# Combining morphological and molecular data to study past foraminiferal communities from a temperate coastal sediment core

Meryem Mojtahid, Magali Schweizer, Damien Le Moigne, Gwendoline

Grégoire, Anne Murat, Isabelle Poirier, Agnès Baltzer, Inge van Dijk,

Marie-Christine Morère-Le Paven, Martin Bourges, et al.

► To cite this version:

Meryem Mojtahid, Magali Schweizer, Damien Le Moigne, Gwendoline Grégoire, Anne Murat, et al. Combining morphological and molecular data to study past foraminiferal communities from a temperate coastal sediment core. Boreas, 2025, 10.1111/bor.70020. hal-05209068

HAL Id: hal-05209068

https://hal.inrae.fr/hal-05209068v1

Submitted on 13 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

**BOREAS**
An international journal
of Quaternary research

# Combining morphological and molecular data to study past foraminiferal communities from a temperate coastal sediment core

MERYEM MOJTAHID (ID), MAGALI SCHWEIZER, DAMIEN LE MOIGNE, GWENDOLINE GRÉGOIRE, ANNE MURAT, ISABELLE POIRIER, AGNÈS BALTZER, INGE VAN DIJK, MARIE-CHRISTINE MORÈRE-LE PAVEN, MARTIN BOURGES, MARIA PIA NARDELLI (ID), CHRISTINE BARRAS, EDOUARD METZGER AND AURELIA MOURET (ID)

BOREAS

<figure><img src="image_3.png" /></figure>

Mojtahid, M., Schweizer, M., Le Moigne, D., Grégoire, G., Murat, A., Poirier, I., Baltzer, A., van Dijk, I., Morère-Le Paven, M.-C., Bourges, M., Nardelli, M. P., Barras, C., Metzger, E. & Mouret, A.: Combining morphological and molecular data to study past foraminiferal communities from a temperate coastal sediment core. *Boreas*. https://doi.org/10.1111/bor.70020. ISSN 0300-9483.

This paper presents the results of a dual approach for assessing fossil benthic foraminiferal communities using both traditional morphology-based analyses and sedimentary ancient DNA (*sedaDNA*) metabarcoding. The main objectives are to test the feasibility of *sedaDNA* analyses to assess foraminiferal biodiversity in temperate shelf sediments (Le Croisic, France) off a major river system through time (Mid- to Late Holocene), and to point out the similarities and differences between classical and molecular methods. Our results show that, in contrast to the high foraminiferal diversity obtained from classic morphological analysis (over 140 taxa), the *sedaDNA* analysis yielded only 20 operational taxonomic units (OTUs), which can be considered as equivalent to species. This strongly suggests a bad preservation of foraminiferal DNA downcore, likely due to the relatively 'high' temperature of the study site (14 °C) and/or to a methodological bias (e.g. insufficient amount of extracted sediment). In the total *sedaDNA*, more than 90% of the reads were assigned to monothalamids (organic-shelled foraminifera). In contrast, only a small number of mineralized taxa, highly dominant when identified using the morphological approach, were detected. This could be due to the naturally higher abundance of monothalamids compared to hard-shelled foraminifera. While this abundance is reflected in *sedaDNA*, it is not preserved in fossil morphological assemblages. In addition, the *sedaDNA* of monothalamids might be easier to extract and their barcode to amplify than hard-shelled foraminifera. The discrepancies between the microfossil data and *sedaDNA* also include several species (e.g. *Ammonia confertitesta* (T6), *Elphidium oceanense* (S3), *Nonionella* sp. T4 and *Nonionella* sp. T6) that were rarely or not at all found in the fossil material which might be an indication of the presence of propagules, morphologically undetected in the >63 µm size fraction used. Finally, the presence of sequences of *A. confertitesta* and fossil specimens in the deep layers of the study cores suggests that this species, considered until now as recently invasive on the European coast, could have been present in the Atlantic coast several thousand years ago, much before any anthropogenic activity involving international shipping and commercial trades.

Meryem Mojtahid (*meryem.mojtahid@univ-angers.fr*), Magali Schweizer, Damien Le Moigne, Inge van Dijk, Martin Bourges, Maria Pia Nardelli, Christine Barras, Edouard Metzger and Aurelia Mouret, *Univ. Angers, Univ. Nantes, Univ. Le Mans, CNRS, LPG, Laboratoire de Planétologie et Géosciences, UMR 6112, 2 Bd Lavoisier, Angers Cedex 49045, France; Gwendoline Grégoire, Anne Murat and Isabelle Poirier, Conservatoire National des Arts et Métiers, Institut National des Sciences et Techniques de la Mer, EPN8, Boulevard Collignon, Tourlaville, Cherbourg en Cotentin 50110, France and LUSAC-Laboratoire Universitaire des Sciences Appliquées de Cherbourg, 60 rue Max-Pol Fouchet, CS 20082, Cherbourg-en-Cotentin 50130, France; Agnès Baltzer, *Univ. Nantes, Laboratoire Géolittomer/IGARUN, UMR6554, Nantes F-44300, France; Marie-Christine Morère-Le Paven, Univ. Angers, INRAE, IRHS, SFR4207 Qua-SaV, Angers 49000, France; received 6th February 2025, accepted 28th May 2025.*

Until very recently, marine sediment cores were mainly studied using the fossil remains (mostly mineral shells) of micro-organisms to assess the biodiversity through time. Because most marine species do not leave fossil remains, the evolution of marine biodiversity in these records is incomplete. Recent advances in molecular techniques offer a new angle to better reconstruct past ecosystem changes and detect shifts in species distributions, including the emergence and spread of invasive taxa. Indeed, even though DNA is predisposed to rapid degradation, it was shown that it may preserve in sediments up to two million years (e.g. Boere et al. 2011; Lejzerowicz et al. 2013; Kirkpatrick et al. 2016; De Schepper et al. 2019; Armbrecht et al. 2022; Kjær et al. 2022). Ancient environmental DNA (eDNA) is referred to as sedimentary ancient DNA (*sedaDNA*) when collected in sediment, which can be analysed through

high-throughput amplicon sequencing (metabarcoding) (e.g. Engel et al. 2020; Nguyen et al. 2023). Recently, *sedaDNA* metabarcoding studies have been increasingly used in Pleistocene marine sediment archives to track environmental changes in bacterial microbiomes (e.g. Ciobanu et al. 2012; Orsi et al. 2017; More et al. 2019) and eukaryotic communities (e.g. Coolen et al. 2013; Lejzerowicz et al. 2013; De Schepper et al. 2019; Armbrecht et al. 2022).

Among the microfossils widely found in marine sediments, foraminifera are a key group used in palaeoclimatic and palaeoenvironmental research. The few existing metabarcoding studies targeting particularly ancient foraminiferal DNA were mostly carried out in cold waters (1–4 °C), favouring an optimal DNA preservation: in the Arctic (Pawłowska et al. 2014, 2016, 2020) and the Antarctic (Demianiuk et al. 2024) oceans for Late Pleistocene sediments, in the South Atlantic abyssal sediments dating back
to the last glacial period (Lejzerowicz et al. 2013) and in the Holocene deep sediments of the Northwest Atlantic (Barrenechea Angeles et al. 2020). All these studies show that (i) foraminiferal *sedaDNA* is well preserved in marine sediment cores, (ii) most *sedaDNA* is assigned to non-fossilizing organic-shelled taxa and (iii) *sedaDNA* can be a meaningful addition to the multi-proxy toolkit usually used in documenting the evolution of past marine ecosystems. Here, we investigate foraminiferal *sedaDNA* in Holocene sediments located off the French coast near the mouth of the Loire River. Although the relatively warm waters could impact DNA conservation, the high sedimentation rates at this location could theoretically help preserve DNA. In temperate waters, some studies have demonstrated good preservation of foraminiferal DNA. For example, Barrenechea Angeles et al. (2023) analysed *sedaDNA* from a sediment core with a 220-year record collected in the Tyrrhenian Sea (Mediterranean). Similarly, Engel et al. (2020) successfully studied foraminiferal DNA in palaeo-tsunami deposits up to 2000 years old from the northwestern coast of Japan. However, only a limited number of studies have simultaneously evaluated and compared the traditional morphology-based approach of foraminifera with environmental DNA metabarcoding in general (e.g. Frontalini et al. 2020; Brinkmann et al. 2023; Nguyen et al. 2023) and *sedaDNA* in particular (Lejzerowicz et al. 2013; Pawłowska et al. 2016, 2020; Barrenechea Angeles et al. 2020; Demianiuk et al. 2024). Here, this dual approach is investigated with the objectives to (i) test the feasibility of using *sedaDNA* for biodiversity palaeoreconstructions in temperate shelf sediments characterized by high sedimentation rates and (ii) highlight the similarities and differences between classical and molecular methods.

## Study area

For this study, we performed micropalaeontological and metabarcoding analyses on marine sediment cores retrieved from the inner continental shelf off the Loire River mouth (Bay of Le Croisic; Fig. 1). This area was selected for its potentially favourable conditions for DNA preservation (i.e. high sedimentation rate off a major river system), well-documented sedimentation characteristics (e.g. Baltzer et al. 2015; Champilou et al. 2019) and extensive previous foraminiferal research (e.g. Thibault de Chanvalon et al. 2015, 2022; Durand et al. 2016, 2018; Mojtahid et al. 2016; Champilou 2020; Fouet et al. 2022, 2024), making it ideal for paleoenvironmental and *sedaDNA* studies.

## Hydrology

Along the French Atlantic coast, wave conditions are influenced by swells predominantly originating from the west-northwest and southwest directions (e.g. Gonçalves et al. 2014). These swells, combined with a

semi-diurnal macrotidal regime, result in intense hydrodynamic conditions in the Bay of Le Croisic.

The Bay of Le Croisic is also highly influenced by the Loire River dynamics (e.g. Proust et al. 2010; Durand et al. 2018) and sediment input (Lazure & Jégou 1998; Delmas et al. 2012). The Loire is the longest river in France (~1020 km), with a current average flow of about 900 m³ s⁻¹. Maximal flow values are reached during winter and early spring (>1800 m³ s⁻¹) whereas minimal values occur during summer and autumn (250 m³ s⁻¹) (http://www.hydro.eaufrance.fr). The Loire River delivers between ~0.5 and 0.8 Mt a⁻¹ of suspended fine sediments to the Bay of Biscay continental shelf via a turbid river plume (Jouanneau et al. 1999; Delmas et al. 2012). Due to the Coriolis effect and the poleward residual tidal current of 2–5 cm s⁻¹ flowing over the shelf (Pingree & Le Cann 1989; Charria et al. 2013), the river plume is dominantly oriented towards the northwest (Lazure & Jégou 1998). The Loire River plume plays a crucial role in shaping the physico-chemical properties of the adjacent continental shelf by delivering freshwater, sediments, and both organic and inorganic materials (e.g. Castaing et al. 1999; Costoya et al. 2017). These inputs significantly influence biological communities, particularly the distribution, abundance, and composition of planktonic and benthic organisms, including benthic foraminifera (e.g. Durand et al. 2018; Rombouts et al. 2019; Champilou 2020; Mertens et al. 2023).

Data from the Molit buoy, deployed at ~10 m depth in the nearby Bay of Vilaine (Retho et al. 2022), indicate that present bottom water temperatures average around 14 °C, ranging from ~7 °C in winter to ~20 °C in summer. Salinity varies seasonally as well, with an average of ~33.5, decreasing to ~30 during peak river discharge in winter and early spring, and rising to ~35 by late summer and early autumn (Retho et al. 2022). Bottom waters remain generally well oxygenated year-round, averaging ~5 mL L⁻¹ and fluctuating seasonally, reaching a minimum of ~2 mL L⁻¹ in summer due to stratification and a maximum of ~7 mL L⁻¹ in winter with enhanced wind-driven mixing (Retho et al. 2022). Phytoplankton blooms typically occur in spring, stimulated by nutrient input from the river, and again in late summer to early autumn, driven by elevated temperatures and stratification (Retho et al. 2022). The Bay of Le Croisic is also notable for its high density of pockmarks (Champilou et al. 2019), where diagenetic processes linked to *Hap-loops* communities may release nutrients such as nitrogen and phosphorus into the water column, thereby stimulating primary production (Judd & Hovland 2007; Champilou 2020).

## Holocene evolution

The Holocene sedimentary record of the Armorican continental shelf reflects three main stages of relative sea level (RSL) rise (Goslin et al. 2015). The first stage,
<figure><img src="image_4.png" /></figure>

Projection: Lambert Conformal Conic (Lambert 93) RS 1980

Geodetic system: RGF93

<caption>**Fig. 1.** Geographical context and sampling locations (maps modified from Durand *et al.* 2018). A. Location of the study area (black rectangle), the Loire River pathway (blue line) and its catchment area (blue area). BoB, Bay of Biscay. B. Digital Elevation Model (DEM) of the Loire outlet and the proximal inner shelf. DEM data are sourced from BD Alti® 75 m (IGN; resolution: 75 m) for land area and from Bathymetric DEM of Atlantic facade (Homonim project, Shom, 2015; resolution: ~110 m) for the sea area. C. Zoom on the study area with the location of the study cores (black dots; 'B', 'D' and 'Anais 3') and the seismic profiles presented in Fig. 2.</caption>

between ~8 and ~5.5 cal. ka BP, was marked by a relatively rapid transgression, with RSL rising from around –10 to –3 m. This was followed by a period of relative stability between ~5.5 and ~2.5 cal. ka BP, when sea level stabilized near –3 to –2 m. A third, more gradual increase has occurred over the most recent millennia. In the nearby Bay of Quiberon, Baltzer *et al.* (2015) report that after the RSL highstand in Mid-Holocene, a noticeable decline in sediment accumulation occurred. This reduction in sedimentation may be due to intensified hydrodynamic activity, which limited deposition and led to poor preservation of upper Holocene deposits.

Beyond RSL rise, Holocene sediment records from the Bay of Biscay continental shelf also reflect millennial-scale climatic variability (e.g. Mojtahid *et al.* 2019; Penaud *et al.* 2020). This variability appears to be driven by fluctuations in precipitation over major French river catchments and by changes in the Atlantic Meridional Overturning Circulation (AMOC) and North Atlantic Oscillation (NAO)-like mechanisms may have played a key role, regulating both regional humidity and wind stress. Solar forcing likely added another layer of variability to these long-term climate patterns.

In addition to natural climate drivers, human activity has significantly shaped the landscape, particularly through deforestation and land-use changes, which in

turn altered sediment supply to the ocean. Evidence of anthropogenic impact in the Loire River catchment dates back at least to ~2.0 cal. ka BP (e.g. Naughton *et al.* 2007), with some indications of influence as early as the Mesolithic–Neolithic transition (~8.0 cal. ka BP) (e.g. Visset *et al.* 2002; Cyprien *et al.* 2004; Arnaud *et al.* 2012). In more recent times, the Loire estuary has been extensively modified to support navigation to the Nantes Harbor, starting with the construction of flood-protection dykes in the 9th century (Moatar & Dupont 2016). During the 19th and 20th centuries, large-scale developments, including river narrowing (e.g. Bona & Prud'homme Lacroix 2009) and the dredging of a channel in 1930 (Dupain 1981), drastically transformed the estuary's morphology (Sanchez *et al.* 2000).

# Material and methods

## Sampling

A geophysical survey was conducted on board of the R/V Haliotis during the POPCORE oceanographic campaign in 2016 (https://doi.org/10.17600/16005300). The survey employed an interferometric sonar (Geoacoustic Geoswath, 250 kHz) coupled with a chirp echosounder profiler (1.7–5.5 kHz), acquiring transects at a speed of
3.5 knots. This provided high-resolution imagery of the sea floor with a vertical resolution of 25 cm and a horizontal resolution of 50 cm (Fig. 2A; Champilou et al. 2019). The geophysical data were used to select coring sites based on criteria such as sediment continuity, the presence of undisturbed horizontal deposits, and overall stratigraphic clarity.

Subsequently, a 4.13 m-long marine core (core D; 47°15.727N, 2°33.373W) was collected in 2018 onboard the R/V Thalia at 22.8 m water depth (Fig. 1). The nearby (1.3 km distance) 4.21 m-long marine core (core B; 47°16.417N, 2°33.283W), collected during the same cruise at 19 m water depth (Fig. 1), is used for ¹⁴C dating and comparison regarding specific analyses (e.g. morphotypes of *Ammonia* spp.). The coring was performed using a Kullenberg corer during the oceanographic campaign HOOPLACOR (https://doi.org/10.17600/18000534). We additionally used a modern sample from a 25 cm-long interface sediment core (core Anais 3; 47°16.443N, 2°34.680W; Fig. 1), retrieved in 2017 by scuba divers at about 19 m water depth during the campaign PLOUF17. This modern sample was collected to establish a reference dataset for comparison with ancient DNA, enabling the evaluation of preservation conditions. Once retrieved, all cores were stored in a cold room at 4 °C.

## Chronological framework and XRF data

No dating was performed on core D. However, the nearby core B benefited from five ¹⁴C dates measured by the Poznań Radiocarbon Laboratory (Poland) and one ¹⁴C date measured at the French national ARTEMIS ¹⁴C AMS facilities on well preserved (not broken and not abraded) bivalve shells and shell fragments (Table 1, Fig. S1). Because the 70.5 cm depth date showed an age inversion relative to the global trend, it was excluded from the calculation of the age model, which was constructed using linear interpolation between the remaining dated levels (Fig. S1). Absolute ¹⁴C dates were calibrated with the calib.org website (Stuiver et al. 2017) using the Marine 20 calibration curve and after applying a $\Delta R = -9\pm45$ published by Tisnérat-Laborde et al. (2010) from Le Croisic.

By analysing the downcore variations of major elements like calcium (Ca) and potassium (K), we made an attempt to correlate the major facies between the two cores (Fig. 2B). K and Ca were selected as they are reliably measured in Atlantic marine sediments, with K reflecting terrigenous input and Ca serving as a proxy for carbonate content (e.g. Govin et al. 2012; Durand et al. 2018). Bulk intensities of major elements (Ca, K) were measured using an AVAATECH X-Ray Fluorescence (XRF) core scanner at 1 cm resolution at EPOC laboratory (Bordeaux, France) for core B and using XRF spectrophotometry (Xsort SPECTRO, Ametek) for core D at ~20 cm resolution. XRF data (counts per

second) were measured using a 10–15 s count, and by setting the voltage to 10–30 kV. The results using Xsort SPECTRO were calibrated using the ‘Enviro-H’ method, which is tailored for rocks and adjusted through ICP-OES/MS analysis of 60 marine samples (Baux et al. 2019).

## Foraminiferal analyses

Cores D and B were sliced in half lengthwise, and one half of each core was sampled. In 2021, samples from both cores were used for morphological analyses, while only core D was sampled for metabarcoding analyses. Whenever possible, samples were taken from fine silty layers (Fig. 2B) to reduce the influence of reworked or transported material, to avoid layers with evidence of discontinuous sedimentation, and to ensure that the foraminifera's DNA is better preserved.

## Morphological analyses

Six samples (~1 cm thick sections) from core D were investigated for morphological analyses of benthic foraminifera, at the same sediment depths as for metabarcoding analyses (cf. below): 35–36, 75–76, 135–136, 315–316, 355–356 and 405–406 cm. A surface sample (1–2 cm depth) was also collected from core Anais 3. This depth differs from the *seda* DNA analysis depth (3.5–4.5 cm; cf. below) because the entire 3.5–4.5 cm sample was used for metabarcoding analysis, leaving no sediment available for morphological analyses. To avoid confusion, both depths from Anais 3 will be labelled as ‘surface sediments’ in the figures. All samples for morphological analyses were first weighed wet, then dried in a 50 °C oven and weighed again. Then, all samples were washed over 63 and 125 µm sieves. The >125 µm fraction was split with a dry Otto microsplitter when necessary, until at least 250–300 specimens were obtained in the final split. Then, specimens from the >125 µm fraction were picked and mounted on Plummer cell slides for taxonomic determination. As species of *Ammonia* are morphologically resembling each other (especially pseudo-cryptic species), they were taxonomically identified using an environmental SEM (Scanning Electron Microscope, Hitachi TM4000). For the distinction between *A. veneta* (T1), *A. aberdoveyensis* (T2) and *A. confertitesta* (T6), identification was performed according to the methodology of Richirt et al. (2019), with a general overview of the spiral sides and a 1000× magnification of the penultimate chamber to investigate pore size. In core B, the same method was applied to identify *Ammonia* specimens (>150 µm fraction) in seven samples (~1 cm thick layers) for comparison: 5–6, 53–54, 65–66, 105–106, 125–126, 276–277 and 365.5–366.5 cm.

We also assessed the composition of the small fraction (63–125 µm) in core D and the surface sample from core Anais 3. These samples were split with a dry Otto
<figure><img src="image_5.png" /><figcaption>Fig. 2. A. Chirp profiles 1 and 2 (cf. Fig. 1) with the location of cores D and B. U4 and U3 represent the main sedimentary units according to Champilou *et al.* (2019). B. Figure showing simplified sedimentary logs of cores D and B, along with the major element curves for Ca and K measured by XRF.</figcaption></figure>
<table><caption>**Table 1.** Radiocarbon dates on core B: all absolute AMS ¹⁴C ages are expressed in radiocarbon years BP (a BP), and calibrated ages are given in calendar years BP (cal. a BP). (*) indicates ¹⁴C age showing age inversion or incoherent results in comparison with the general tendency. Accordingly, this radiocarbon date was excluded from the calculation of the age model. Reservoir correction is inferred from Tisnérat-Laborde *et al.* (2010) (Le Croisie site).</caption><thead><tr><th>Sample reference</th><th>Laboratory</th><th>Dated material</th><th>Sample depth (cm)</th><th>AMS ¹⁴C ages (radiocarbon a BP)</th><th>ΔR</th><th>Calibration curve</th><th>Calibrated ages 2σ range (cal. a BP)</th><th>Median probability (cal. a BP)</th></tr></thead><tbody><tr><td>Hooplacor B1S1 53–54</td><td>Poznań</td><td>Complete bivalve shell</td><td>53.5</td><td>5290±35</td><td>-9±45</td><td>Marine 20</td><td>5293 5642</td><td>5472</td></tr><tr><td>Hooplacor B1S1 65–66</td><td>Poznań</td><td>Complete bivalve shell</td><td>65.5</td><td>5335±35</td><td>-9±45</td><td>Marine 20</td><td>5321 5693</td><td>5517</td></tr><tr><td>Hooplacor B1S1 70–71</td><td>CEA Artemis</td><td>Shell fragments</td><td>70.5</td><td>4565±35</td><td>-9±45</td><td>Marine 20</td><td>4398* 4806*</td><td>4592*</td></tr><tr><td>Hooplacor B1S2 25–26</td><td>Poznań</td><td>Shell fragments</td><td>125.5</td><td>5780±35</td><td>-9±45</td><td>Marine 20</td><td>5811 6201</td><td>6006</td></tr><tr><td>Hooplacor B1S3 76–77</td><td>Poznań</td><td>Complete bivalve shell</td><td>276.5</td><td>6680±35</td><td>-9±45</td><td>Marine 20</td><td>6778 7179</td><td>6992</td></tr><tr><td>Hooplacor B1S4 65–66</td><td>Poznań</td><td>Complete bivalve shell</td><td>366</td><td>7170±35</td><td>-9±45</td><td>Marine 20</td><td>7312 7629</td><td>7477</td></tr></tbody></table>

microsplitter until a minimum of 250–300 individuals was obtained in the final split. One out of the seven samples was picked, put on Plummer cell slides, and determined. For the rest of the samples, foraminifera were counted and determined taxonomically directly under the stereomicroscope. Due to the small size of individuals, the characteristic features of *Ammonia* spp. (pore size, height of sutures, etc.) could not be used to distinguish between species.

The absolute densities are expressed as the number of individuals cm⁻³ of sediment, and the relative abundances (% of total abundance) were calculated for all samples.

### Metabarcoding analysis of foraminifera

To prevent cross-contamination due to the disruption of the core structure, the tube of core D was drilled carefully without touching the sediment (2 cm wide holes using a hole saw), directly after sediment coring in 2018 and at the same sediment depths sampled later for morphological analyses (cf. above). Approximately 5 g of sediment were collected for each subsample using a sterile 10 mL truncated syringe and transferred into sterile plastic containers. To avoid contamination, samples were taken from the inner part of the core. Outer sediment layers that had contact with the core lining were discarded. Once sediment was sampled for metabarcoding, the hole was filled with polystyrene pieces and tightly closed with tape.

The interface core Anais 3 was sliced using a push core. The 3.5–4.5 cm sediment depth was sampled using a sterile 10 mL truncated syringe after removing the part that was in contact with the sampling tube. The sample was entirely used for *sedaDNA* and transferred into a sterile plastic container. All *sedaDNA* subsamples were frozen at -20 °C directly after sampling until further analysis.

In 2021, the total DNA content of each subsample was extracted from sediment using the DNeasy PowerMax Soil Kit (Qiagen) following the manufacturer protocol. To avoid extraneous DNA contamination, all extractions and amplifications were performed in a clean room (Moltech laboratory, Angers) or a molecular laboratory (IRHS, Angers) free from any foraminiferal DNA or PCR product. To monitor cross-contamination, a negative control was added during the DNA extraction (named negative_extraction) and another one during the DNA amplification (named PCR8neg) (extraction kit with no sediment and PCR mix with no added DNA). The first amplification was performed with AccuPrime™ Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific) and an initial denaturation of 94 °C for 1 min followed by 60 cycles at 94 °C for 30 s (denaturation), at 50 °C for 30 s (primer annealing) and at 72 °C for 1 min (extension) plus a final extension at 72 °C for 3 min. Foraminifera-specific primers s14F1 (AAGGGCACCACAAGAACGC; Pawlowski 2000) and s15r (CCACCTATCACAYAATCATG; Lejzero-wicz *et al.* 2014) were used to amplify a 135–190 bp fragment situated at the 3' end of the SSU rRNA gene in the V9 region, which is the barcode for foraminifera (Pawlowski & Holzmann 2014). Amplicons, including two negative controls (one for extraction and one for amplification step), were purified by magnetic beads. A second amplification was performed with the same polymerase (first denaturation step at 94 °C (1 min), followed by 12 cycles at 94 °C (1 min), 55 °C (1 min) and 68 °C (1 min), and a final elongation at 68 °C for 10 min) to incorporate Illumina adapters and tags to distinguish the samples. Second PCR amplicons were also purified by magnetic beads, quantified with the QuantIT Pico-Green dsDNA Assay Kit (ThermoFisher Scientific) and pooled in equimolar concentrations. The concentration of the pool was monitored with quantitative PCR (KAPA SYBR FAST, Merck). Amplicon libraries were
mixed with 10% PhiX and sequenced with MiSeq reagent
kit v2 500 cycles. Library constructions, MiSeq sequenc-
ing and demultiplexing were performed at the ANAN
platform (SFR QUASAV, INRAE Beaucouzé, France).

Raw reads were demultiplexed to samples with DADA2 v. 1.16 (Callahan et al. 2016) and MiSeq overhangs and primers were removed with Cutadapt v. 3.5 (Martin 2011). Reads were then processed using DADA2 v. 1.16 (Callahan et al. 2016) in R v. 4.1.2 (R Core Team 2021). Raw reads were quality controlled by truncating the reads (forward and reverse length of 200 bp) and filtering to a maximum number of ‘expected errors’ of two. Amplicon Sequence Variants (ASV) were dereplicated if identical, clustered and pair-end reads merged using a minimum overlap of 12 bp and maximum mismatch of 0 bp. Chimeras were removed using the ‘pooled’ method. The ASVs were taxonomically assigned using PR² v. 4.14.0 (Guillou et al. 2013). Demultiplexed raw reads were deposited in the Sequence Read Archive (SRA) at NCBI (https://www.ncbi.nlm.nih.gov/sra/PRJNA1104619).

Non-foraminiferal ASVs (i.e. not recognized as fora-
minifera in the DADA2 analysis) were removed. The per-
centages of reads present in the negative controls
(negative_extraction & PCR8neg) versus the total of
samples were calculated for the different ASVs, and
the ASVs with percentages of contamination above 5%
were removed. The taxonomic assignment of each
ASV was manually checked by sequence alignment
and comparisons with reference databases, and ASVs
belonging to the same taxon were pooled into opera-
tional taxonomic units (OTUs).

Results

Sedimentological characteristics and chronological
framework

The lithological characteristics of cores D and B are gen-
erally comparable, consisting primarily of fine silts to
silty sands rich in mollusc shells, including both intact
specimens and shell debris (Fig. 2B). Based on XRF data
and lithological descriptions, both cores show silty sed-
imentation at the bottom (~420–290 cm for core D and
~420–380 cm for core B) and at the top (~0–100 cm for
both cores). In between, the sediment is coarser, consist-
ing of sandy silts enriched with shell debris, which is also
marked by the highest concentrations of Ca (Fig. 2B).
Furthermore, the cores recovered sediments from the
same sedimentary unit as shown from the seismic profiles
(Fig. 2A).

¹⁴C dates of core B show that sediments below 50 cm depth are mostly of Mid-Holocene age (7.5–5.5 cal. ka BP) (Fig. S1). Above, the sedimentation rate either slowed considerably or sedimentation faced high hydrodynamics, resulting in a poor preservation of upper Holocene sediments (e.g. Baltzer *et al.* 2015). We suggest

that the chronology of core B can be applied to core D
based on the overall observed lateral continuities in lith-
ological units (Fig. 2B). However, this should only be
considered as an approximate general chronological
framework and not as a definitive determination of the
age of the sedimentary units in core D.

Morphological data

The absolute densities of benthic foraminifera from the >125 µm fraction vary between 137 ind. cm⁻³ at the surface and 7728 ind. cm⁻³ at 75–76 (Fig. 3). A total number of 146 taxa were identified, among which 24 showed relative abundances >2.5%, and 14 showed relative abundances >5% in at least one sample. Among these 14 abundant species, *Planorbulina mediterranensis* (14% on average between all samples), *Gavelinopsis praegeri* (12.5%) and *Quinqueloculina* spp. (5.6%) are continuously dominant along the record. *Lobatula lobatula* (6.1%) and *Ammonia aberdoveyensis* (7%) are highly present between 136 and 35 cm. *Adelosina* spp. (5%) and *Haynesina germanica* (5%) are mostly present in samples below 135 cm depth. *Ammonia beccarii*, *Cibicides refulgens* and *Elphidium* species (E. advenum, E. gerthi, E. margaritaceum, E. selseyense) do not show any pattern. *Eggerelloides scaber* (10%) is only present at the surface.

The absolute densities of benthic foraminifera from the 63 to 125 µm fraction vary between 5616 ind. cm⁻³ at the surface and 63 680 ind. cm⁻³ at 355–356 cm depth (Fig. 3). A total number of 140 taxa were identified, among which 18 show relative abundances >2.5% (Fig. 3), and 12 show relative abundances >5% in at least one sample. All these taxa are also found in the >125 µm fraction. Similarly to the >125 µm fraction, *G. praegeri* (13.4%) is dominant at all depths. *Ammonia* spp. (4%), cibicidids (including *Lobatula*, *Cibicoides* and *Cibicides*) (13.2%), *Elphidium* spp. (3%), *Haynesina* spp. (6.5%) and *Quinqueloculina* spp. (2.3%) do not show any pattern. The difference with the >125 µm fraction is the high dominance of *Bolivina* spp. (17% in the 63–125 µm vs. 0.3% in the >125 µm) all along the record and the more important contribution of *Cassidulina* spp. (4.2% in the 63–125 µm vs. 0.3% in the >125 µm) and *Discorbis* spp. to the assemblage (3.1% in the 63–125 µm vs. 0.9% in the >125 µm).

All *Ammonia* specimens from the coarse fraction were identified using SEM images. In both cores D and B, *A. aberdoveyensis*, *A. beccarii* and *A. falsobeccarii* dominate (Fig. 4). *Ammonia veneta* is present in both cores but in lower numbers. *Ammonia confertitesta* is absent in core D and present in very low numbers in some samples of core B (5–6, 105–106, 125–126 cm).

SedaDNA analyses

The High Throughput Sequencing of the six samples of core D and the surface sample of core Anais 3 and the two
<figure><img src="image_6.png" /><figcaption>Fig. 3. Relative abundances of benthic foraminiferal species present with >2.5% in at least one sample in the 63–125 µm and >125 µm fractions of cores D and Anais 3. The absolute densities (ind. 50 cm⁻³) are noted on the right side of the histograms (N = the total number when multiplying by the split). The differentiation between *Ammonia* morphotypes was not possible for the 63–125 µm fraction. For the >125 µm fraction, the percentages of the *Ammonia* species were summed together for a better comparison with the small fraction. The details of the *Ammonia* species are shown in Fig. 4.</figcaption></figure>

controls gave 676 703 raw reads among which 375 577 reads (representing 55.5% of the total reads) were retained after the cleaning process with DADA2. Among the 108 ASVs obtained with the DADA2 analysis, five were not assigned to Foraminifera. Seventeen additional ASVs had a contamination rate above 5%. These 22 ASVs were removed. The remaining 86 ASVs, comprising 354 654 reads, were checked manually one by one, comparing them with alignments of sequences from known identity. It was decided to keep all remaining ASVs, including the four with less than 10 reads, although their low number of reads was less significant than more abundant OTUs. These ASVs could be merged into 20 OTUs (Table 2). Five of six ASVs with a contamination <5% were grouped in one OTU identified as *Nonionella* sp. T6. The sixth one was identified as *Epistominella* sp. and was part of another OTU without contamination. The nine OTUs belonging to Rotaliida (hyaline calcareous foraminifera) were identified at the

species level and represented 35% of the retained reads (Table 2). Among the 10 OTUs belonging to monothalamids (soft-shelled foraminifera) and representing 65% of the reads, only 2 were identified as known species. Seven others were identified as environmental clades, and one was unidentified (Table 2). A final OTU, with only four reads, was attributed to Miliolida.

The number of reads downcore varies between a minimum of 5489 at 135–136 cm sediment depth and a maximum of 87 755 at 75–76 cm sediment depth (Fig. 5). In all samples, the soft-shelled foraminifera are highly dominant (>95%), except for the surface sediment and at 75–76 cm depth where they are nearly absent (Fig. 5). All OTUs from hard-shelled foraminifera could be identified to the genus or species level. Among them, *A. aberdoveyensis*, *A. veneta*, *L. lobatula*, *E. selseyense* and *E. oceanense* are also common in the morphological assemblage (Fig. 6). *A. confertitesta* is detected in *seda*DNA at 35–36 cm (only 3 reads) and 75–76 cm (1547 reads)
<figure><img src="image_7.png" /><figcaption>Fig. 4. Relative abundances of species determined using SEM imaging within the *Ammonia* group in core Anais 3, core D (>125 µm fraction) and core B (>150 µm fraction). *N* represents the counted number of picked specimens (not the total number when multiplying by the split).</figcaption></figure>

depths (Figs 5, 6) whereas it was not identified using the
morphological approach in core D (Fig. 4). *Epistomi-
nella* sp. detected in *sedaDNA* is found in the morpholog-
ical analyses of the small fraction (63–125 µm), although
in very low numbers. *Nonionella* spp. found in *sedaDNA*
are not present in the morphological analysis (Fig. 3).

Discussion

*SedaDNA preservation and composition*

Our study achieved an acceptable number of good reads
(over 350 000), with an average of about 43 750 reads per
sample. However, the *sedaDNA* analysis identified only
86 ASVs, which were grouped into 20 OTUs (Table 2).
These numbers are low compared to other environmental
*sedaDNA* studies targeting foraminifera (169 OTUs out
of 256 925 reads in Lejzerowicz *et al.* (2013); 394 OTUs
out of 8 700 815 reads in Pawłowska *et al.* (2014); 1383
OTUs out of 4 227 450 reads in Demianiuk *et al.* 2024).
Moreover, except OTU1, which was present
in high numbers in four different levels, the other OTUs
represented by more than 10 reads (OTU2-OTU15) were
only abundant in one level each (Table 2). These two
points strongly suggest a bad preservation of foraminif-
eral DNA downcore (only the most abundant taxa would
have their DNA preserved over time) in our study site
and/or methodological biases (e.g. amount of extracted
sediment). Although Dell'Anno *et al.* (2005) suggested
that large amounts of DNA may be preserved in benthic
systems characterized by a relatively high temperature
(~13 °C), such as the bottom waters of the Bay of Le
Croisic (14 °C as yearly average; Retho *et al.* 2022), many
studies reported that extracellular DNA preservation in

sedimentary deposits over geological time scales is
favoured by low temperatures and anoxic conditions
(e.g. Coolen & Overmann 1998; Willerslev *et al.* 2003).
This is supported by three studies conducted in cooler
environments (Svalbard, the Ross Sea and the South
Atlantic deep sea) which analysed *sedaDNA* of forami-
nifera and which reported higher numbers of OTUs com-
pared to our study (Lejzerowicz *et al.* 2013; Pawłowska
*et al.* 2014; Demianiuk *et al.* 2024). In addition to these
taphonomic constraints, *sedaDNA* preservation in the
Bay of Le Croisic Bay should be considered in light of
the Mid-Holocene environmental context, when most
of the sediments in our core were likely deposited. For
instance, elevated sea temperatures during the Holocene
Thermal Maximum (e.g. Mary *et al.* 2017) may have
enhanced microbial activity and enzymatic degradation,
contributing to the bad preservation of *sedaDNA*.

Besides, storing sediments at -20 °C for nearly
3 years before extracting DNA may have altered DNA
preservation. Although -20 °C reduces enzymatic and
microbial activity that can degrade sedimentary DNA,
it is less effective than ultra-low temperatures, such as
-80 °C, in maintaining DNA integrity. Finally, the
amount of sediment used (~5 g) may be insufficient to
obtain enough *sedaDNA*, especially if a significant por-
tion has degraded due to high water temperatures and/or
non-ideal storage temperature. To address this, it is cru-
cial to maximize the amount of sediment extracted.
Increasing the sediment quantity would help recover
more *sedaDNA*, ensuring a more comprehensive repre-
sentation of the site's genetic diversity. Moreover, mod-
ern eDNA studies showed that 10 g of sediment might be
necessary for the sample's DNA content to be
representative of an environment's microbial commu-
<table><caption>Table 2. Table listing the OTUs obtained through the high throughput sequencing and their taxonomical assignments for the six samples of core Anais 3. Soft-shelled foraminifera are highlighted in grey. ASVs with asterisks indicate merged ASVs representing the same species. The numbers of reads <10 are given in italics.</caption><thead><tr><th>OTU</th><th>ASV</th><th>Class</th><th>Order</th><th>Family</th><th>Genus</th><th>Species</th><th>Surface</th><th>35–36 cm</th><th>75–76 cm</th><th>135–136 cm</th><th>315–316 cm</th><th>355–356 cm</th><th>405–406 cm</th></tr></thead><tbody><tr><td>OTU001</td><td>ASV001*</td><td>Monothalamids</td><td>NA</td><td>Saccaminidae</td><td>NA</td><td>Saccaminid</td><td>0</td><td>71 362</td><td>0</td><td>0</td><td>10 388</td><td>24 330</td><td>75 986</td></tr><tr><td>OTU002</td><td>ASV002*</td><td>Globothalamea</td><td>Rotalida</td><td>Nonionidae</td><td>Nonionella</td><td>Nonionella sp. T6</td><td>1</td><td>0</td><td>62 924</td><td>1</td><td>0</td><td>0</td><td>2</td></tr><tr><td>OTU003</td><td>ASV004*</td><td>Globothalamea</td><td>Rotalida</td><td>Epistominellidae</td><td>Epistominella</td><td>Epistominella sp.</td><td>31 813</td><td>0</td><td>1</td><td>0</td><td>1</td><td>30</td><td>2</td></tr><tr><td>OTU004</td><td>ASV019*</td><td>Monothalamids</td><td>NA</td><td>NA</td><td>NA</td><td>Monothalamid</td><td>0</td><td>0</td><td>1</td><td>0</td><td>24 923</td><td>34</td><td>1</td></tr><tr><td>OTU005</td><td>ASV010*</td><td>Globothalamea</td><td>Rotalida</td><td>Cibicididae</td><td>Lobatula</td><td>ICEMON7</td><td>0</td><td>0</td><td>23 279</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>OTU006</td><td>ASV016*</td><td>Monothalamids</td><td>NA</td><td>Saccaminidae</td><td>NA</td><td>Saccaminid id.</td><td>1</td><td>0</td><td>0</td><td>0</td><td>3</td><td>14 391</td><td>0</td></tr><tr><td>OTU007</td><td>ASV028*</td><td>Monothalamids</td><td>NA</td><td>NA</td><td>NA</td><td>ADEMéd-ASV1422</td><td>0</td><td>0</td><td>0</td><td>5390</td><td>0</td><td>0</td><td>0</td></tr><tr><td>OTU008</td><td>ASV031*</td><td>Monothalamids</td><td>NA</td><td>NA</td><td>NA</td><td>Monothalamid sp. 4008</td><td>0</td><td>3234</td><td>0</td><td>27</td><td>0</td><td>0</td><td>0</td></tr><tr><td>OTU009</td><td>ASV049*</td><td>Globothalamea</td><td>Rotalida</td><td>Ammoniidae</td><td>Ammonia</td><td>Uncult foram IC19 (AY452796)</td><td>0</td><td>3</td><td>1547</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>OTU010</td><td>ASV053*</td><td>Globothalamea</td><td>Rotalida</td><td>Nonionidae</td><td>Nonionella</td><td>Ammonia confertitesta (T6)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>734</td></tr><tr><td>OTU011</td><td>ASV058*</td><td>Monothalamids</td><td>NA</td><td>NA</td><td>NA</td><td>Nonionella sp. T4</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>356</td><td>0</td></tr><tr><td>OTU012</td><td>ASV087*</td><td>Globothalamea</td><td>Rotalida</td><td>Ammoniidae</td><td>Ammonia</td><td>id. IB21-ASV262</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>72</td></tr><tr><td>OTU013</td><td>ASV084*</td><td>Globothalamea</td><td>Rotalida</td><td>Ammoniidae</td><td>Ammonia</td><td>Aberdoveyensis (T2b)</td><td>0</td><td>0</td><td>0</td><td>67</td><td>1</td><td>0</td><td>0</td></tr><tr><td>OTU014</td><td>ASV083*</td><td>Monothalamids</td><td>Monothalamids</td><td>NA</td><td>Psammophaga</td><td>Ammonia veneta (T1)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>63</td><td>0</td></tr><tr><td>OTU015</td><td>ASV094</td><td>Globothalamea</td><td>Rotalida</td><td>Elphidiidae</td><td>Elphidium</td><td>crystallifera</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>14</td><td>0</td></tr><tr><td>OTU016</td><td>ASV097</td><td>Monothalamids</td><td>NA</td><td>NA</td><td>Cedhagenia</td><td>Elphidium oceanense (S3)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>2</td></tr><tr><td>OTU017</td><td>ASV096</td><td>Monothalamids</td><td>NA</td><td>Saccaminidae</td><td>NA</td><td>Cedhagenia saltata</td><td>3</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td></tr><tr><td>OTU018</td><td>ASV104</td><td>Monothalamids</td><td>NA</td><td>NA</td><td>NA</td><td>Saccaminid sp. 2399</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>0</td></tr><tr><td>OTU019</td><td>ASV100</td><td>Tubothalamea</td><td>Miliolida</td><td>NA</td><td>Elphidium</td><td>Vellaria or Psammophaga?</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>2</td><td>0</td></tr><tr><td>OTU020</td><td>ASV105</td><td>Globothalamea</td><td>Rotalida</td><td>Elphidiidae</td><td>Elphidium</td><td>NA</td><td>0</td><td>1</td><td>3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>Elphidium selsevense (S5)</td><td>3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>Total</td><td>31 821</td><td>74 600</td><td>87 755</td><td>5489</td><td>35 321</td><td>39 220</td><td>76 800</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>Fig. 5. Number of reads per sediment depth, and the relative abundances and the OTUs according to their taxonomic assignments in cores D and Anais 3. The asterisks refer to soft-shelled foraminifera.</figcaption></figure>

nity, including microeukaryotes such as foraminifera (e.g. Brandt *et al.* 2020; Brinkmann *et al.* 2023). With smaller quantities of sediment, eDNA derived from propagules is proportionally more abundant as there are fewer adult specimens (Brinkmann *et al.* 2023). In our *seda*DNA samples, this was probably indicated by the abundance of *Elphidium oceanense* (S3), *Nonionella* sp. T4 and *Nonionella* sp. T6 which are not found in the morpho-assemblage (Fig. 6) (see below).

In our samples, the number of reads attributed to monothalamids accounted for >90% of the total diversity. Comparatively, multi-chambered hard-shelled taxa belonging to Globothalamea (Rotaliida) and Tubothalamea (Miliolida), although detected in most samples, are only represented by a few reads (<10%), except in the surface sediment and at 75–76 cm depth (Fig. 5). The dominance of monothalamids was also observed in previous foraminiferal research based on molecular methods, either on *seda*DNA (e.g. Lejzerowicz *et al.* 2013; Pawłowska *et al.* 2014; Barrenechea Angeles *et al.* 2023; Demianiuk *et al.* 2024) or modern eDNA (e.g. Lejzerowicz *et al.* 2014; Al-Enezi *et al.* 2022; Cao *et al.* 2022; Brinkmann *et al.* 2023; Singer *et al.* 2023; Maeda *et al.* 2024). These studies suggested several factors to explain the high proportion of monothalamids: (i) a naturally higher abundance of monothalamids compared to hard-shelled foraminifera, (ii) monothalamids might be easier to extract as they have an organic test and (iii) the shorter barcode size in some monothalamids (i.e. saccaminids) favours

their amplification and sequencing compared to taxa with a longer barcode. Among the OTUs assigned to monothalamids, only two minor species (<1% contribution to the total assemblage) were identified as known species, *Cedhagenia saltata* and *Psammophaga crystallifera*. A large part of monothalamids could not be assigned to any known reference sequence because relatively few species from this group have been both morphologically identified and DNA barcoded in reference databases (Singer *et al.* 2023; Holzmann *et al.* 2024), despite their high overall diversity. This foraminiferal group is also usually overlooked by micropaleontologists because their soft shells get destroyed during routine morphological investigations of modern material or are not preserved in fossil assemblages (e.g. Gooday *et al.* 1996; Duros *et al.* 2017; Fontanier *et al.* 2022). Contrary to monothalamous foraminifera, most of the rotaliids found in the molecular assemblage of core D could be assigned to the species level (Fig. 5, Table 2). There is currently a higher number of sequences available for Globothalamea (78%) than for monothalamids (16%) in the reference public database GenBanks (Singer *et al.* 2023; Holzmann *et al.* 2024). Metabarcoding data strongly reinforce the important contribution that monothalamids make to foraminiferal communities but also reveal that several OTUs could not be assigned to any foraminiferal taxon. This emphasizes the importance of single-cell foraminiferal barcoding to improve the database of DNA sequences, especially for monothalamous species.
<figure><img src="image_9.png" /><figcaption>Fig. 6. A. Relative abundances of taxa present with >2.5% in at least one sample in the >63 µm fraction (63–125 µm + >125 µm fractions) of cores D and Anais 3. The absolute densities per 50 cm² (N = the total number when multiplying by the split) are indicated on the side of the histograms. B. The relative abundances of reads of the fossilizing hard-shelled foraminifera (N = the total number of reads of the hard-shelled taxa). The asterisks indicate sediment levels (35–36 and 315–316 cm) with very low read counts making percentage-based histograms unreliable: 4 (3 reads for *Ammonia confertitesta* (T6) and 1 read for an undetermined miliolid), and 1 (*Epistominella* sp.), respectively.</figcaption></figure>

### Comparison of foraminiferal communities in molecular and morphological data

The *sedaDNA*-s taxonomic composition differs from the microfossil assemblage at different levels:

* Only Rotaliida, Miliolida and Textulariida were observed as microfossils while the molecular data was dominated by sequences of non-fossilized monothalamids (Figs 5, 6). Here, the absence of monothalamids is due to their bad preservation in the fossil record (Gooday *et al.* 1996). This was also observed in other studies comparing foraminiferal

morphological and *sedaDNA* metabarcoding data (e.g. Lejzerowicz *et al.* 2013; Pawłowska *et al.* 2014; Demianiuk *et al.* 2024).

* Despite the high number of reads sequenced in the different sediment levels of cores Anais 3 and D, only very few of the mineralized morphotaxa were identified as OTUs: *Ammonia aberdoveyensis* (T2), *Ammonia veneta* (T1), *Lobatula lobatula* (B), *Elphidium oceanense* (S3), *Epistominella* spp. In contrast, the most common morphotaxa, such as *Gavelinopsis praegeri*, *Bolivina* spp., *Cassidulina* spp., *Bulimina* spp., *Planorbulina mediterranensis*, *Quinqueloculina* spp., were not identified by *sedaDNA* (Fig. 6). This
divergence between morphological and metabarcoding data was also observed in other studies with fossil (e.g. Lejzerowicz et al. 2013; Pawłowska et al. 2014; Demianiuk et al. 2024) and modern material (e.g. Cavaliere et al. 2021; Al-Enezi et al. 2022; Brinkmann et al. 2023). Several factors could explain such a result. As discussed above, the overall bad preservation of DNA in our study core could be one of the main reasons. The volume of sediment samples was probably not large enough to reveal the total diversity (Pawlowski et al. 2022). Furthermore, monothalamids are more frequently found in *seda*DNA than hard-shelled foraminifera (e.g. Lejzerowicz et al. 2013; Pawłowska et al. 2014; Demianiuk et al. 2024; present study). This observation may indicate that monothalamids are naturally more abundant or that their DNA is released into sediments more readily. Once released, their DNA might bind to sediment grains, providing protection from bacterial decay (Demianiuk et al. 2024).

* The discrepancies between the microfossil data and *seda*DNA also include the species that were rarely or not found in the fossil material of core D (*Ammonia confertitesta* (T6), *Epistominella* sp., *Elphidium oceanense* (S3), *Nonionella* sp. T4 and *Nonionella* sp. T6) (Fig. 6). This might be an effect of the presence of propagules, morphologically undetected in the >63 µm size fraction we used. Most of these never grow to maturity, and thus, they are lost in the fossil record but inflate the DNA record (Alve & Goldstein 2010; Goldstein & Alve 2011). Interestingly, *Ammonia confertitesta* (T6), *Nonionella* sp. T4 and T6 (as *N. stella*) and *Elphidium oceanense* (S3) were also dominant in the environmental DNA data of the Gullmar fjord but rarely or not reported as morphospecies (Brinkmann et al. 2023). The authors interpret their presence as propagules in the sediment. The same was observed for *A. confertitesta* on the French Atlantic coast (Fouet et al. 2024). For *Epistominella*, it could be due to the small size of adult specimens, as they were detected morphologically in the small fraction, but not the large one.

* Although the DNA sequences of some of the species dominating the fossil record (e.g. *Lobatula lobatula*, *Elphidium oceanense*) are present in one or two core levels, their abundance and distribution do not reflect those in microfossil assemblages (Fig. 6). The dissimilarity of taxon composition and abundances between morphological and *seda*DNA-based analyses of the same samples is common in ancient and modern DNA investigations of foraminifera (e.g. Pawłowska et al. 2014; Cao et al. 2022; Barrenechea Angeles et al. 2023; Fouet et al. 2024). Pawłowska et al. (2014) explain such a discrepancy by a methodological bias involving a differential effectiveness of DNA extractions and PCR amplification, making the quantification of species occurrence diffi-

cult (e.g. Lloyd *et al.* 2010). Another explanation is that some species are widely present in the samples but in low numbers in each sample. More sediment is examined for morphological analyses than for molecular ones, and the quantity of extracted DNA is not sufficient to detect these species properly (Pawlowski *et al.* 2014; Fouet *et al.* 2024). Here, in addition, the dissimilarity in distribution can be explained by the bad preservation of *seda*DNA. Also, there is a wide intragenomic variability in foraminifera, changing the number of DNA genomic copies across species (Weber & Pawlowski 2014).

* The taxonomic groups Miliolida and Textulariida are absent (or nearly absent) from the *seda*DNA data while several species of these groups are present in the fossil record with >5% (e.g. *Miliolinella subrotunda*, *Quinqueloculina* spp., *Textularia conica*; Fig. 6). For Miliolida, only one OTU belonged to this group and no species could be assigned (Table 2). The miliolids are usually underrepresented in metabarcoding data (e.g. Pawlowski *et al.* 2016; Shi *et al.* 2021; Brinkmann *et al.* 2023; Singer *et al.* 2023) likely due to the divergence in the primer regions (Pawlowski *et al.* 2014). For the textulariids, the number of OTUs can also be lower compared to the morphological data (e.g. Pawlowski *et al.* 2016), which can be explained by the lack of reference data in some cases (Pawlowski *et al.* 2014), but does not apply here as all the OTUs were identified at the order level.

*Is *Ammonia confertitesta* (T6) an invasive species on the French Atlantic coast?*

In this study, the combination of morphological and molecular approaches is tentatively applied to detect the emergence of *Ammonia confertitesta* (T6). In the European coast, *A. confertitesta* (T6) was suggested to be an invasive species by several studies (e.g. Pawlowski & Holzmann 2008; Schweizer *et al.* 2011). This is due to the fact that there is a biogeographical gap between Japan and northeast China, its presumed original habitat (Hayward *et al.* 2004; Toyofuku *et al.* 2005) and European coasts (Schweizer *et al.* 2011; Bird *et al.* 2020; Richirt *et al.* 2021). It has been reported that this species arrived in the Baltic Sea around AD 2000 where no *Ammonia* species were present before (Polovodova *et al.* 2009; Schweizer *et al.* 2011). An anthropogenic introduction via ship ballast water or with imported Japanese oysters was hypothesized (Pawlowski & Holzmann 2008). More recently, the investigation of a long sediment core from the Loire estuary (near our study area; Fig. 1) with morphological analysis estimated its introduction back at least to the 18th century, most likely via shipping and the increase of trade with China and India from the 16th and 17th centuries (Fouet 2022).

Our morphological data from core D did not detect the presence of *A. confertitesta* (T6), at least not in the
>125 µm size fraction (Fig. 3) following the methodology of Richirt *et al.* (2019). However, this species was detected in the *sedaDNA* analysis, at a sediment depth of 75–76 cm (Fig. 5). To estimate the age of this depth interval in core D, we compared it with the well-dated nearby core B, suggesting an approximate age of several thousand years (Fig. 5). Based on the observed lateral continuity in lithological units between the two cores (Fig. 2), we propose that the chronology of core B can be tentatively applied to core D. However, in the absence of direct chronological control, this correlation should be treated with caution, as local variations in sedimentation rates or depositional conditions could affect age estimates. Despite these limitations, the inferred age provides a useful context for considering the possible early presence of *A. confertitesta*.

The DNA sequences of *A. confertitesta* (T6) found in core D might originate from propagules, which can be transported over long distances. Therefore, their presence alone does not conclusively prove that *A. confertitesta* was present on the French coast, most likely since the Mid-Holocene. However, morphological analysis of core B reveals adult specimens of *A. confertitesta* coexisting with the more dominant *A. aberdoveyensis* at depths of 125–126 cm, dated to 6000 cal. years BP. This finding supports the inferred age model correspondence between cores B and D (Fig. 4). Although it could be argued that these specimens were also transported, their size (>150 µm) and intact shell structure indicate that they were unlikely to have been transported from very distant locations. Thus, despite the need for caution due to chronological approximation and the rather bad preservation of *sedaDNA* in core D, our results from both cores B and D indicate that *A. confertitesta* (T6) might have been present on the Atlantic coast several thousand years ago. This predates any anthropogenic activities involving shipping or trade with Asia, supporting the conclusion that it is an autochthonous species. The fact that this species is spreading out and is supplanting *Ammonia aberdoveyensis* and *Ammonia veneta* in several European coastal settings (e.g. Bird *et al.* 2020; Richirt *et al.* 2021; Fouet 2022) since at least the 18th century (Fouet 2022) may suggest more recent changes in environmental conditions due to human activities to which *Ammonia aberdoveyensis* and *Ammonia veneta* are more vulnerable. These preliminary findings highlight the need for more detailed investigations using well-dated, long-term records. This approach would allow for accurately determining both the timing of this species' presence and the onset of its invasiveness along the French coast.

## Conclusions

Previous studies in colder environments have shown that *sedaDNA* is a valuable tool for detecting both fossilized and non-fossilized taxa. However, our research suggests

that foraminiferal DNA preservation might be limited in oxic, 'warm-water' sediments. Additionally, future investigations of *sedaDNA* in the area will require larger sediment volumes to improve the detection of the entire diversity, a more rapid DNA extraction after sediment sampling and storage at lower temperatures (i.e. -80 °C). All these parameters may account for the significant discrepancies between morphological data and *sedaDNA* results in the presence and distribution of taxa. On the other hand, as in previous studies, our *sedaDNA* data suggest that monothalamous foraminifera are a dominant group in these environments and might provide valuable environmental information in the study area, which has remained inaccessible until now.

The dual approach reported here, combining morphological and DNA analyses, seems to be a good strategy to obtain a more complete overview of the foraminiferal communities since both approaches present biases. This dual approach showed that *Ammonia confertitesta* (T6) could potentially be present on the Atlantic coast several thousand years ago, way before any anthropogenic activity involving international shipping and commercial trades.

*Acknowledgements.* – The study was funded by the HOOPLA project (WEAMEC marine energy; PI: Edouard Metzger) and by the HOOPLACOR project (The French Oceanographic Fleet; PI: Aurélia Mouret). ¹⁴C dating was funded by the TRESSE project (the Office français de la biodiversité; PI: Bernadette Tessier). The authors warmly thank Fabio Gauvin, Alexandra Faitout and Sophie Quinchard (LPG), Hayley Melville and Philippe Blanchard (MOLTECH-Anjou), and Thibault Clochard (IRHS) for their help with foraminifera picking and DNA analyses. Coralie Marais, Muriel Bahut and Sandrine Balzergue (IRHS) are acknowledged for the High-Throughput Sequencing. We would also like to thank all the crew members of the R/V Thalia for their valuable assistance during the oceanographic cruise. The authors declare that there are no known financial, personal or other relationships that could be viewed as potential conflicts of interest. We thank the two anonymous reviewers for their thorough and constructive feedback. We are also grateful to Jan A. Piotrowski (Editor-in-Chief) for his valuable suggestions, which helped improve the manuscript.

*Author contributions.* – MM: conception and design of the study, data acquisition, analysis and interpretation of data, writing the original draft, reviewing and editing. MS: data acquisition, analysis and interpretation of data, revising and reviewing. AM, EM, MPN, CB and AB: conception and design of the HOOPLA and HOOPLACORE projects, revising the article critically. MCLP, IVD and GG: data acquisition, revising the article critically. DLM, IP, AM and MB: data acquisition.

*Data availability statement.* – Raw data is available on SEANOE data repository (https://doi.org/10.17882/104522) and the Sequence Read Archive (SRA) with accession number PRJNA1104619.

## References

Al-Enezi, E., Francescangeli, F., Balassi, E., Borderie, S., Al-Hazeem, S., Al-Salameen, F., Boota Anwar, A., Pawlowski, J. & Frontalini, F. 2022: Benthic foraminifera as proxies for the environmental quality assessment of the Kuwait Bay (Kuwait, Arabian gulf): morphological and metabarcoding approaches. *Science of the Total Environment* 833, 155093. https://doi.org/10.1016/j.scitotenv.2022.155093.
Alve, E. & Goldstein, S. T. 2010: Dispersal, survival and delayed growth of benthic foraminiferal propagules. *Journal of Sea Research* **63**, 36–51.

Armbrecht, L. and 33 others 2022: Ancient marine sediment DNA reveals diatom transition in Antarctica. *Nature Communications* **13**, 5787. https://doi.org/10.1038/s41467-022-33494-4.

Arnaud, F., Révillon, S., Debret, M., Revel, M., Chapron, E., Jacob, J., Giguet-Covex, C., Poulenard, J. & Magny, M. 2012: Lake Bourget regional erosion patterns reconstruction reveals Holocene NW European Alps soil evolution and paleohydrology. *Quaternary Science Reviews* **51**, 81–92. https://doi.org/10.1016/j.quascirev.2012.07.025.

Baltzer, A., Cassen, S., Walter-Simonnet, A.-V., Clouet, H., Lorin, A. & Tessier, B. 2015: Variations du niveau marin Holocène en Baie de Quiberon (Bretagne sud): marqueurs archéologiques et sédimentologiques. *Quaternaire. Revue de l'Association française pour l'étude du Quaternaire* **26**, 105–115. https://doi.org/10.4000/quaternaire.7201.

Barrenechea Angeles, I., Lejzerowicz, F., Cordier, T., Schepplitz, J., Kucera, M., Ariztegui, D., Pawlowski, J. & Morard, R. 2020: Planktonic foraminifera eDNA signature deposited on the seafloor remains preserved after burial in marine sediments. *Scientific Reports* **10**, 20351. https://doi.org/10.1038/s41598-020-77179-8.

Barrenechea Angeles, I., Romero-Martínez, M. L., Cavaliere, M., Varrella, S., Francescangeli, F., Piredda, R., Mazzocchi, M. G., Montresor, M., Schirone, A., Delbono, I., Margiotta, F., Corinaldesi, C., Chiavarini, S., Montereali, M. R., Rimauro, J., Parrella, L., Musco, L., Dell'Anno, A., Tangherlini, M., Pawlowski, J. & Frontalini, F. 2023: Encapsulated in sediments: eDNA deciphers the ecosystem history of one of the most polluted European marine sites. *Environment International* **172**, 107738. https://doi.org/10.1016/j.envint.2023.107738.

Baux, N., Murat, A., Faivre, Q., Lesourd, S., Poizot, E., Méar, Y., Brasselet, S. & Dauvin, J.-C. 2019: Sediment dynamic equilibrium, a key for assessing a coastal anthropogenic disturbance using geochemical tracers: application to the eastern part of the Bay of Seine. *Continental Shelf Research* **175**, 87–98. https://doi.org/10.1016/j.csr.2019.02.002.

Bird, C., Schweizer, M., Roberts, A., Austin, W. E. N., Knudsen, K. L., Evans, K. M., Filipsson, H. L., Sayer, M. D. J., Geslin, E. & Darling, K. F. 2020: The genetic diversity, morphology, biogeography, and taxonomic designations of Ammonia (Foraminifera) in the North-east Atlantic. *Marine Micropaleontology* **155**, 101726. https://doi.org/10.1016/j.marmicro.2019.02.001.

Boere, A. C., Rijpstra, W. I. C., Lange, G. J. D., Damsté, J. S. S. & Coolen, M. J. L. 2011: Preservation potential of ancient plankton DNA in Pleistocene marine sediments. *Geobiology* **9**, 377–393. https://doi.org/10.1111/j.1472-4669.2011.00290.x.

Bona, P. & Prud'homme Lacroix, B. 2009: Amélioration du fonctionnement hydro-sédimentaire de l'estuaire de la Loire. *Journées de l'hydroalique* **31**, 1–10.

Brandt, M. I., Trouche, B., Henry, N., Liautard-Haag, C., Maignien, L., de Vargas, C., Wincker, P., Poulain, J., Zeppilli, D. & Arnaud-Haond, S. 2020: An assessment of environmental metabarcoding protocols aiming at favoring contemporary biodiversity in inventories of Deep-Sea communities. *Frontiers in Marine Science* **7**, 2020. https://doi.org/10.3389/fmars.2020.00234.

Brinkmann, I., Schweizer, M., Singer, D., Quinchard, S., Barras, C., Bernhard, J. M. & Filipsson, H. L. 2023: Through the eDNA looking glass: responses of fjord benthic foraminiferal communities to contrasting environmental conditions. *Journal of Eukaryotic Microbiology* **70**, e12975. https://doi.org/10.1111/jeu.12975.

Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A. & Holmes, S. P. 2016: DADA2: high-resolution sample inference from Illumina amplicon data. *Nature Methods* **13**, 581–583. https://doi.org/10.1038/nmeth.3869.

Cao, Y., Lei, Y., Fang, J. K. & Li, T. 2022: Molecular diversity of foraminiferal eDNA in sediments and their correlations with environmental factors from the Yellow Sea. *Ecological Indicators* **142**, 109294. https://doi.org/10.1016/j.ecolind.2022.109294.

Castaing, P., Froidefond, J. M., Lazure, P., Weber, O., Prud'homme, R. & Jouanneau, J. M. 1999: Relationship between hydrology and seasonal distribution of suspended sediments on the continental shelf of the Bay of Biscay. *Deep Sea Research Part II: Topical Studies in Oceanography* **46**, 1979–2001. https://doi.org/10.1016/S0967-0645 (99)00052-1.

Cavaliere, M., Barrenechea Angeles, I., Montresor, M., Bucci, C., Brocani, L., Balassi, E., Margiotta, F., Francescangeli, F., Bouchet, V. M. P., Pawlowski, J. & Frontalini, F. 2021: Assessing the ecological quality status of the highly polluted Bagnoli area (Tyrrhenian Sea, Italy) using foraminiferal eDNA metabarcoding. *Science of the Total Environment* **790**, 147871. https://doi.org/10.1016/j.scitotenv.2021.147871.

Champilou, J.-B. 2020: Etude multidisciplinaire de l'habitat benthique à Haploops spp. au large du Croisic (Loire Atlantique). Suivi de l'impact d'un câble électrique sur cet habitat. These de doctorat, Angers.

Champilou, J.-B., Baltzer, A., Murat, A., Reynaud, M., Maillet, G. M., Nardelli, M. P. & Metzger, É. 2019: New evidence of perfect overlapping of Haploops and pockmarks field: is it a coincidence? *Marine Geology* **415**, 105961. https://doi.org/10.1016/j.margeo.2019.105961.

Charria, G., Lazure, P., Le Cann, B., Serpette, A., Reverdin, G., Louzel, S., Batifoulier, F., Dumas, F., Pichon, A. & Morel, Y. 2013: Surface layer circulation derived from Lagrangian drifters in the Bay of Biscay. *Journal of Marine Systems* **109–110** (Supplement), S60–S76. https://doi.org/10.1016/j.jmarsys.2011.09.015.

Ciobanu, M.-C., Rabineau, M., Droz, L., Révillon, S., Ghiglione, J.-F., Dennielou, B., Jorry, S.-J., Kallmeyer, J., Etoubleau, J., Pignet, P., Crassous, P., Vandenabeele-Trambouze, O., Laugier, J., Guégan, M., Godfroy, A. & Alain, K. 2012: Sedimentological imprint on sub-seafloor microbial communities in Western Mediterranean Sea Quaternary sediments. *Biogeosciences* **9**, 3491–3512. https://doi.org/10.5194/bg-9-3491-2012.

Coolen, M. J. L. & Overmann, J. 1998: Analysis of subfossil molecular remains of purple sulfur bacteria in a Lake sediment. *Applied and Environmental Microbiology* **64**, 4513–4521. https://doi.org/10.1128/AEM.64.11.4513-4521.1998.

Coolen, M. J. L., Orsi, W. D., Balkema, C., Quince, C., Harris, K., Sylvia, S. P., Filipova-Marinova, M. & Giosan, L. 2013: Evolution of the plankton paleome in the Black Sea from the Deglacial to Anthropocene. *Proceedings of the National Academy of Sciences of the United States of America* **110**, 8609–8614. https://doi.org/10.1073/pnas.1219283110.

Costoya, X., Fernández-Nóvoa, D., deCastro, M. & Gómez-Gesteira, M. 2017: Loire and Gironde turbid plumes: characterization and influence on thermohaline properties. *Journal of Sea Research* **130**, 7–16. https://doi.org/10.1016/j.seares.2017.04.003.

Cyprien, A.-L., Visset, L. & Carcaud, N. 2004: Evolution of vegetation landscapes during the Holocene in the central and downstream Loire basin (Western France). *Vegetation History and Archaeobotany* **13**, 181–196.

De Schepper, S., Ray, J. L., Skaar, K. S., Sadatzki, H., Ijaz, U. Z., Stein, R. & Larsen, A. 2019: The potential of sedimentary ancient DNA for reconstructing past sea ice evolution. *The ISME Journal* **13**, 2566–2577. https://doi.org/10.1038/s41396-019-04571-1.

Dell'Anno, A., Corinaldesi, C., Stavrakakis, S., Lykousis, V. & Danovaro, R. 2005: Pelagic-benthic coupling and diagenesis of nucleic acids in a Deep-Sea continental margin and an open-slope system of the eastern Mediterranean. *Applied and Environmental Microbiology* **71**, 6070–6076. https://doi.org/10.1128/AEM.71.10.6070-6076.2005.

Delmas, M., Cerdan, O., Cheiron, B., Mouchel, J.-M. & Eyrolle, F. 2012: Sediment export from French rivers to the sea. *Earth Surface Processes and Landforms* **37**, 754–762. https://doi.org/10.1002/esp.3219.

Demianiuk, E., Baca, M., Popović, D., Angeles, I. B., Nguyen, N.-L., Pawlowski, J., Anderson, J. B. & Majewski, W. 2024: Sedimentary ancient DNA insights into foraminiferal diversity near the grounding line in the western Ross Sea, Antarctica. *EGUSphere* **2824**, 1–30. https://doi.org/10.5194/egusphere-2024-2824.

Dupain, R. 1981: Effets de l'endiguement de la rive nord de l'estuaire de la Loire. *Revue Française de Géotechnique* **14**, 28–33.
Durand, M., Mojtahid, M., Maillet, G. M., Baltzer, A., Schmidt, S., Blet, S., Marchès, E. & Howa, H. 2018: Late Holocene record from a Loire River incised paleovalley (French inner continental shelf): insights into regional and global forcing factors. *Palaeogeography, Palaeoclimatology, Palaeoecology* **511**, 12–28. https://doi.org/10.1016/j.palaeo.2018.06.035.

Durand, M., Mojtahid, M., Maillet, G. M., Proust, J.-N., Lehay, D., Ehrhold, A., Barré, A. & Howa, H. 2016: Mid-to late-Holocene environmental evolution of the Loire estuary as observed from sedimentary characteristics and benthic foraminiferal assemblages. *Journal of Sea Research* **118**, 17–34. https://doi.org/10.1016/j.seares.2016.08.003.

Duros, P., Silva Jacinto, R., Dennielou, B., Schmidt, S., Martinez Lamas, R., Gautier, E., Roubi, A. & Gayet, N. 2017: Benthic foraminiferal response to sedimentary disturbance in the Capbreton canyon (Bay of Biscay, NE Atlantic). *Deep Sea Research Part I: Oceanographic Research Papers* **120**, 61–75. https://doi.org/10.1016/j.dsr.2016.11.012.

Engel, M., Schön, I., Patel, T., Pawłowski, J., Szczuciński, W., Dawson, S., Garrett, E. & Heyvaert, V. M. A. 2020: Chapter 20 – paleogenetic approaches in tsunami deposit studies. *In Engel, M., Pilarczyk, J., May, S. M., Brill, D. & Garrett, E. (eds.): Geological Records of Tsunamis and Other Extreme Waves*, 427–442. Elsevier, Amsterdam.

Fontanier, C., Deflandre, B., Rigaud, S., Mamo, B., Dubosq, N., Lamarque, B., Langlet, D., Schmidt, S., Lebleu, P., Poirier, D., Cordier, M. A. & Grémare, A. 2022: Live (stained) benthic foraminifera from the West-Gironde Mud Patch (Bay of Biscay, NE Atlantic): assessing the reliability of bio-indicators in a complex shelf sedimentary unit. *Continental Shelf Research* **232**, 104616. https://doi.org/10.1016/j.csr.2021.104616.

Fouet, M. 2022: Répartition des communautés de foraminifères dans les estuaires de la façade atlantique. Ph.D. thesis, University of Angers, 259 pp.

Fouet, M. P. A., Schweizer, M., Singer, D., Richirt, J., Quinchard, S. & Jorissen, F. J. 2024: Unravelling the distribution of three Ammonia species (Foraminifera, Rhizaria) in French Atlantic Coast estuaries using morphological and metabarcoding approaches. *Marine Micro-paleontology* **188**, 102353. https://doi.org/10.1016/j.marmicro.2024.102353.

Fouet, M. P. A., Singer, D., Coynel, A., Héliot, S., Howa, H., Lalande, J., Mouret, A., Schweizer, M., Tcherkez, G. & Jorissen, F. J. 2022: Foraminiferal distribution in two estuarine intertidal mudflats of the French Atlantic Coast: testing the Marine Influence Index. *Water* **14**, 645. https://doi.org/10.3390/w14040645.

Frontalini, F., Cordier, T., Balassi, E., Armynot du Chatelet, E., Cermakova, K., Apothéloz-Perret-Gentil, L., Martins, M. V. A., Bucci, C., Scantamburlo, E., Treglia, M., Bonamin, V. & Pawlowski, J. 2020: Benthic foraminiferal metabarcoding and morphology-based assessment around three offshore gas platforms: congruence and complementarity. *Environment International* **144**, 106049. https://doi.org/10.1016/j.envint.2020.106049.

Goldstein, S. T. & Alve, E. 2011: Experimental assembly of foraminiferal communities from coastal propylene banks. *Marine Ecology Progress Series* **437**, 1–11. https://doi.org/10.3354/meps09296.

Gonçalves, M., Martinho, P. & Guedes Soares, C. 2014: Wave energy conditions in the western French coast. *Renewable Energy* **62**, 155–163. https://doi.org/10.1016/j.renene.2013.06.028.

Gooday, A. J., Bowser, S. S. & Bernhard, J. M. 1996: Benthic foraminiferal assemblages in Explorers Cove, Antarctica: a shallow-water site with deep-sea characteristics. *Progress in Oceanography* **37**, 117–166. https://doi.org/10.1016/S0079-6611(96)00007-9.

Goslin, J., Van Vliet Lanoë, B., Spada, G., Bradley, S., Tarasov, L., Neill, S. & Suanez, S. 2015: A new Holocene relative sea-level curve for western Brittany (France): insights on isostatic dynamics along the Atlantic coasts of north-western Europe. *Quaternary Science Reviews* **129**, 341–365. https://doi.org/10.1016/j.quascirev.2015.10.029.

Govin, A., Holzwarth, U., Heslop, D., Ford Keeling, L., Zabel, M., Mulitza, S., Collins, J. A. & Chiessi, C. M. 2012: Distribution of major elements in Atlantic surface sediments (36°N–49°S): imprint of terrigenous input and continental weathering.

Geochemistry, Geophysics, Geosystems **13**, Q01013. https://doi.org/10.1029/2011GC003785.

Guillou, L. and 31 others 2013: The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. *Nucleic Acids Research* **41**, D597–D604. https://doi.org/10.1093/nar/gks1160.

Hayward, B. W., Sabaa, A. & Grenfell, H. R. 2004: Benthic foraminifera and the late Quaternary (last 150 ka) paleoceanographic and sedimentary history of the Bounty Trough, east of New Zealand. *Palaeogeography, Palaeoclimatology, Palaeoecology* **211**, 59–93. https://doi.org/10.1016/j.palaeo.2004.04.007.

Holzmann, M., Nguyen, N.-L., Angeles, I. B. & Pawlowski, J. 2024: BFR2: a curated ribosomal reference dataset for benthic foraminifera. *Scientific Data* **11**, 1292. https://doi.org/10.1038/s41597-024-04137-8.

Jouanneau, J. M., Weber, O., Cremer, M. & Castaing, P. 1999: Fine-grained sediment budget on the continental margin of the Bay of Biscay. *Deep Sea Research Part II: Topical Studies in Oceanography* **46**, 2205–2220. https://doi.org/10.1016/S0967-0645(99)00060-0.

Judd, A. & Hovland, M. 2007: Pockmarks, shallow gas, and seeps: an initial appraisal. *In Judd, A. & Hovland, M. (eds.): Seabed Fluid Flow: The Impact on Geology, Biology and the Marine Environment*, 7–44. Cambridge University Press, Cambridge.

Kirkpatrick, J. B., Walsh, E. A. & D'Hondt, S. 2016: Fossil DNA persistence and decay in marine sediment over hundred-thousand-year to million-year time scales. *Geology* **44**, 615–618. https://doi.org/10.1130/G37933.1.

Kjær, K. H. and 37 others. 2022: A 2-million-year-old ecosystem in Greenland uncovered by environmental DNA. *Nature* **612**, 283–291. https://doi.org/10.1038/s41586-022-05453-y.

Lazure, P. & Jégou, A. M. 1998: 3D modelling of seasonal evolution of Loire and Gironde plumes on Biscay Bay continental shelf. *Oceanologica Acta* **21**, 165–177.

Lejzerowicz, F., Esling, P. & Pawlowski, J. 2014: Patchiness of deep-sea benthic Foraminifera across the Southern Ocean: insights from high-throughput DNA sequencing. *Deep Sea Research Part II: Topical Studies in Oceanography* **108**, 17–26. https://doi.org/10.1016/j.dsr2.2014.07.018.

Lejzerowicz, F., Esling, P., Majewski, W., Szczuciński, W., Decelle, J., Obadia, C., Arbizu, P. M. & Pawlowski, J. 2013: Ancient DNA complements microfossil record in deep-sea sub-surface sediments. *Biology Letters* **9**, 20130283. https://doi.org/10.1098/rsbl.2013.0283.

Lloyd, K. G., MacGregor, B. J. & Teske, A. 2010: Quantitative PCR methods for RNA and DNA in marine sediments: maximizing yield while overcoming inhibition. *FEMS Microbiology Ecology* **72**, 143–151. https://doi.org/10.1111/j.1574-6941.2009.00827.x.

Maeda, A., Nishijima, M., Iguchi, A., Ota, Y., Suzumura, M. & Suzuki, A. 2024: Environmental DNA metabarcoding of foraminifera for biological monitoring of bottom water and sediments on the Takuyo-Daigo Seamount in the northwestern Pacific. *Frontiers in Marine Science* **10**, 1243713. https://doi.org/10.3389/fmar.2023.1243713.

Martin, M. 2011: Cutadapt removes adapter sequences from high-throughput sequencing reads. *EMBnet.Journal* **17**, 10–12. https://doi.org/10.14806/ej.17.1.200.

Mary, Y., Eynaud, F., Colin, C., Rossignol, L., Brocheray, S., Mojtahid, M., Garcia, J., Peral, M., Howa, H., Zaragosi, S. & Cremer, M. 2017: Changes in Holocene meridional circulation and poleward Atlantic flow: the Bay of Biscay as a nodal point. *Climate of the Past* **13**, 201–216. https://doi.org/10.5194/cp-13-201-2017.

Mertens, K. N., Retho, M., Manach, S., Zoffoli, M. L., Doner, A., Schapira, M., Bilien, G., Séchet, V., Lacour, T., Robert, E., Duval, A., Terre-Terrillon, A., Derrien, A. & Gernez, P. 2023: An unprecedented bloom of *Lingulodinium polyedra* on the French Atlantic coast during summer 2021. *Harmful Algae* **125**, 102426. https://doi.org/10.1016/j.hal.2023.102426.

Moatar, F. & Dupont, N. 2016: *La Loire fluviale et estuarienne - Un milieu en évolution*. 320 pp. FMA5292, Editions Quae, Versailles.

Mojtahid, M., Durand, M., Coste, P.-O., Toucanne, S., Howa, H., Nizou, J., Eynaud, F. & Penaud, A. 2019: Millennial-scale Holocene
hydrological changes in the northeast Atlantic: new insights from 'La Grande Vasière' mid-shelf mud belt. *The Holocene* 29, 467–480.
https://doi.org/10.1177/0959683618816478.

Mojtahid, M., Geslin, E., Coynel, A., Gorse, L., Vella, C., Davranche, A., Zozzolo, L., Blanchet, L., Bénéteau, E. & Maillet, G. 2016: Spatial distribution of living (Rose Bengal stained) benthic foraminifera in the Loire estuary (western France). *Journal of Sea Research* 118, 1–16.
https://doi.org/10.1016/j.seares.2016.02.003.

More, K. D., Giosan, L., Grice, K. & Coolen, M. J. L. 2019: Holocene paleodepositional changes reflected in the sedimentary microbiome of the Black Sea. *Geobiology* 17, 436–448. https://doi.org/10.1111/gbi.12338.

Naughton, F., Sanchez Goñi, M. F., Desprat, S., Turon, J.-L., Duprat, J., Malaizé, B., Joli, C., Cortijo, E., Drago, T. & Freitas, M. C. 2007: Present-day and past (last 25 000 years) marine pollen signal off western Iberia. *Marine Micropaleontology* 62, 91–114. https://doi.org/10.1016/j.marmicro.2006.07.006.

Nguyen, N.-L., Pawłowska, J., Angeles, I. B., Zajaczkowski, M. & Pawłowski, J. 2023: Metabarcoding reveals high diversity of benthic foraminifera linked to water masses circulation at coastal Svalbard. *Geobiology* 21, 133–150. https://doi.org/10.1111/gbi.12530.

Orsi, W. D., Coolen, M. J. L., Wuchter, C., He, L., More, K. D., Irigoien, X., Chust, G., Johnson, C., Hemingway, J. D., Lee, M., Galy, V. & Giosan, L. 2017: Climate oscillations reflected within the microbiome of Arabian Sea sediments. *Scientific Reports* 7, 6040. https://doi.org/10.1038/s41598-017-05590-9.

Pawłowska, J., Lejzerowicz, F., Esling, P., Szczuciński, W., Zajaczkowski, M. & Pawlowski, J. 2014: Ancient DNA sheds new light on the Svalbard foraminiferal fossil record of the last millennium. *Geobiology* 12, 277–288. https://doi.org/10.1111/gbi.12087.

Pawłowska, J., Wollenburg, J. E., Zajaczkowski, M. & Pawlowski, J. 2020: Planktonic foraminifera genomic variations reflect paleoceanographic changes in the Arctic: evidence from sedimentary ancient DNA. *Scientific Reports* 10, 15102. https://doi.org/10.1038/s41598-020-72146-9.

Pawłowska, J., Zajaczkowski, M., Łacka, M., Lejzerowicz, F., Esling, P. & Pawlowski, J. 2016: Palaeoceanographic changes in Hornsund Fjord (Spitsbergen, Svalbard) over the last millennium: new insights from ancient DNA. *Climate of the Past* 12, 1459–1472. https://doi.org/10.5194/cp-12-1459-2016.

Pawlowski, J. & Holzmann, M. 2008: Diversity and geographic distribution of benthic foraminifera: a molecular perspective. *Biodiversity and Conservation* 17, 317–328. https://doi.org/10.1007/s10531-007-9253-8.

Pawlowski, J. & Holzmann, M. 2014: A plea for DNA barcoding of foraminifera. *Journal of Foraminiferal Research* 44, 62–67.
https://doi.org/10.2113/gsjfr.44.1.62.

Pawlowski, J. 2000: Introduction to the molecular systematics of foraminifera. *Micropaleontology* 46, 1–12.

Pawlowski, J. and 34 others 2022: Environmental DNA metabarcoding for benthic monitoring: a review of sediment sampling and DNA extraction methods. *Science of the Total Environment* 818, 151783. https://doi.org/10.1016/j.scitotenv.2021.151783.

Pawlowski, J., Esling, P., Lejzerowicz, F., Cedhagen, T. & Wilding, T. A. 2014: Environmental monitoring through protist next-generation sequencing metabarcoding: assessing the impact of fish farming on benthic foraminifera communities. *Molecular Ecology Resources* 14, 1129–1140. https://doi.org/10.1111/1755-0998.12261.

Pawlowski, J., Esling, P., Lejzerowicz, F., Cordier, T., Visco, J. A., Martins, C. I. M., Kvalvik, A., Staven, K. & Cedhagen, T. 2016: Benthic monitoring of salmon farms in Norway using foraminiferal metabarcoding. *Aquaculture Environment Interactions* 8, 371–386. https://doi.org/10.3354/aei00182.

Penaud, A., Ganne, A., Eynaud, F., Lambert, C., Coste, P. O., Herlédan, M., Vidal, M., Goslin, J., Stéphan, P., Charria, G., Pailler, Y., Durand, M., Zumaque, J. & Mojtahid, M. 2020: Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic). *Quaternary Science Reviews* 229, 106135. https://doi.org/10.1016/j.quascirev.2019.106135.

Pingree, R. D. & Le Cann, B. 1989: Celtic and Armorican slope and shelf residual currents. *Progress in Oceanography* 23, 303–338.

Polovodova, I., Nikulina, A., Schönfeld, J. & Dullo, W.-C. 2009: Recent benthic foraminifera in the Flensburg Fjord (Western Baltic Sea). *Journal of Micropaleontology* 28, 131–142. https://doi.org/10.1144/jm.28.2.131.

Proust, J.-N., Renault, M., Guennoc, P. & Thinon, I. 2010: Sedimentary architecture of the Loire River drowned valleys of the French Atlantic shelf. *Bulletin de la Société Géologique de France* 181, 129–149.

R Core Team. 2021: *R: A language and environment for statistical computing*. R Foundation for Statistical Computing, Vienna. Available at: https://www.R-project.org/.

Retho, M., Quemener, L., Le Gall, C., Repecaud, M., Souchu, P., Gabellec, R. & Manach, S. 2022: *COAST-HF– Data and Metadata from the MOLIT Buoy in the Vilaine Bay*. Seanoe, Ifremer, Brest. https://doi.org/10.17882/46529.

Richirt, J., Schweizer, M., Bouchet, V. M. P., Mouret, A., Quinchard, S. & Jorissen, F. J. 2019: Morphological distinction of three ammonia phylotypes occurring along European coasts. *Journal of Foraminiferal Research* 49, 76–93. https://doi.org/10.2113/gsjfr.49.1.76.

Richirt, J., Schweizer, M., Mouret, A., Quinchard, S., Saad, S. A., Bouchet, V. M. P., Wade, C. M. & Jorissen, F. J. 2021: Biogeographic distribution of three phylotypes (T1, T2 and T6) of ammonia (foraminifera, Rhizaria) around Great Britain: new insights from combined molecular and morphological recognition. *Journal of Micropaleontology* 40, 61–74. https://doi.org/10.5194/jm-40-61-2021.

Rombouts, I., Simon, N., Aubert, A., Cariou, T., Feunteun, E., Guérin, L., Hoebeke, M., McQuatters-Gollop, A., Rigaut-Jalabert, F. & Artigas, L. F. 2019: Changes in marine phytoplankton diversity: assessment under the Marine Strategy Framework Directive. *Ecological Indicators* 102, 265–277. https://doi.org/10.1016/j.ecolind.2019.02.009.

Sanchez, M., Grovel, A. & Hosseini, K. 2000: Impact sédimentaire des travaux d'aménagement de l'estuaire de la Loire. *Bulletin de Engineering Geology and the Environment* 59, 239–246. https://doi.org/10.1007/s100640000047.

Schweizer, M., Polovodova, I., Nikulina, A. & Schönfeld, J. 2011: Molecular identification of Ammonia and Elphidium species (Foraminifera, Rotaliida) from the Kiel Fjord (SW Baltic Sea) with rDNA sequences. *Helgoland Marine Research* 65, 1–10. https://doi.org/10.1007/s10152-010-0194-3.

Shi, J., Lei, Y., Li, H. & Li, T. 2021: NGS-metabarcoding revealing novel foraminiferal diversity in the Western Pacific Magellan Seamount sediments. *Journal of Oceanology and Limnology* 39, 1718–1729. https://doi.org/10.1007/s00343-021-0427-0.

Singer, D., Fouet, M. P. A., Schweizer, M., Mouret, A., Quinchard, S. & Jorissen, F. J. 2023: Unlocking foraminiferal genetic diversity on estuarine mudflats with eDNA metabarcoding. *Science of the Total Environment* 902, 165983. https://doi.org/10.1016/j.scitotenv.2023.165983.

Stuiver, M., Reimer, P. J. & Reimer, R. W. 2017: CALIB 7.1 [WWW program]. Available at: http://calib.org (accessed 03.04.2017).

Thibault de Chanvalon, A., Geslin, E., Mojtahid, M., Métais, I., Méléder, V. & Metzger, E. 2022: Multiscale analysis of living benthic foraminiferal heterogeneity: ecological advances from an intertidal mudflat (Loire estuary, France). *Continental Shelf Research* 232, 104627. https://doi.org/10.1016/j.csr.2021.104627.

Thibault de Chanvalon, A., Metzger, E., Mouret, A., Cesbron, F., Knoery, J., Rozuel, E., Launeau, P., Nardelli, M. P., Jorissen, F. J. & Geslin, E. 2015: Two-dimensional distribution of living benthic foraminifera in anoxic sediment layers of an estuarine mudflat (Loire estuary, France). *Biogeosciences* 12, 6219–6234. https://doi.org/10.5194/bg-12-6219-2015.

Tisnérat-Laborde, N., Paterne, M., Métivier, B., Arnold, M., Yiou, P., Blamart, D. & Raynaud, S. 2010: Variability of the northeast Atlantic sea surface Δ14C and marine reservoir age and the North Atlantic Oscillation (NAO). *Quaternary Science Reviews* 29, 2633–2646. https://doi.org/10.1016/j.quascirev.2010.06.013.
Toyofuku, M. S., Kitazato, H. & Tsuchiya, M. 2005: Phylogenetic relationships among genus ammonia (foraminifera) based on ribosomal DNA sequences, which are distributed in the vicinity of the Japanese Islands. *Frontier Research on Earth Evolution* 2, 1–9.

Visset, L., Cyprien, A.-L., Carcaud, N., Ouguerram, A., Barbier, D. & Bernard, J. 2002: Les prémices d’une agriculture diversifiée à la fin du Mésolithique dans le Val de Loire (Loire armoricaine, France). *Comptes Rendus Palevol* 1, 51–58. https://doi.org/10.1016/S1631-0683(02)00007-6.

Weber, A. A.-T. & Pawlowski, J. 2014: Wide occurrence of SSU rDNA intragenomic polymorphism in foraminifera and its implications for molecular species identification. *Protist* 165, 645–661. https://doi.org/10.1016/j.protis.2014.07.006.

Willerslev, E., Hansen, A. J., Binladen, J., Brand, T. B., Gilbert, M. T. P., Shapiro, B., Bunce, M., Wiuf, C., Gilichinsky, D. A. & Cooper, A. 2003: Diverse plant and animal genetic records from Holocene and Pleistocene sediments. *Science* 300, 791–795. https://doi.org/10.1126/science.1084114.

## Supporting Information

Additional Supporting Information to this article is available at http://www.boreas.dk.

Fig. S1. Calibrated radiocarbon ages (cal. a BP) on core B. (*) indicate $^{14}$C age showing age inversion in comparison with the general tendency. The horizontal bars around each dated point correspond to the 2σ (two standard deviations) range of the calibrated radiocarbon ages. The dashed blue line represents the linear age model.