera OTUs taxonomically annotated and assigned to the different groups? Please provide a detailed description, and also apply a phylogenetic method for assigning the taxonomy.
lines 115-116: These sentences appear to be fragments or incompletely edited sections of text. Please revise or remove.
Figure 2: Do these cells have a stain applied to them (rose bengal)? Please specify in the legend.
figure 5: It is unclear to me what the "Chord diagram" is representing. Please add more information to the legend. What do the numbers on the ring represent? How are the different pieces of the ring connected and what do the connections represent? Does it imply that only Monothalamiids and Rotaliida and "others" were detected with eDNA but not Textulariida? What does it mean that the connecting strands only fit to a smaller or large piece of the outer rims?
line 171: How was this done? Were phylogenetic trees used for this ? I didn't see any trees presented, and no text is in the methods regarding how taxonomy was assigned to the OTUs.
line 204: Please provide a citation
line 228: Please cite the figure showing the decline in the abundance of foraminifera. The manuscript does not show any figure or table displaying the total abundance of foraminifera in the different salinities. Fig 3B only shows species diversity, is Fig 3A showing the total numbers of foarms counted on a relative scale? The authors must have the total numbers of forms that they found (they counted 9000, at least this is what they write). This should be displayed somehow so readers can see how the total abundance of foraminifera increased or decreased across the gradient.
line 230: Figure 3B does not show this, rather there is only 1 species of Miliolida that is constant across all salinities. Please clarify.
line 249: The DNA does not identify species it identifies OTUs, please correct.
line 262: The morphological data show also very low diversity of Milioliida, only one species (in Fig 3B).
lines 263-265: where is the result of this statistical analysis presented in the manuscript? Please cite it here in the text. What do you mean "their numbers are substantial"? Is this the total number of observed tests? The authors should provide a figure showing the quantitative data on total numbers of tests observed. Or is this what is displayed in Fig 3A on a relative scale? Its unclear
lines 277-280: You should also cite these recent papers that used RNA (eRNA) to study active benthic foraminifera, to provide a more comprehensive overview :
DOI: 10.1126/sciadv.adt2147
doi.org/10.1038/s41396-020-0708-1
General comments: Check the manuscript for typos, grammatical mistakes, and capitalize "in situ".