<figure><img src="image_1.png" /></figure>

# High resolution 3D images of sediment cores as powerful tool for exploring foraminiferal microhabitats

M.P. Nardelli<sup>a, *, 1</sup>, N. Vanderesse<sup>b, *, 1</sup>, M. Moller<sup>a, b</sup>, C. Guilhermic<sup>a</sup>, A. Mouret<sup>a</sup>

## ARTICLE INFO

**Keywords:**

µCT-scan

FLEC

Proxy

Epifaunal

Infaunal

Sub-millimetric scale

## ABSTRACT

Benthic foraminifera are marine protists largely used as bioindicators and proxies of paleo-environments. Epifaunal species are supposed to live at or above the sediment surface and are therefore used as proxies for bottom water conditions, while infaunal inhabit the sediment column, thus tracing porewater chemistry. Traditional analytical methods based on core slicing, however, have a low resolution that does not allow to precisely characterise the preferential microhabitat(s) of indicator species.

In this study we performed microtomographic analyses on an experimental sediment core, to observe the life-position of living foraminifera of two surface-dwelling species *Ammonia confertitesta* and *Haynesina germanica*, reported both as epifaunal or shallow infaunal. The images we obtained offered for the first time the possibility to observe each individual in 3D space with a numerical resolution of 13 µm/voxel.

The results revealed that the two species are never located above or at the sediment surface and have their preferential microhabitats in a sub-superficial sediment layer constrained in the 0–500 µm interval below the surface. Rapid decrease of abundances below this layer suggests that their microhabitat could be even more specific than previously thought.

µCT-scan of sediment cores is also a valuable tool to obtain high-resolution information about foraminiferal ecology. The described method is useful to assess the effective microhabitat of all foraminiferal species that are usually used as proxies for paleorecords, to ensure that the information we can obtain from them is attributable to bottom water or to porewater conditions at a specific sediment depth.

## 1. Introduction

Benthic foraminifera are largely used for paleoenvironmental and paleoceanographic reconstructions. Most of the proxies are based on the geochemistry of the calcareous shell of certain target species. The assumption behind is that the shell is biomineralized in equilibrium with the surrounding seawater and roughly reflects the ions concentrations of the seawater at that time. Generally, for the reconstruction of bottom water characteristics the geochemical proxies are measured on target species that are supposed to live above or at the sediment-water interface, the so-called epifaunal (sensu stricto) species. They are also referred to as elevated epifaunal foraminifera (EEF, Lutze and Thiel, 1989; Schönfeld, 1997; Schönfeld et al., 2011). Typical epifaunal genera used for geochemical analyses are *Cibicidoides*, *Nuttallides*, *Oridorsalis*, and *Planulina* (e.g., Katz et al., 2010; Rathburn et al., 2018). However,

the term epifaunal in a sensu lato often include “surface dwelling” species that can have both epifaunal sensu stricto and shallow infaunal behavior. This assimilation can be source of bias for paleoenvironmental reconstructions. Based on observations of modern faunas, both in situ and in culture, when geochemical calibrations are performed, it is evident that a shift (more or less important according to the species) exists between the expected signal, based on seawater characteristics, and the actual record. This is often attributed either to cellular interference in biomineralization processes (e.g., isotope fractionation, selection on ions, increased pH in the inner vacuoles...) or to ontogenic causes (the geochemical signal of the shell can vary during the growth of a same specimen in a same environment; e.g. Schumacher et al., 2010; Theodor et al., 2016). In a more generalized way this is referred to as the vital effect (e.g., Erez, 1978; Wit et al., 2012). In paleorecords, moreover, additional processes can contribute to alter the signal, as for

<footnote><sup>a</sup> Université Angers, Nantes Univ., Le Mans Univ, CNRS, LPG, Laboratoire de planétologie et géosciences, UMR CNRS 6112, 49000 Angers, France</footnote>

<footnote><sup>b</sup> University of Bordeaux, CNRS, PACEA UMR 5199, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: mariapia.nardelli@univ-angers.fr (M.P. Nardelli), nicolas.vanderesse@u-bordeaux.fr (N. Vanderesse).</footnote>

<footnote n="1">¹ Maria Pia Nardelli and Nicolas Vanderesse equally contributed to this manuscript and should both be considered as first authors.</footnote>
example diagenesis and taphonomy.

Part of the bias could be also due to the microhabitat of the species, which is often attributed on the basis of ecological studies performed at low vertical resolution in the sedimentary columns.

In fact, benthic foraminiferal faunas are usually studied after sub-sampling sediment cores in 0.5 or 1 cm-thick slices (e.g., Mojthahid et al., 2010; Schönfeld et al., 2012) or, more rarely, at 0.2 cm (e.g. Thibault de Chanvalon et al., 2015). This procedure hampers to exactly identify the life position of each specimen, in a sediment whose geochemical conditions change at submillimeter scale (e.g. Deborde et al., 2008; Cesbron et al., 2016; Thibault de Chanvalon et al., 2015). Some alternative methods have been explored to detect more precisely the actual life position of target species, but they generally are limited to qualitative analyses. For example, Bernhard et al. (2003) developed the optical analysis of embedded cores to observe submillimeter life positions of bacteria, protists, and metazoans in laminated sediments of the Santa Barbara Basin. More recently Bernhard et al. (2023) used the same method to observe the millimeter scale distribution of meio- and nanobenthos in sediment around hydrothermal vent chimneys. Jesus et al. (2023) used a similar approach to study species-specific strategies for photo-regulation and photo-acclimation of microphytobenthic diatoms.

In the present study we quantitatively analyzed the microhabitat distribution of a foraminiferal assemblage to challenge the general assumption that epifaunal (sensu lato) foraminifera live at the sediment surface and test the hypothesis that part of the available ecological information on foraminiferal microhabitats is biased by the low resolution of traditional analytical methods. To test our hypothesis, we performed a 3D microtomographic analysis of a sediment core sampled in a simplified microcosm containing two foraminiferal species: *Ammonia confertitesta* (often referred to as T6 or *Ammonia tepida* in literature, Pavard et al., 2023) and *Haynesina germanica*.

X-ray CT scanning, or microtomography, is a non-destructive analysis technique that allows the visualization of the inner structure of materials. It is based on the differential attenuation of an X-ray beam transmitted through a sample. Depending on the local density and atomic number of the material, x-rays are more or less absorbed, hence producing a radiographic projection very similar to a medical radiography. The transmission power of X-rays is conditioned by the flux and energy of the beam. In a conventional lab CT scanner, a planar sensor converts the x-ray signal into an image in which contrast reflects the variable absorption of the material. Since the sample under investigation is imaged at increasing angles, the corresponding radiographic projections can be combined to quantify local attenuation in the volume. The data produced by this reconstruction is a 3D image in which the voxels (the 3D equivalent of the pixels in a 2D image) carry values that correspond to the local density of the material. The size of the voxels in the real space, usually expressed in µm/voxel, is directly related to the magnifying power of the setup. It is sometimes referred to as the numerical resolution. Smaller voxel size enables smaller details to be imaged (e.g. Kimoto et al., 2023; Chen et al., 2023).

The two species used in our experiment can share similar habitats (i.e., intertidal mudflats) but have substantial differences in feeding strategies and motility. Indeed, *A. confertitesta* (expanded to *A. tepida* morphogroup) has a generalist feeding behavior (Wukovits et al., 2018), while *H. germanica* is a mixotroph selectively feeding on large diatoms (Jauffrais et al., 2016; Wukovits et al., 2018). *Haynesina germanica* was shown to have positive geotaxis, and both species are able to intensely move for food search (Seuront and Bouchet, 2015). Both species are considered as surface-dwellers, reported to live both at the sediment surface (e.g., Bouchet et al., 2009; Thibault de Chanvalon et al., 2015) or as shallow infauna (e.g., Petersen et al., 2019; Deldicq et al., 2020), few millimeters to centimeters down in the sediment in natural environments. The duality of their preferred life position was attributed either to the presence of large bioturbators or seasonal variability of environmental conditions or high motility of the specimens in the sediment

column (e.g., Bouchet et al., 2009; Thibault de Chanvalon et al., 2015; Deldiq et al., 2020).

In our study, the obtained CT-scan images allowed to visualize the 3D distribution and the microhabitat preferences of these two species with an unprecedented resolution of 13 µm/voxel, in an experimental environment where bioturbation and seasonal variability were intentionally absent.

Foraminifera in the sediment cores were manually indexed on each image and their coordinates in the volume were recorded. It was not always possible to trustfully distinguish the two species because they are both spiral and the resolution of the images of the cross-sections are often difficult to attribute doubtlessly. We therefore decided to pool the species.

The analytical process of the images obtained on our experimental core requested methodological development which is also detailed in the study.

## 2. Materials and methods

### 2.1. Sediment sampling and experimental setting

To obtain the sediment core we sampled fresh sediment from the intertidal mudflat of Bourgneuf Bay (French Atlantic coast, 47°005700 N, 2°102900 W). At low tide, we carefully took off the first centimeter of sediment, supposed to be richer in living foraminifera and other fauna, and collected about 5 L of sediment (between 1 and 10 cm of depth) from the site. In order to limit bioturbation effects due to macrofauna, the sediment was frozen (−20 °C) to kill all the eventual living faunas still present then unfrozen, homogenized and an approximately 10 cm-thick layer was added to an aquarium. The aquarium was filled with 0.45 µm-filtered seawater from an unpolluted site (Dinard, North of France), the salinity was stabilized at 34 and the temperature at 15 °C, matching the in-situ conditions in Bourgneuf Bay at the time of sampling (February 2021). Seawater was recycled (>70 %) every two days, to avoid accumulation of toxic elements released by the sediment (ammonium, nitrate, see Guilhermic et al., 2023 for more details).

Superficial sediment (0–0.5 cm) from the same sampling site was manually collected and wet sieved in-situ. Only the 125–500 µm was retrieved, transferred to the laboratory within a few hours, and stored at 15 °C with air bubbling until the addition to the experimental setting.

Seven days after the settling of the sediment in the aquarium, the 125-500 µm sieved sediment, containing living foraminifera, meiofauna and organic residuals of microphytobenthos was added at the sediment surface with a Pasteur pipette. Aliquots of the sample were observed under a microscope to estimate the composition of the added community. It was mainly constituted of two dominant planispiral species (*Ammonia confertitesta* and *Haynesina germanica*, > 95 %) with the rare presence of another planispiral species, *Elphidium selseyense* (<5 %). All these species are generally described as surface dwellers in the literature (e.g., Murray and Alve, 2000; Alve, 2001; Bouchet et al., 2009; Cesbron et al., 2016; Papaspyrou et al., 2013; Thibault de Chanvalon et al., 2015).

### 2.2. Oxygen profiles

Dissolved oxygen measurements were performed on the sediment in the aquarium all along the experimental duration and on the day of the sampling using 50 µm tip diameter Clark-type Unisense™ microelectrodes, mounted on an automated micro-manipulator (Revsbech, 1989) taking measurements with a 50 µm vertical step.

### 2.3. Sampling preparation

After two weeks of acclimatization, foraminifera were sampled by coring inside the aquarium with a cut syringe of about 2.5 cm in diameter and 8 cm long, paying attention not to disturb the sediment
interface. Following the protocol for FLEC (Fluorescently Labeled Embedded Core) described by Bernhard et al. (2003), the core was incubated with Cell Tracker Green for 12 h minimum, at experimental temperature conditions, then resin embedded. Several steps were necessary for embedding. Samples were firstly fixed with glutaraldehyde 3 % and sodium cacodylate trihydrate 0.1 M, then dehydrated with successive addition of ethanol at different concentrations (30 %, 50 %, 70 %, 90 %, 95 %, 100 %) under vacuum to improve percolation. Two successive additions of acetone 100 % preceded the gradual introduction of ultra-low viscosity resin (Spurr, Sigma-Aldrich; Spurr, 1969) in the core using graded series of resin / acetone mixtures (50 %, 60 %, 70 %, 80 %, 90 %, 100 %) and two additional baths with resin 100 % at the end. The core was then transferred to oven at 50 °C until hardening (about 2 months in our case, but this can vary depending on the volume of the sample).

The core was analyzed under µCT-scan and the foraminiferal tests indexed in the 3D images (more details in the next section). After 3D scanning, a longitudinal slice, about 1 mm thick, was cut from the embedded core using a diamond wheel saw, and then wet-polished with silicon carbide powders of decreasing particle size (1200, 800, 600, 320 and 180 µm) (modified after Bernhard et al., 2023). Once polished, the vertical sections were observed for visual analysis of living (i.e., CTG-labeled) specimens using a Leica TCS SP8 AOBS confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) equipped with a HCX PL Fluotar 10×/ON 0.30 or a HC PL APO CS2 20×/NA 0.75 dry objective and gateable hybrid detectors (GaAsP). Images were acquired in the format 1024 × 1024 pixels, bit depth of 8, and a scanspeed of 400 Hz. Excitation was performed with a 488-nm Argon laser (40 mW) and the band-pass for the detection of the emitted light was set between 491 and 750 nm. Z-series optical sections were collected with a step-size of 3 µm using a Super Z Galvo Type H stage and were displayed as maximum z-projections using the LAS X software.

## 2.4. CT-scan analysis

The sediment core was analyzed in a v|tome|x s 240 CT scanner using the following parameters: Voltage: 110 kV; intensity: 200 µA; exposure time: 500 ms; number of projections for 360° rotation: 2550; voxel size: 13 µm/voxel.

At each rotation step, three radiographic projections were acquired and averaged to reduce noise. The radiographic projections were processed by the Phoenix datos|x reconstruction 2 program to produce a 3D image with dimensions 2024x2024x2024 voxels. Several scans were stitched together to cover a 5 cm depth starting from the interface. In total, the acquisition and the reconstruction took ca. 5 h. The syringe body was removed before acquisition to ease the fastening of the core in the fixture system.

The 3D image was analyzed with the programs Fiji/ ImageJ and visualized with Dragonfly.

It was possible, with this software, to produce virtual cross-sections

<figure><img src="image_2.png" /><figcaption>Fig. 1. 3D view of the core at µCT-scan. a) Core with virtual horizontal cut plane; b) Virtual vertical cross-section.</figcaption></figure>

at arbitrary angles and positions. Fig. 1 presents a 3D view with cut plane (Fig. 1a) and a vertical cross-section of the core (Fig. 1b).

## 2.5. Image treatments and counting

The numerical resolution of the scan allowed us to easily point out the foraminiferal shells and localise them in 3D space. The positions of the foraminifera were manually recorded with Fiji software in slices at increasing depths below the interface. Only the first 10 mm of the core were analyzed because no or very few living foraminifera were reported below this depth in twin cores from the same aquarium analyzed with traditional slicing and picking techniques (data reported by Guilhermic et al., 2023 and in Fig. 8a).

For an optimal visualization of the foraminiferal specimens in the complex sedimentary matrix, the 3D image was processed with a Non-Local Mean filter. This filter allows a noise reduction of the image without altering its texture.

To reduce possible bias on the edges of the core, the foraminifera were detected in a subset of the volume, on a 15 × 15 mm region of interest, centered with the axis of the core (Fig. 2a).

The analysis took about half a day, for a total of ca. 2000 detected forams. The exact position (x,y) and the z coordinate of the horizontal slice corresponding to the observation were recorded. As the same foraminifer can be visible on multiple slices, we grouped the points with the same (x, y) coordinates and positioned each specimen in the middle of the cloud of the obtained points.

In Figs. 1 and 3a, it can be observed that the interface (i.e., sediment surface) was not flat. Thus, the measured z coordinates of the foraminifera in the reference frame of the 3D image did not accurately reflect their actual positions with respect to the interface. The coordinates were therefore corrected to take into account the irregularities of the interface (Fig. 3b). Interface was first segmented, then coded according to the depth of each of its pixels with respect to the first slice of the 3D image (Fig. 3a). The actual depth coordinate of each foraminifer with respect to the interface, Zinterf ace, was then corrected according to the following formula:

$$Z_{\text{corrected}} = Z_{\text{measured}} - Z_{\text{interface}} \quad (1)$$

where Zmeasured corresponds to the depth with respect to slice 0, which corresponds to the first image where the highest part of the sediment surface is visible.

The analysis of CT-scan images does not allow to discriminate living and dead foraminifera. The sediment used to fill the microcosm was not sieved, and even if it was sampled from 1 to 10 cm below the surface, where concentrations of living and dead foraminifera are not the highest, that dead specimens were probably present in the experimental set up. However, as the sediment was homogenized before being added to the aquarium, we can suppose that the vertical distribution of the dead specimens eventually present should be homogeneous. We therefore estimated the potential number of dead foraminifera on the base of the mean concentrations we counted below 8 mm, where Guilhermic et al. (2023) reported zero or close to zero living individuals in the analyzed replicates (Fig. 8a).

## 2.6. 3D distribution of foraminifera

Once all the foraminifera in the core were pointed out, their distribution with respect to the vertical distance to the interface was plotted. In addition, we carried out statistical tests to assess if their spatial distribution in the horizontal plane (x,y) was either random, dispersed, or aggregated. These tests relied on Ripley's G function, which quantifies the proportion of nearest neighbor points to each point of the pattern at various distance thresholds. By comparing the cumulative histogram of nearest neighbor's numbers at increasing distance values to histograms corresponding to generate random patterns, it is possible to assess the
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** a) Z cross section with region of interest (ROI = red) and circled forams (yellow); b) Close-up views of sediment inside the ROI; c) Example of close-up view forams. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** a) Real bathymetry of the core surface (scale unit = mm); b) Scheme used for foraminiferal position toward an irregular interface.</figcaption></figure>

randomness of the observed pattern. These tests were carried out with the pointpats package of the Python Spatial Analysis Library (Kang et al., 2023; Rey and Anselin, 2007).

# 3. Results

## 3.1. Porewater geochemistry

Nine oxygen profiles obtained on the aquarium where the core was sampled are shown in Fig. 4.

Oxygen concentrations in the overlying water approximately ranged from 238 to 258 µmol/L. A strong decreasing gradient was observed in the first few hundreds of micrometers below the sediment surface, and concentrations reached values down to 135 to 150 µmol/L at 500 µm depth. A weaker gradient was recorded in the last micrometers before reaching zero oxygen value.

The oxygen penetration depth (as the depth where oxygen concentration is zero) varied between 1.2 and 1.6 mm at the sampling time.

## 3.2. Vertical distribution of foraminifera

The obtained counting of benthic foraminifera (all species together) in the region of interest is resumed in the vertical profile of Fig. 5, with a vertical resolution of 52 µm (i.e., 4 × 13 µm-sized voxel), as close as

possible to the 50 µm step size of the oxygen profiles.

We can observe that the maximum peak of abundance, based on the CT-scan images, was not at the very surface of the sediment, but rather in the first 500 µm below the surface.

Just below the peak, abundances rapidly decreased toward near zero within few hundreds of micrometers and only rare specimens were observed below 2 mm of depth.

The results of the observation of the longitudinal slices under confocal microscopy (Fig. 6) clearly showed that the large majority of the specimens located at the top of the core (0–500 µm interval) were alive (i.e. CTG-labeled) at the sampling time. Rare dead specimens were also observed at these sediment depths (Fig. 6d, e, j). Moreover, the images also showed that, due to the test-size of the foraminifera (about 250–500 µm in average in our case), part of the shell of specimens pointed at 200–300 µm depth (depth of maximum abundance, Fig. 4) were sometimes reaching or passing the sediment surface (Fig. 6c, h). The specimens we observed did not show a preferential orientation toward the surface, sometimes they had vertical position (Fig. 6a,b), sometimes on a side (Fig. 6c), sometimes dorsal (Fig. 6g), sometimes umbilical (Fig. 6f).

## 3.3. 3D distribution of foraminifera

The 3D distribution of foraminifera after correction for interface
<figure><img src="image_5.png" /><figcaption>Fig. 4. Dissolved oxygen profiles measured in the same aquarium at the sampling time. Each colour corresponds to a replicated profile.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 5. Vertical distribution of identified foraminifera with an averaged 52 µm vertical resolution. Shadowed area represents the average number of putatively dead individuals observed below 8 mm of depth (following Guilhermic et al., 2023). The light-blue spots around each mean point (black) represent the maximum size of foraminiferal tests (500 µm). Red diamonds represent an average oxygen profile. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

orientation is reported in Fig. 7a. We tested the Ripley's G function both on faunas from the whole core and from the first 500 µm of sediment, where abundances of living specimens were the highest. In Fig. 7b and c the results for the 0–500 µm are reported. For both tests, the results showed no significant differences of x,y coordinates between the observed foraminifera distribution and a random pattern.

# 4. Discussion

## 4.1. Define a precise microhabitat for ecological and geochemical proxies' improvement

Benthic foraminifera are largely used as paleoceanographical and paleoecological proxies, both based on assemblages and calcareous shell composition. Their preferential microhabitat is supposed to be mostly driven by oxygen and organic matter availability (Jorissen et al., 1995) and is a key parameter to reconstruct past conditions at the seabed. Their life position is also supposed to influence the chemical composition of their calcareous shells, as they are supposed to calcify in equilibrium with the surrounding seawater. This is therefore an important parameter in the choice of pertinent indicator species to reconstruct bottom water or porewater chemistry of past environments.

As an example, paleoxygenation and paleoproduction are estimated through the use of $\Delta\delta^{13}C$ on benthic foraminiferal shells (e.g., Hoogakker et al., 2015; Theodor et al., 2016). McCorkle and Emerson (1988) proposed the $\Delta\delta^{13}C$ between bottom water and pore-water at the sediment depth where oxygen approaches zero as a proxy for oxygen content of the bottom water. As oxygen consumption is related to organic matter availability, then this proxy was extended to productivity (e.g., McCorkle and Emerson, 1988; McCorkle et al., 1990). However, $^{13}C_{DIC}$ concentration is highly variable among different sediment intervals and even a small shift in preferential life position of a species with respect to the putative microhabitat can completely bias the results. Indeed, Theodor et al. (2016) highlighted that the application of this proxy is ambiguous due to, among other factors, a slight species-specific difference in microhabitat that can result in significant $\delta^{13}C$ shifts in epifaunal species.

Therefore, as Murray (2003) already underlined, "it is important to gather data on microhabitat in order to define the limits of variability so that it might be applied to the interpretation of the fossil record".

The vertical microhabitats of living benthic foraminifera were deeply investigated only starting from 1990s (after Corliss, 1985). Since then, living benthic foraminifera were traditionally analyzed on sliced sediment cores, with a resolution step of 0.5 to 1 cm in the large majority of cases and, atbest, down to 0.2 cm. Because of this methodological limitation, Corliss (1991) extended the use of the term epifaunal to the species living in the topmost 1 cm of the sediment. Thereafter, foraminiferal species that show their maximum peak at the topmost layers of sediment (roughly the first cm) were referred to as epifaunal (sensu lato) and the rest as shallow to deep infaunal (see Jorissen, 1999 et references therein).

This dichotomy in terms of definition of epifaunal microhabitats generates two opposite situations. On one hand, if we strictly consider the sensu stricto definition, none of the foraminiferal species possibly living inside the sediment, even at shallow depth, could be referred to as epifaunal, as already suggested by Murray (2003).

Eventually, the only exception could be some species belonging to genera that are often found in microhabitats above the sediment surface (e.g., leaves or gravel/rocks) such as for example *Cibicidoides*, *Cornuspira*, and *Planulina* or large symbiont-bearing tropical foraminifera (e.g., *Amphistegina*, *Operculina*). However, as already evoked by Gottschalk et al. (2016) many of these genera are not exclusively reported to live above or at the sediment surface and have also been found within the topmost centimeters of the sediment (e.g., Fontanier et al., 2008a, 2008b; Venturelli et al., 2018). This means that even if they could realistically occupy epifaunal sensu stricto microhabitats (Lutze and Thiel, 1987) they can also have infaunal behavior.

On the other hand, if we enlarge the definition of epifaunal microhabitats to the topmost centimeter(s) of the sediment, we implicitly accept to assume that the environmental characteristics are nearly homogeneous within this interval and can be assimilated to the bottom water conditions (in terms of oxygen, organic matter availability and (pore)water chemistry, for example), which is unreliable. As an
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Confocal microscopy images of living (CTG labeled, a,b,c,d,f,g,h) and dead (d, e, j) foraminiferal specimens on selected sections of the core. The white line redraws the sediment surface. Scale bar = 100 µm.</figcaption></figure>

example, our results show that oxygen concentrations can vary from saturation to zero on a (sub-)millimetric spatial range (Fig. 4). Indeed, some recorded offsets in geochemical proxies among a same species or between species were already attributed to a possible shift of micro-habitat (e.g. Gottschalk et al., 2016; Theodor et al., 2016). It is therefore mandatory to obtain high-resolution information about the microhabitat of indicator species to trustfully apply geochemical paleoproxies.

As a first attempt, in this study, we performed a µCT-scan analysis of a sediment core to quantitatively observe, at a 13 µm spatial resolution, the distribution in 3D space of the two species, *Ammonia confertitesta* and *Haynesina germanica*, that are generally reported in microhabitats corresponding to the definition of epifaunal sensu lato and/or shallow infaunal (e.g., Bouchet et al., 2009; Cesbron et al., 2016; Murray and Alve, 2000; Papaspyrou et al., 2013; Thibault de Chanvalon et al., 2015).

The profiles we obtained (Fig. 5) show that nearly no specimens were located on the sediment surface and that the peak of abundance was rather in the first 300 to 500 µm below the interface. Below this peak of abundances, the number of specimens rapidly decreased by more than 70 % until 2 mm depth. The putatively living individuals below this depth were rare and this is probably the reason why we were not able to catch any of them in the core sections observed at confocal microscopy.

It is interesting to compare the vertical profile we obtained for foraminifera in our experiment (Fig. 5), where a sub-superficial peak of abundance was observed, with the ones (reported in Fig. 8a) obtained by Guilhermic et al. (2023) through the traditional analysis of 2 replicated twin cores collected in the same conditions from the same experiment, sliced at 2 mm resolution step. Despite a difference in absolute abundances in the two replicates, the results of Guilhermic et al. (2023) showed in both cores the peaks of abundance in the first layer of sediment (0–0.2 cm), and less abundant but still present living specimens down to 0.8 to 1.2 mm.

If the general shapes of the vertical profiles are similar and fit with the “Type a” vertical distribution described in Jorissen (1999) for epifaunal and shallow infaunal species, the ecological information obtained is not the same with the two methods. Our results, in fact, allow to exclude that this distribution includes epifaunal (sensu stricto) species and additionally suggest that the significant presence of living individuals down to several millimeters could be an artefact due to the slicing method. Indeed, on the µCT-scan images we were able to correct the position of each specimen in relation to the real topography of sediment surface. On the contrary, when a sediment core whose surface

is inclined is sliced along a horizontal plan, specimens from different absolute depth intervals are pooled together, artificially increasing abundances of surface dwellers in deep layers (Fig. 8b).

In summary, our results allow to exclude that the observed species have an epifaunal (sensu stricto) behavior. If the switch from one to the other microhabitat is a question of few tens or hundreds of micrometers, in an optic of using benthic foraminifera shells as reliable proxies for paleoenvironment reconstruction, this information is fundamental. Indeed, at the depth where we observed the peak of abundance, we actually measured undersaturated dissolved oxygen conditions (135 to 150 µmol/L against 240 to 260 µmol/L above the sediment surface, Fig. 4). As this undersaturation is the result of organic matter consumption, presumably also food availability in this interval was much lower than at the sediment surface. The strong gradient in redox fronts and nutrient concentrations on a sub-millimetric spatial scale in superficial layers of sediment column is something often reported in geochemical studies including analyses at high spatial resolution, at least in coastal (e.g. Robertson et al., 2008; Metzger et al., 2016) and shelf environments (e.g., Rabouille et al., 2021). This means that paleo-oxygenation and paleoproductivity proxies recorded in the shells of foraminifera preferentially or even occasionally living the first 500 µm below the sediment surface are probably very far from being representative of bottom water conditions. On the other hand, the very narrow infaunal microhabitat we observed for the studied species, suggest that their ecological preferences could be even more specific than previously thought, which could represent an advantage for the use of foraminifera as bioindicators in present environments and past records, as long as their preferential microhabitat is known on an accurate spatial scale. We therefore suggest that high-resolution µCT-scan analyses on sediment cores should be used to explore the real average life-position of putative epifaunal or infaunal species to validate their use as proxies for bottom water or pore-water respectively. Our approach proved to be a useful tool to this aim, offering the possibility to observe foraminifera in their life position with a resolution two to three orders of magnitude better than the analysis of sliced sediment cores.

Additionally, µCT-scan analysis offers the opportunity to observe the 3D distribution and specific microenvironment occupied by specimens. This can be useful to test ecological hypothesis such as the possible influence of microhabitat patchiness or specific structures (e.g. burrows, roots, organic matter patches, etc.) on faunal distribution.

In our case, we tested the hypothesis of an aggregated distribution of living specimens in a homogenized and unbioturbated sediment column.
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** a) 3D distribution of foraminifera (dead and alive without distinction) identified in the core. b) X, Y distribution of foraminifera in the first 500 µm of sediment below the surface (zone of maximal abundance of living specimens). Z_Corr_foram_mm means interface-corrected position on z axis (in mm) c) Ripley's G function of the corresponding pattern.</figcaption></figure>

The results did not show a significant aggregation on a X, Y space (Fig. 7c), which is coherent with the results reported by Schönfeld et al. (2023) for a given cohort of data on a 10 cm spatial scale. However, this result could be influenced by the peculiar experimental conditions. In natural conditions, aggregation of specimens could be enhanced, for example, by the presence of large burrows (e.g. Bouchet et al., 2009; Thibault de Chanvalon et al., 2015) which were intentionally absent in our set up. The µCT-scan approach should be further applied in (close to) natural conditions to unravel this kind of questions.

## 4.2. *Limits of the study*

The results of the present study represent a first step in new applications of modern analytical tools that could revolutionize future research in the domain of micropaleontology and ecology of benthic species. As all explorative works, much effort is still needed to optimize

methodological protocols and obtain more representative results.

In our study, as a first attempt, we preferred to analyze resin-embedded experimental cores instead of natural ones sampled at sea. The idea was to slightly simplify the analytical effort by reducing the variability in sediment grain-size (mainly silt at the sampling site and homogenized before the experiment) and avoiding the presence of large meiofauna and macrofauna, susceptible to complexify the structures by burrowing. This could have at least partially influenced our observations, due for example to the absence of predators or large burrowers, the absence of day/night cycles, or limited food sources. It is therefore probable that in our experimental set up the natural vertical mobility of the two species is less expressed than in natural habitats. We also artificially increased the number of foraminiferal specimens in the core (about 800–1200 ind./10 cm² as reported by Guilhermic et al. (2023) for our experiment vs about 150 ind./10 cm² after Choquel (2021) at the same sampling site, i.e. Bourgneuf Bay mudflat). Our aim was to be sure
<figure><img src="image_9.png" /><figcaption>**Fig. 8.** a) Vertical foraminiferal distribution reported by Guilhermic et al. (2023) on two replicated cores sampled in the same aquarium at the same sampling time as the present study. Modified after Guilhermic et al. (2023) b) Explicative scheme of the potential bias introduced by the traditional slicing of a core with a non-horizontal flat surface.</figcaption></figure>

to have enough living specimens to easily differentiate them from background “noise” represented by dead ones, as at present we are not able to discriminate the two on the obtained images.

Also, the embedding of sediment cores with resin is laborious and requires several steps of manipulation. Even with a maximum of attention and carefulness, the occurrence of disturbance and artefacts cannot be totally excluded. The resin embedding needs dehydration steps that can shrink the sediment. In some cases, cores can crack under the desiccation process by progressively replacing porewater with acetone as a preliminary step for final embedding procedure. These cracks are generally easily recognizable (they are visible in Fig. 1 as an example), but they add complexity to the images and can therefore represent an additional difficulty for their analysis.

Nevertheless, concerning the possibility of physical disturbance during embedding, the existent literature on FLEC procedures, including the CTG labeling steps, shows that the sediment interface is perfectly preserved during these procedures. If the experimental conditions are respected during the labeling time, there is no reason for the results to be biased by the incubation time. For example, Jesus et al. (2023), using the same protocol as in our study, reached to show the change in spatial orientation of benthic diatoms in the microphytobenthos at the sediment interface with respect to the lighting conditions.

Finally, the application of this method to natural environments with higher biodiversity would need the development of specific taxonomical keys, based on a section view instead of external view as usual, to identify the target species for geochemical or ecological proxies. Both in coastal and bathyal habitats, in fact, target species can differ only for small morphological details that cannot be easily seen on a cross section at present (e.g. spines, ribs, pustules...).

**5. Conclusions and perspectives**

The applications of microtomography span a wide range of research and engineering fields such as biomedical, materials science, archaeology, geoscience, life science, to name a few.

In our study, µCT-scanning of a sediment core belonging to an experimental microcosm allowed to observe and quantify the spatial distribution of a foraminiferal assemblage represented by two foraminiferal species, *Ammonia confertitesta* and *Heynesina germanica*. The study proves the usefulness of µCT-scan of sediment cores as a tool for ecological and palaeoecological purposes. Indeed, the results revealed that the two species that are generally reported as epifaunal (sensu lato) in fact live only below the sediment surface, in a shallow infaunal microhabitat restricted to the first 500 µm of depth. Our observations underline the importance of knowing the exact preferential microhabitat of species used as proxies for reliable reconstructions of past bottom water or pore-water conditions and the potential bias introduced by the core slicing in determining their vertical distribution.

Moreover, the same approach could allow to unravel ecological questions about feeding or interspecific competition strategies, or about the organisation of the assemblages in presence of sediment reworking, bioturbation, roots or other factors influencing the 3D space.

Finally, in a context of AI development for the automatization of foraminifera identification (e.g., Hsiang et al., 2019; Johansen and Sorensen, 2020; Marchant et al., 2020; Luijmes et al., 2024), the possibility to obtain automatised counting, living vs dead discrimination and taxonomical recognition of species on a 3D view as offered by CT-scan, should represent a major target for the next future.

**CRediT authorship contribution statement**

**M.P. Nardelli:** Conceptualization, Methodology, Investigation, Funding acquisition, Project administration, Writing first draft and reviews editing. **N. Vanderesse:** Conceptualization, Methodology, Investigation, Software, Validation, Data curation, Visualization, Writing first draft and reviews editing. **M. Moller:** Investigation, Resources, Writing – review & editing. **C. Guilhermic:** Investigation, Resources, Writing – review & editing. **A. Mouret:** Conceptualization, Methodology, Investigation, Project administration, Writing – review & editing.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgements

The study was funded by INSU, LEFE (BEGIN project) and LPG laboratory. The authors thank Rodolphe Perrot (SCIAM Angers) for the technical help in image acquisition at confocal microscopy and Laurent Lenta (LPG Nantes) for the preparation of longitudinal sections of the core for microscope analyses. They also thank Damien Le Moigne, Hélène Howa and Sophie Sanchez for their help in the experimental setting up and Sophie Quinchard and Eric Bénéteau for technical support in resin-embedding procedures. Many thanks also to Dr. Christine Barras and Pr. Meryem Mojtahid for their useful suggestions and comments on a first draft and to Pr. Gerhard Schmiedl and an anonymous reviewer for their useful suggestions to improve this final version.

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