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# An illustrated protocol for extracting palynomorphs from Early Pleistocene pollen-poor sediments using LST Fastfloat

Valérie Andrieu, Belinda Gambin

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Valérie Andrieu, Belinda Gambin. An illustrated protocol for extracting palynomorphs from Early Pleistocene pollen-poor sediments using LST Fastfloat. Review of Palaeobotany and Palynology, 2025, 341, pp.105372. 10.1016/j.revpalbo.2025.105372. hal-05165979

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Research papers

# An illustrated protocol for extracting palynomorphs from Early Pleistocene pollen-poor sediments using LST Fastfloat

Valérie Andrieu<sup>a, *</sup>, Belinda Gambin<sup>b</sup>

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

**Keywords:**

Protocol

Palynology

LST Fastfloat

Pollen-poor sediments

Mediterranean

## ABSTRACT

This method for extracting palynomorphs is specifically dedicated for Early Pleistocene pollen-poor carbonate or siliceous sediments in the Mediterranean, which are characterised by prolonged summer droughts. It utilises LST Fastfloat, a non-toxic, low viscosity sodium polytungstate heavy liquid. Due to its physical qualities, particularly its low viscosity, LST Fastfloat enables more efficient pollen concentration compared to other methods, including those that rely on alternative dense liquids.

This approach was applied to fluviatile and lacustrine deposits in both the western (Marseille) and eastern (Acıgöl, Kocabaş) Mediterranean. Unlike standard method, LST Fastfloat produced highly concentrated pollen residues and clear pollen slides, reducing analysis time, which is an important advantage in a labour-intensive discipline such as palynology. The primary drawback of this method is its high cost, although it is partially recyclable.

Our article is designed to be didactic and richly illustrated, featuring figures and photographs to facilitate its application, particularly for beginners. The positive results presented here may encourage researchers to sample sediments previously considered pollen-poor or devoid of palynomorphs, especially from understudied regions of major biogeographical or archaeological interest, such as the arid regions of the tropics and the Mediterranean.

## 1. Introduction

The analysis of pollen, spores and Non-Pollen Palynomorphs (NPPs) in sediments enables the reconstruction of the past vegetation and climate across most regions of the world, provided that natural (e.g. peat bogs, lakes, rivers, caves, oceans) or anthropogenic (e.g. archaeological sites, pitch from ancient ships) pollen-bearing sediments have been preserved in non-oxidising conditions. Standard pollen extraction methods (Erdtman, 1943; Faegri and Iversen, 1989; Moore et al., 1991) rely on the sequential application of acids (HCl, HF) to dissolve mineral components, followed by bases (KOH or NaOH) and acetolysis to remove organic matter. The resulting pellet is sieved through a 200-µm (or finer) mesh and filtered through a 10-µm filter. This method is most commonly used in humid temperate regions, where pollen-bearing sediments are well preserved.

In Mediterranean and arid environments, as well as some oceanic contexts where pollen concentrations may be low due to the remoteness of pollen sources, the most effective extraction technique involves density separation. This method isolates lighter organic particles,

including palynomorphs, from heavier mineral fractions. Several dense liquids are available for this process, each with specific advantages and limitation, including toxicity, water solubility, viscosity, and cost. These include Thoulet's solution, zinc chloride, bromoform, sodium polytungstate (SPT) and lithium heteropolytungstate (LST) (Goeury and de Beaulieu, 1979; Savage, 1988; Nakagawa et al., 1998; Traverse, 2007; Caffrey and Horn, 2013; Leipe et al., 2019).

Caffrey and Horn (2013) were unsuccessful in extracting pollen from sediments in an arid environment, possibly due to the viscosity or quality of the LST used, the small sediment volume they processed (2 cm³), or post-depositional taphonomic factors such as oxidation. Eskola et al. (2021) compared the effectiveness of hydrofluoric acid (HF) treatment and LST Fastfloat for pollen extraction from a Finnish glacial sequence spanning the Pleistocene to Holocene. Both methods successfully extracted pollen and yielded comparable results, except for differences in the representation of *Betula* and *Pinus*, which are abundant in boreal ecosystems.

Unlike previous studies on LST Fastfloat in palynology, our study does not aim to compare pollen extraction techniques, because this has

<footnote><sup>a</sup> Aix-Marseille Univ., CNRS, IRD, INRAE, UM 34 CEREGE, Aix-en-Provence, France</footnote>

<footnote><sup>b</sup> University of Malta, Institute of Earth Systems, Msida, Malta</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: andrieu@cerege.fr (V. Andrieu), belinda.gambin@um.edu.mt (B. Gambin).</footnote>
<caption>**Table 1**</caption>

Marseille pollen data parameters. APF: Absolute Pollen Frequencies; NPP: Non Pollen Palynomorphs; PS: Pollen Sum.

<table><caption>**Table 2**</caption><thead><tr><th></th><th>Minimum</th><th>Maximum</th><th>Mean</th></tr></thead><tbody><tr><td>Taxa sum with NPP</td><td>23</td><td>59</td><td>32</td></tr><tr><td>Taxa sum without NPP</td><td>20</td><td>52</td><td>28</td></tr><tr><td>APF with NPP</td><td>11</td><td>468</td><td>97</td></tr><tr><td>APF without NPP</td><td>2</td><td>16</td><td>8</td></tr><tr><td>PS with NPP</td><td>234</td><td>27,096</td><td>4707</td></tr><tr><td>PS without NPP</td><td>87</td><td>1673</td><td>637</td></tr><tr><td>Mass g</td><td>51</td><td>451</td><td>222</td></tr></tbody></table>

Kocabaş pollen data parameters. APF: Absolute Pollen Frequencies; NPP: Non Pollen Palynomorphs; PS: Pollen Sum.

<table><thead><tr><th></th><th>Minimum</th><th>Maximum</th><th>Mean</th></tr></thead><tbody><tr><td>Taxa sum with NPP</td><td>41</td><td>46</td><td>43</td></tr><tr><td>Taxa sum without NPP</td><td>36</td><td>42</td><td>39</td></tr><tr><td>APF with NPP</td><td>2</td><td>10</td><td>7</td></tr><tr><td>APF without NPP</td><td>2</td><td>8</td><td>4</td></tr><tr><td>PS with NPP</td><td>363</td><td>604</td><td>462</td></tr><tr><td>PS without NPP</td><td>333</td><td>507</td><td>399</td></tr><tr><td>Mass g</td><td>250</td><td>250</td><td>250</td></tr></tbody></table>

already been documented (Peck, 1974; Lentfer and Boyd, 2000; Caffrey and Horn, 2013; Leipe et al., 2019; Eskola et al., 2021). Instead, we focus on the extraction of pollen and spores from Mediterranean Early Pleistocene carbonate or siliceous sediments, which are typically pollen-poor. The Mediterranean region is characterised by strong summer droughts, which are not conducive to pollen preservation, particularly in shallow depositional environments subject to significant bathymetric (lakes) or flow (rivers) fluctuations. However, these environments often provide the only availbale sedimentary archives for reconstructing past Mediterranean environments.

Our objective is to present a pollen extraction protocol using LST fastfloat which, unlike the standard method, has allowed us to successfully extract pollen from Early Pleistocene sites in western (France) and eastern (western Turkey) Mediterranean regions, previously considered pollen-poor or devoid of palynomorphs. We hope that our findings will encourage further palaeoecological studies in environments considered low in pollen preservation, such as warm arid regions, including the Mediterranean and dry tropics.

## 2. The samples analysed

We applied this protocol to Mediterranean sediments dated between 1 and 2.3 Ma which, after treatment with standard pollen extraction methods (Erdtman, 1943; Faegri and Iversen, 1989; Moore et al., 1991), were found to be devoid of or extremely poor in pollen. These sediments include silts and tufa (cold travertine) at the site of Marseille, France (Andrieu et al., 2024) dated to 1 Ma, conglomerate and travertine (cold and hydrothermal) in Kocabaş (western Turkey) dated to 1.2–1.6 Ma (Lebatard et al., 2014) and a core 600 m long made of lacustrine chalk, silts, and clays (with gypsum in evaporitic layers) in Acıgöl (western Turkey) (Andrieu-Ponel et al., 2021) dated to present to 2.3 Ma.

In Marseille, our protocol enabled the extraction of pollen from a site considered non-polliniferous by palynologists (Nicol-Pichard, 1982). Prior environmental reconstructions for this site were based on plant macrofossil studies conducted in the 19th century (Saporta, 1867). Of these 23 samples selected, 7 yielded palynomorphs. Given that travertines and tufa are well known for their low pollen preservation potential (Bertini et al., 2014), we deliberately processed large sediment masses, ranging from 51 and 451 g (Table 1).

The pollen assemblages obtained are ecologically reliable, as indicated by their taxonomic diversity, which ranges from 23 to 52 taxa (Table 1). According to McAndrews and King (1976), the identification of at least 20 ecologically significant pollen taxa is sufficient to validate an assemblages. Furthermore, the pollen assemblages are consistent with modern ecosystems along the western Mediterranean (Quézel and Médail, 2003; Pires and Pavon, 2018).

The pollen sum (PS), including NPPs, ranges from 234 to 27,096 (with a high concentration of charcoal microparticles in one sample and from 87 to 1673 when excluding NPPs. These results align with the conclusions of Djamali and Cilleros (2020), who indicate that ecologically significant pollen assemblages do not necessarily require high pollen counts, but that a pollen sum of <150 grains can be statistically valid, provided taxonomic richness is high (≥ 20 taxa, McAndrews and King, 1976). In Marseille, the mean taxonomic richness was 32 taxa when including NPPs and 28 when excluding them (Table 1). The most pollen-rich and taxonomically diverse samples originated from compact fluviate silty sediments, whereas samples found to be pollen-free or contaminated by modern pollen were from porous and oxidised carbonate tufa.

In the Kocabaş travertine complex, western Turkey (Ozkul et al., 2002), our objective was to analyse sediments near the stratigraphic level where the remains of Turkey’s oldest hominin were discovered (Kappelman et al., 2008). After several unsuccessful attempts to extract pollen using the standard method, we employed LST FastFloat for density separation.

Samples were collected from the lower and upper conglomerates flanking the travertine layer that contained the hominin remains. The pollen extraction process focused on the micritic cement of the conglomerate, which was separated from the coarser fraction (pebbles, gravel, sand) after crushing. This separation was performed under a binocular magnifying glass using a mounted needle. A substantial amount of sediment (250 g) was processed (Table 2).

This protocol yielded pollen assemblages with high taxonomic richness (mean count: 39–43) and ecological consistency with eastern Mediterranean ecosystems (Davis, 1965-1988; Akman et al., 1978). These assemblages complement previous palaeoenvironmental insights derived from the study of Kocabaş macroremains (Rausch et al., 2019), and have allowed for the characterisation of local and regional vegetation before and after the hominin occupation (Andrieu-Ponel et al., 2021).

In the Acıgöl sedimentary sequence (Andrieu-Ponel et al., 2021 and in progress), western Turkey, 107 out of 126 processed samples yielded pollen-rich assemblages (Plate 1) using the LST Fastfloat method. In contrast, the previously applied standard method produced suboptimal results, with slides that were difficult to analyse (Plate 1).

The sediments consist of evaporitic or deep-water deposits, depending on climatic and tectonic conditions (Demory et al., 2020). In evaporitic and shallow-water environments, taphonomic processes may lead to poor pollen preservation, explaining the observed variation in taxonomic richness and Absolute Pollen Frequency (APF) (Table 2). Overall, mean taxonomic diversity is high (36 to 41, Table 3), with APF values ranging from 4140 to 15,859 (Table 3). In contrast, sediments deposited in deeper-water environments exhibit excellent sporopollenin preservation. The resulting pollen assemblages align well with Eastern Mediterranean ecosystems (Davis, 1965-1988; Akman et al., 1978).

Samples found to be non-polliniferous or pollen-poor were primarily associated with evaporitic or shallow lacustrine deposits, characterised by algal maxima (e.g. Botryococcus or Chrysophyceae) (Fig. 1), or with layers contemporaneous with fire events, as indicated by peaks in microcharcoal concentration (Fig. 1).

Both for the Marseille site and the western Turkey sites (Kocabaş and Acıgöl), the LST protocol yielded clear, high-quality pollen slides that were easy to analyse, significantly reducing processing time.
<figure><img src="image_5.png" /><figcaption>Plate I. Photographs of Acıgöl pollen slides. Photo. 1–4: standard method to extract pollen was used. Photo. 5–8: LST Fastfloat was used.</figcaption></figure>
<table><caption>**Table 3**
Acigöl pollen data parameters. APF: Absolute Pollen Frequencies; NPP: Non Pollen Palynomorphs; PS: Pollen Sum.</caption><thead><tr><th></th><th>Minimum</th><th>Maximum</th><th>Mean</th></tr></thead><tbody><tr><td>Taxa sum with NPP</td><td>20</td><td>68</td><td>41</td></tr><tr><td>Taxa sum without NPP</td><td>16</td><td>63</td><td>36</td></tr><tr><td>APF with NPP</td><td>189</td><td>282,205</td><td>15,859</td></tr><tr><td>APF without NPP</td><td>118</td><td>27,122</td><td>4140</td></tr><tr><td>PS with NPP</td><td>361</td><td>15,902</td><td>2799</td></tr><tr><td>PS without NPP</td><td>206</td><td>2143</td><td>587</td></tr><tr><td>Mass g</td><td>2.8</td><td>16.5</td><td>12</td></tr></tbody></table>

### 3. Palynomorphs extraction protocol

LST Fastfloat is commerically available at a density of 2.82 with a viscosity of 10 cP; however, for our palynological studies, we adjusted the density to 2.0, or 1.9 in cases where extremely pollen-poor material was suspected. Unlike other dense liquids, LST is easily diluted with water and can be recycled. Some loss occurs due to residual liquid trapped in core pellets after centrifugation, but its efficiency, particularly in reducing pollen slide analysis time, offsets its high cost.

For effective chemical processing, a large sample mass is recommended. The mean sample mass was 222 g at Marseille (Table 1), 250 g at Kocabaş (Table 2) and 12 g at Acigöl (Table 3). When sample weight exceeds 10–15 g, depending on the sediment type (minerogenic or biogenic), material should be distributed across multiple 150 ml centrifuge tubes to optimise chemical action. Coarse sediments require sieving to remove large fractions (e.g., sand, pebbles) and maximise palynomorph concentration.

For indurated conglomerates suspected to be polliniferous or archaeologically significant (e.g. the Kocabaş hominin series; Andrieu-Ponel et al., 2021), pollen must be extracted from the cement matrix after the rock is crushed and sorted under a binocular microscope. In the case of indurated travertine or tufa, large field samples should be collected and subsequently cut using a circular saw (Fig. 2) to eliminate sediments exposed to current pollen pollution.

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** A sample of travertine from Marseille after cutting with a circular saw.</figcaption></figure>

**Precautions:**

* Water reacts violently with acids and may cause bodily harm, therefore always ensure that tubes, rods etc. are clean and dried well before use, as well as remove all water sources from the fume cupboard area.

* Hydrofluoric acid (HF) is very dangerous, therefore correct handling and efficient fume extraction is essential.

<figure><img src="image_7.png" /><figcaption>**Fig. 1.** Pollen diagram of Acigöl, selected taxa. NPP: Non Pollen Palynomorphs, APF: Absolute Pollen Frequencies.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 3. Illustrated LST Fastfloat pollen extraction process.</figcaption></figure>

• All references to H₂O refer to demineralised water.

The protocol's required steps are outlined below, accompanied by a simplified diagram illustrating the pollen extraction process (Fig. 3).

## 1. Sample preparation

a. Weigh samples, then sieve at 250 µm to remove large particles.

b. Place in 150 ml conical-bottom centrifuge tubes. (NOTE: If the samples are large, use 250 ml glass beakers).

c. Add Lycopodium tablets (optional).

## 2. Deflocculation (Na hexametaphosphate)

a. If the samples are clayey or to improve HCl action, add Na hexametaphosphate 50% (ratio 1/3 pellet: 2/3 defloculant) and mix with a clean glass rod.

b. Stir with a Vortex mixer or a mechanical stirrer for at least 1 h.

c. Centrifuge at 2500 rpm for 5 min.

d. Invert tubes (Fig. 4) on absorbent paper to partially dry (30 mins). (NOTE: There may be some dripping as the pellet is deflocculated. This can be corrected with a last rince of H₂O with a few drops of HCl).
<figure><img src="image_9.png" /><figcaption>Fig. 4. Invert to drain.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 5. Addition of KOH 10%.</figcaption></figure>

### 3. HCl (Acid) treatment

a. Add cold pure HCl (37%); ratio 1/3 pellet: 2/3 HCl.

b. Leave for 4 h minimum or overnight.

c. Repeat HCl treatment if necessary (especially carbonate-rich sediments).

d. Rinse twice with H₂O.

e. Decant supernatant into appropriate chemical waste container.

### 4. KOH (Base) treatment

a. Add KOH 10% or NaOH 10% (Fig. 5).

b. Heat in a bain-marie at 50 °C for 10 min (stirring occasionally), allow to cool.

c. Centrifuge at 2500 rpm for 5 min.

d. Decant supernatant into the appropriate chemical waste container.

e. Rinse twice with H₂O, or until supernatant is clear.

(NOTE: For sediment rich in humic acid (releases a black liquid), rinse 5 times with H₂O, then stop the reaction by adding a few drops of HCl to the last rinse).

### 5. Deflocculation (Na hexametaphosphate)

a. Add deflocculant (Na hexametaphosphate 50%) and mix with a clean glass rod.

b. Stir with a Vortex mixer (3 min) or use a mechanical stirrer for 1 h (Fig. 6).

c. (NOTE: If the sediment is not sticky, this step can be omitted; however, it is recommended because the deflocculation optimises the LST action).

d. Centrifuge at 2500 rpm for 5 min.

e. Turn tubes upside down onto absorbent paper to partially dry (30 mins). (NOTE: There may be some dripping as the pellet is deflocculated).

### 6. LST Fastfloat treatment

a. Add LST density 2 (1.9 if sediment is pollen-poor; Fig. 7); ratio 1/3 pellet: 2/3 LST.

b. Mix the LST and the pellet with a clean glass rod, followed by manual or mechanical agitation (5 min to 4 h, depending if the pellet is sticky (clayey).

(NOTE: This step is vital because the more successful the deflocculation, the better the separation of the palynomorphs from the mineral particles).

c. Centrifuge at 2500 rpm (5/5) for 10 min.

d. Decant supernatant into new 150 ml centrifuge tubes.

e. Fill with H₂O to lower the density.

f. Centrifuge at 3000 rpm for 10 min.

g. Collect the supernatant (made from diluted LST) in a flask to recycle the dense liquid (see instructions at the end of this protocol).

h. Rinse again with H₂O, centrifuge at 2500 rpm (5/5) for 10 min.

i. Final rinse with H₂O and a few drops of HCl, and centrifuge at 3000 rpm (5/5) for 10 min.

j. Decant supernatant into the appropriate chemical waste container.

### 7. Acetolysis (siliceous fossils will be lost), optional

a. For 8 samples with a small pellet: acetolysing liquid = 90 ml acetic anhydride + 10 ml sulphuric acid.

b. Use a ratio of 1/3 pellet: 2/3 glacial acetic to dehydrate the sample, stir with a clean glass rod and vortex for 60 s.

c. Centrifuge at 2500 rpm for 5 min.

d. Decant supernatant into the appropriate chemical waste container.

e. Add acetolysis solution (1/3 pellet: 2/3 mix) and stir with a dry clean glass rod.

f. Heat in a bain-marie at 55 °C (or a saucepan on a hot plate) for 5 min.

g. Remove and add glacial acetic acid or alcohol up to half way to stop reaction.

(NOTE: Leave to cool slightly (about 10 mins) to avoid centrifuging hot tubes, otherwise they will become deformed).

h. Centrifuge at 2500 rpm for 5 min.
<figure><img src="image_11.png" /><figcaption>Fig. 6. Mechanical stir for 1 h.</figcaption></figure>

i. Decant supernatant into the appropriate chemical waste container.

j. Rinse with H₂O.

k. Centrifuge at 2500 rpm for 5 min.

l. Decant supernatant into the appropriate chemical waste container.

8. HF treatment (avoid fumes/skin contact/glass equipment), optional

a. If minerals are still present (whitish haze at base of tubes): use pure HF (70%). (NOTE: This may result in the loss of some palynomorphs; however, the slides will be free of particles that could interfere with preparation or slow down analysis).

b. Add HF to tubes, ratio 1/3 pellet: 2/3 HF and stir with a clean dry teflon rod.

c. Leave for 4 h or overnight (HF is no longer active after 4 h). Repeat this step if residual silica sand remains because these mineral particles can interfere with the proper mounting of pollen between the slide and coverslip.

NOTE: HF treatment may make the tubes pinkish in colour (Fig. 8).

d. Decant supernatant into the appropriate chemical waste container.

e. Use new conical tubes (15 ml if the pellet is small), carefully rinse with H₂O (add up to 50 ml mark).

f. Centrifuge at 2500 rpm for 5 min.

g. Decant the supernatant into the appropriate chemical waste container.

h. Rinse using hot H₂O + a few drops of HCl to remove neoformed fluorides.

i. Centrifuge at 2500 rpm for 5 min.

j. Decant the supernatant into the appropriate chemical waste container.

(NOTE: If the pellet is small, transfer the sediment to new 15 ml conical tubes).

<figure><img src="image_12.png" /><figcaption>Fig. 7. Check density before use.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>Fig. 8. HF treatment can change the tube colour.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>(a) Sieve 10 µm then 7µm</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>(b) Grease screw top area</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>(c) Press down to seal</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>(d) Vacuum filter at 5µm to clean LST</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>(e) Evaporate to clean LST</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>(f) Measure density</figcaption></figure>

<caption>**Plate II.** LST recycling process.</caption>

## 9. Sieving

a. If the pellet is dirty / full of debris, deflocculate again with Na hexametaphosphate and then sieve (use plenty of H₂O to wash the pellet).

b. For samples from temperate and boreal regions use a 10 µm filter, or a 7 µm filter for tropical regions (pollen <10 µm).

c. Rinse the filter with a wash bottle in a beaker to recover the fraction >10 µm. (NOTE: Do not add too much water because the sample must fit into a 15 ml conical tube).

d. Centrifuge at 2500 rpm for 5 min.

e. Using a disposable plastic pasteur pipette, transfer the sediment into labelled 1.5 ml microtubes with conical bottoms.

f. Centrifuge at 2500 rpm for 5 min.

g. Add some alcohol (to accelerate evaporation).

h. Centrifuge at 2500 rpm for 5 min.

i. Let the alcohol evaporate a little and add glycerine.

j. Microscope observation is now possible using the clear slides (Plate I, photo. 5–8).

## LST recycling (Plate II)

1. Allow the dense liquid to settle for several days, so that the debris falls to the bottom of the jar naturally (under gravity).

2. Using a vacuum pump, sieve through a 10 µm and then a 7 µm filter to remove the largest particles, followed by a 5 µm filter. Usually, the step 10 µm to 7 µm is enough.

3. Evaporate the diluted LST in a one-litre beaker on a hot plate until the density is greater than or equal to 2.

4. If there are still particles floating in the liquid (generally LST crystals), sieve again through a 10 µm filter.

5. Adjust the LST to the correct density using H₂O.
## 4. Method evaluation

The LST Fastfloat method offers several important benefits, including but not limited to:

* Reduced toxicity: minimises or eliminates the use of HF, thereby making the palynomorph extraction procedure safer.

* Soluble in water.

* By multiplying sievings, improve slide clarity, produces easy-to-read slides and reduces analysis time.

* Higher spore-pollen concentration: Enhances the visibility of palynomorphs, which allows pollen slides to be analysed more quickly.

However, this method has two main limitations to consider:

* High cost: LST is expensive but recyclable.

* Potential palynomorph loss: as with all extraction methodologies, some material may be lost during processing.

## 5. Conclusion

This method is intended for researchers investigating early Quaternary palynomorph-poor sediments, particularly in Mediterranean and tropical dry regions, where ecological and climatic histories remain poorly understood due to the scarcity of pollen-rich archives. While initial results are highly promising, further validation would require the analysis of a larger sample set. Notably, non-oxidised (orange, reddish) samples should be prioritised, a substantial sample mass (≥ 10 g and no upper limit) is necessary, and it is important to multiply the steps of deflocation followed by sievings.

These findings emphasise the potential for re-examining sedimentary sequences of biogeographical or archaeological significance, even though previously considered pollen-poor, in order to enhance our understanding of past environmental conditions in these regions.

## Credit author statement

Both authors have written, read and agreed to the published version of the manuscript.

## Funding

This work received financial funding from ANR FOOD-RE (ANR-21-CE03-0019) V. Andrieu leader 1 April 2022 (https://www.cerege.fr/fr/sciences/terre-et-planetes/projets-terre-et-planete/anr-food-re/).

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Andrieu Valerie reports financial support was provided by French National Research Agency. Andrieu Valerie reports a relationship with French National Research Agency that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We would particularly like to thank the Micropaleontological Preparation Laboratory (CEREGE) and its staff (Jean-Charles Mazur, Dr. Christine Pailles and Sandrine Conrod). We thank Aix-Marseille University for the funding of 1) the FIR program for the invitation of Dr Belinda Gambin (University of Malta) to CEREGE (Aix-en-Provence,

France) for one month in 2024, 2) the open-access of this article.

## Data availability

No data was used for the research described in the article.

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