M Albignac

E Maria

T De Oliveira

C Roux

D Goudouneche

A F Mingotaud

G Bordeau

Alexandra Ter Halle
email: alexandra.ter-halle@cnrs.fr

Assessment of nanoplastic extraction from natural samples for quantification purposes

Assessment of nanoplastic extraction from natural samples for quantification purposes

Introduction

Plastic pollution is a growing topic of public concern. To address the questions of society, research has intensified to better evaluate the effects of plastic on ecosystems and human health. Analytical developments were proposed to characterize plastic pollution at the micro-and nano-scales [START_REF] Filella | Questions of size and numbers in environmental research on microplastics: methodological and conceptual aspects[END_REF]. The analysis of nanoplastic (NP, particles below 1 µm) is still very challenging. Technological developments in terms of characterization and detection were recently reviewed [START_REF] Schwaferts | Methods for the analysis of submicrometer-and nanoplastic particles in the environment[END_REF]. Physicochemical characterization and investigation of NP behavior in the environment are also better understood [START_REF] Besseling | Fate of nano-and microplastic in freshwater systems: A modeling study[END_REF][START_REF] Gangadoo | Nano-plastics and their analytical characterisation and fate in the marine environment: from source to sea[END_REF][START_REF] Pradel | Deposition of environmentally relevant nanoplastic models in sand during transport experiments[END_REF][START_REF] Wu | Effect of salinity and humic acid on the aggregation and toxicity of polystyrene nanoplastics with different functional groups and charges[END_REF].

To study NP fate and behavior, many laboratory tests have been performed with model particles [START_REF] Gangadoo | Nano-plastics and their analytical characterisation and fate in the marine environment: from source to sea[END_REF] and typically with polystyrene nanospheres (also called polystyrene latex, PSL). PSLs are used as model particles because they are commercially available in a wide range of sizes and with various functionalizations [START_REF] Chae | Effects of micro-and nanoplastics on aquatic ecosystems: Current research trends and perspectives[END_REF]. PSL is usually available as formulated aqueous dispersions and the presence of surfactants leads to very stable dispersions, preventing aggregation and sedimentation. To proceed with tests, PSLs are classically diluted in water; or sometimes in DMSO [START_REF] Jeong | Nanoplastic Ingestion Enhances Toxicity of Persistent Organic Pollutants (POPs) in the Monogonont Rotifer Brachionus koreanus via Multixenobiotic Resistance (MXR) Disruption[END_REF]. After dilution, the dispersions are usually vortexed, less frequently sonicated [START_REF] Dong | Combinational effect of titanium dioxide nanoparticles and nanopolystyrene particles at environmentally relevant concentrations on nematode Caenorhabditis elegans[END_REF]. Often, the dispersions are used as they are in exposure media. To remove the preservatives, it was sometimes proposed that the PSL dispersions be centrifuged [START_REF] Cole | Ingestion of Nanoplastics and Microplastics by Pacific Oyster Larvae[END_REF], filtered [START_REF] Greven | Polycarbonate and Polystyrene Nanoplastic Particles Act as Stressors to the Innate Immune System of Fathead Minnow (Pimephales Promelas)[END_REF] or dialyzed [START_REF] Mattsson | Altered Behavior, Physiology, and Metabolism in Fish Exposed to Polystyrene Nanoparticles[END_REF][START_REF] Mattsson | Brain damage and behavioural disorders in fish induced by plastic nanoparticles delivered through the food chain[END_REF][START_REF] Pikuda | Toxicity Assessments of Micro-and Nanoplastics Can Be Confounded by Preservatives in Commercial Formulations[END_REF]. After these preparation steps, PSL concentrations were sometimes controlled by nanoparticle tracking analysis (NTA) to provide concentrations (expressed in number of particles/mL) [START_REF] Mattsson | Altered Behavior, Physiology, and Metabolism in Fish Exposed to Polystyrene Nanoparticles[END_REF][START_REF] Mattsson | Brain damage and behavioural disorders in fish induced by plastic nanoparticles delivered through the food chain[END_REF] or by fluorescence measurements [START_REF] Pikuda | Toxicity Assessments of Micro-and Nanoplastics Can Be Confounded by Preservatives in Commercial Formulations[END_REF] but the concentrations of the particles were seldom monitored during these operations.

Even if polystyrene nanosphere purchased from market, might be far away from the environmental samples, the use of PSL offers a fair number of advantages in evaluation tests and will certainly still be heavily used in the future. However, very few studies have investigated more environmentally relevant systems, such as polydisperse and polymorphic mixtures of model particles [START_REF] Baudrimont | Ecotoxicity of polyethylene nanoplastics from the North Atlantic oceanic gyre on freshwater and marine organisms (microalgae and filter-feeding bivalves)[END_REF][START_REF] Pradel | Deposition of environmentally relevant nanoplastic models in sand during transport experiments[END_REF]. The use of PSL allows us to draw an outline regarding NP fate and behavior or interaction with organisms in the natural habitat while keeping in mind that NP occurring in the environment are highly complex systems [START_REF] Galloway | Interactions of microplastic debris throughout the marine ecosystem[END_REF][START_REF] Garvey | Molecular-Scale Understanding of the Embrittlement in Polyethylene Ocean Debris[END_REF][START_REF] Gigault | Current opinion: What is a nanoplastic?[END_REF][START_REF] Rowenczyk | Microstructure Characterization of Oceanic Polyethylene Debris[END_REF][START_REF] Ter Halle | Nanoplastics: A Complex, Polluting Terra Incognita[END_REF].

There are only very few studies reporting NP analysis in natural samples [START_REF] Davranche | Nanoplastics on the coast exposed to the North Atlantic Gyre: Evidence and traceability[END_REF][START_REF] Mintenig | Closing the gap between small and smaller: towards a framework to analyse nano-and microplastics in aqueous environmental samples[END_REF][START_REF] Sullivan | Detection of trace sub-micron (nano) plastics in water samples using pyrolysis-gas chromatography time of flight mass spectrometry (PY-GCToF)[END_REF][START_REF] Ter Halle | Nanoplastic in the North Atlantic Subtropical Gyre[END_REF][START_REF] Wahl | Nanoplastic occurrence in a soil amended with plastic debris[END_REF][START_REF] Zhou | Cloud-Point Extraction Combined with Thermal Degradation for Nanoplastic Analysis Using Pyrolysis Gas Chromatography-Mass Spectrometry[END_REF]. The sampling strategies proposed to extract and concentrate NP were never properly evaluated and the possible losses overlooked.

Ter Halle et al. proposed the pre-filtration of seawater on 5 µm polyethersulfone (PES) membranes to isolate the colloidal fraction, which was then concentrated by frontal ultrafiltration (20 kDa PES membranes) for DLS characterization [START_REF] Ter Halle | Nanoplastic in the North Atlantic Subtropical Gyre[END_REF]. The dispersion then underwent lyophilization. NP were trapped in the remaining sea salts and directly analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). This approach was only applied to detection, and the possible losses during this multiple step sample preparation were not evaluated. The evaluation of NP recovery during sample preparation was only reported once upon ultrafiltration on PES [START_REF] Mintenig | Closing the gap between small and smaller: towards a framework to analyse nano-and microplastics in aqueous environmental samples[END_REF]; recoveries were limited to 54% with PES membrane. Sullivan et al. recently analyzed NP in river water [START_REF] Sullivan | Detection of trace sub-micron (nano) plastics in water samples using pyrolysis-gas chromatography time of flight mass spectrometry (PY-GCToF)[END_REF]. Without pre-filtration, they directly proceeded to frontal filtration of 200 mL of river water on a polytetrafluoroethylene (PTFE) membrane (0.45 µm porosity). The particles trapped on the filters were introduced, after cryomilling, into the pyrolysis chamber for semiquantitative analysis by pyrolysis-gas chromatography time of flight mass spectrometry (Py-GC-TOF). But the absence of prefiltration did not enable to established the size range of the analyzed particles. PTFE membranes were often used in pre-filtration step to isolate the NP from microplastics [START_REF] Davranche | Nanoplastics on the coast exposed to the North Atlantic Gyre: Evidence and traceability[END_REF][START_REF] Materić | Micro-and Nanoplastics in Alpine Snow: A New Method for Chemical Identification and (Semi)Quantification in the Nanogram Range[END_REF][START_REF] Wahl | Nanoplastic occurrence in a soil amended with plastic debris[END_REF] but the retention capacity of the PTFE membrane was never estimated. As the number of studies quantifying nanoplastic in natural sample is increasing [START_REF] Cai | Analysis of environmental nanoplastics: Progress and challenges[END_REF], future work must include the evaluation of NP recoveries during sample preparation.

The present study aims at evaluating PSLs (from 50 to 1000 nm) behavior upon various preparation protocols, such as filtration, ultrafiltration or dialysis. The method to monitor PSL was based on convenient, nondestructive and easy-to-handle fluorescence measurements. As a pre-filtration step, nylon single strand tissue and stainless-steel were compared to polymer-based membranes. In order to trap and collect NP, we evaluated glass fiber filters. Finally, a two-step protocol was proposed and tested with a river sample and the colloidal content was monitored by NTA.

Materials and methods

Materials and chemicals.

Polybead® polystyrene nanospheres (1000, 750, 500, 350, 200, 100 and 50 nm) were purchased as aqueous solutions at 2.6% w/v (Polysciences, Warrington, USA). The membranes used were 47 mm diameter and of different polymer-based natures: polycarbonate, PC (Fischer Scientific, Illkirch-Graffenstaden, France), PES (Sterlitech, USA), cellulose acetate, CA (Sterlitech, USA), nylon single strand (Sefar Nitex®, VWR, Fontenay-sous-Bois, France), hydrophilic PTFE membranes (Omnipore, Merck Millipore, Molsheim, France), stainless-steel grid "plain dutch weave" (Negofiltre, Moret-Loing-et-Orvanne, France) and glass fiber filters (GF/F Whatman®, Merck Millipore, Molsheim, France). Ultra-pure water (18.2 mΩ.cm, Milli-Q filtration unit, Merck Millipore, USA) was used for solution preparation. Glass filtration units were purchased from Verre Wagner (Toulouse, France). Frontal ultrafiltration measurements were carried out in Amicon® 400 mL stirred cells (Aldrich, Saint Quentin Fallavier, France). Tangential ultrafiltration was carried out on Akta® flux S (GE Healthcare, Vélizy-Villacoublay, France). For frontal ultrafiltration, the membrane used was PES type BU50 at 50 kDa (Alting, Hoerdt, France). Tangential ultrafiltration was carried out on autoclavable PES at 50 kDa, hollow fiber cartridge UFP-50-C-MM01A (GE Healthcare, Vélizy-Villacoublay, France). Dyalysis cells were from the brand Tube-O-Dialyze mini dialysis system with membrane presenting a cut off at 1 kDa (Merck Molsheim, France).

Filter calcination. The glass fiber filters were prepared by calcination to remove any polymer or organic compound traces with a heating ramp from room temperature to 500 °C at a rate of 80 °C/hour with a hold of 30 hours at 500 °C in a furnace (Nabertherm® LV052K1RN1). Stainless steel grids were calcined at 500°C for 3 h with a heating ramp of 200°C/h from room temperature in the same furnace.

PSL dispersion preparation.

The unfunctionalized PSL were named PS, and the carboxyl-and amino-terminated PSL particles were named PSCOOH and PSNH2, respectively. The size was indicated afterward (PS-50 for 50 nm unfunctionalized beads). PSL dispersions were obtained after diluting the commercial formulations to reach 10 ppm by three successive dilution steps (dilution factor of 2600). Before any characterization or manipulation (filtration, dialysis, ultrafiltration), the diluted dispersion was sonicated for 30 minutes at 25°C. We ensured that sonication, in our experimental setup, did not alter the fluorescence emission of the beads and that the bead sizes were not altered by diffusion light scattering (DLS) measurements for PSCOOH-500, PSCOOH-350 and PSCOOH-50. The nominal size stated by the manufacturer were confirmed by DLS measurements. The z-averages were measured at 50.3 ± 1.1 nm, 97.7 ± 0.6 nm, 361.3 ± 5.5 nm and 520.5 ± 25.7 nm for PSCOOH-50 PSCOOH-100, PSCOOH-350, and PSCOOH-500, respectively. Tests were systematically run with freshly diluted dispersions. When we run the stability evaluation, they were stored at 4°C.

Gravimetric filtration.

The filtrations were carried out under vacuum with a glass unit equipped with 0 porosity sintered glass (corresponding to pores between 160 and 250 µm) with a diameter of 47 mm to hold the membranes. For the filtration, systematically a volume of 10 mL was used and filtered in 3 minutes. Membranes were tested with nanospheres bigger or smaller than their reported pore sizes. Nanospheres concentrations were measured using the calibrations curves presented in figure SI2 and SI3 before and after each operation. The retention capacity was calculated by the difference of concentration measured before and after filtration. Tests performed with 6 replicates presented retention capacities with a RSD of 10%.

Dyalysis. The cells used had a volume of 5 mL with 1 kDa cut off membrane (Merck Molsheim, France). A volume of 3 ml of nanosphere dispersion was placed in the dialysis cell which was placed in a 5 L beaker for 48 h. The retentate was analyzed by fluorescence in order to estimate how much nanospheres were recovered after the operation.

Ultrafiltration. Frontal ultrafiltration was carried out with 100 mL of dispersion at 1 bar.

Tangential ultrafiltration was carried out with 100 mL of dispersion at 50 mL.min -1 , with transmembrane pressure at 0.5 bar and delta pressure at 0.22 bar Dynamic light scattering (DLS). DLS was carried out at 25°C on a Malvern (Orsay, France) Zetasizer NanoZS. Solutions were analyzed in triplicate without being filtered to characterize the plain samples. Data were analyzed using the general-purpose nonnegative least squares (NNLS) method. DLS measurements were carried out at 1000 ppm for the carboxyl functionalized particles with sizes from 50 to 1000 nm.

Scanning electron microscopy (SEM). After filtration, the membrane was dried at room temperature for 48 hours. A piece of the membrane was cut and fixed on a microscope stub with double-sidedtape, then sputtered with platinum. Membranes were examined on an FEI Quanta 250 FEG scanning electron microscope at an accelerating voltage of 5-10 kV.

Fluorescence. Fluorescence measurements were performed at 25°C using a HORIBA Jobin Yvon Fluorolog 2 IHR equipped with a temperature-controlled cuvette holder, with an excitation wavelength of 250 nm and an emission wavelength of 310 nm. The 6 points calibration curves were obtained from the 10 ppm solution after proper dilution.

Calibration points were recorded with fresh solutions and systematically recorded the same day as the filtration tests. In order to ensure that the signal was systematically properly integrated, we monitored PSL concentrations above a limit of quantification set at 1 ppm regardless of the nanosphere size or functionalization.

Sampling of river water. The water sample from Garonne River was collected on 16 September 2021 directly from the bank of the River (gps coordinates: 43°34'38.1"N 1°26'08.8"E) in a 2 L glass bottle. The water temperature was around 22°C, and the turbidity around 21 NTU. The river sample was processed with the two steps filtration on 5 µm stainless steel grid and glass fiber filter directly after sampling without storage delaying.

Nanoparticle tracking analysis (NTA). Colloidal contents and size distribution of river water samples were assessed by NTA using a Malvern NanoSight NS300® (Malvern Instruments Ltd, UK) at 25°C. The experiments were performed in triplicate under a flow of 0.1 mL.min -1 during 90 sec. The conditions were checked to have an optimal particle concentration in the microscope field of view between 10 7 and 10 9 particles.mL -1 . The presented result consisted in the merging of the triplicats.

Results and discussion

Fluorescence measurements

Standard PSL presents an intrinsic fluorescence upon UVC excitation. Although this fluorescence is far less intense compared to biolabeled beads, the difference in cost makes this method useful and convenient. After dilution at 10 pmm, when repeatedly analyzing a given PSL dispersion, the fluorescence emission presented a relative standard deviation (RSD) of 2 % (n=8 with PSCOOH-50). The repeated dilution from a commercial formulation was also reproducible; the RSD were below 8% (n=6) with the PSL tested (PSCOOH-50, PSCOOH-100, PSCOOH-500, PSNH2-500 and PS-500).

Using polystyrene intrinsic fluorescence emission as a basis for quantification, we evaluated if there was a linear relationship with PSL concentration. The calibration curves presented excellent linearity with correlation factors greater than 0.97 with all tested PSL (Figure SI2 and Figure SI3). The reproducibility of the calibration curves was very good when repeating the standards preparation (use of PSCOOH-100, n=5, no significant differences with Student's t-test at 5%).

The PSL intensities of fluorescence were size-dependent; the bigger beads being more fluorescent. This interesting property was recently observed by Konemann et al. but was very difficult to rationalize [START_REF] Konemann | Characterization of steady-state fluorescence properties of polystyrene latex spheres using off-and online spectroscopic methods[END_REF]. One suggestion would be that there was more fluorescence loss with smaller particles due to increasing light scattering. It is important to underline that we have designed this set of experiments with a conservative mass with an initial concentration set at 10 ppm, whereas due to the particle size differences, the number of particles were different from on test to another when particle size varied.

Finally, when comparing nanospheres of the same size with different functionalization, we observed differences. With the nanospheres at 100 nm, the intensity of fluorescence decreased as: PSNH2>PSCOOH>PS (Figure SI3). The differences were less marked with 500 nm beads (Figure SI4). The functionalization of PS leading to variations in the benzyl moiety spatial organization certainly impact the fluorescence properties.

The supplier indicates a 6 months stability of the commercial dispersion. But there was no indication about the stability after aqueous dilution. We evidenced that the diluted dispersions at 10 ppm were not stable. After 7 days of storage at +4°C, PS-500 evidenced a 50 % decrease of the fluorescence (RSD=2.5%, n=3). The signal was even weaker after 15 days. The DLS measurements did not indicate any changes in the PS-500 hydrodynamic diameter. The same decay was measured after dilution with PSCOOH-500 and PS-100. PS-50 appeared more stable with variations below 10% over 7 days. After this first evaluation, our first recommendation is to systematically use freshly diluted dispersion and avoid storage.

Membrane filtration

The purpose of this series of test was to provide a protocol to isolate the colloidal fraction in a natural sample for quantification; in other word a procedure that would allow to retain particles larger than 1 µm but quantitatively recover the smaller ones in the filtrate. Membranes present distinct physicochemical characteristics implying electrostatic interactions with particles together with variations in pore size uniformity due to membrane construction and tortuosity. For all these reasons, retention cutoffs are not absolute [START_REF] Hernandez | Pore size distributions in microporous membranes. A critical analysis of the bubble point extended method[END_REF] and the experimental tests presented here appeared necessary. The retention capacities were compared with strictly the same operating conditions in terms of volume filtered and flow rate at the same mass concentrations (10 ppm). This implies that, if comparing the conditions between the 50 and 500 nm diameter beads for example, the 50 nm beads were 1000 times more numerous.

PS-500, PSCOOH-500 and PSNH2-500 were retained in the same proportions for a given membranes. In total, CA, PVDF, PES, PC and PTFE membranes were tested at pH 6 (Figure 1 to 3, difference in retention capacities were below 10%). The same results were obtained with PS-100, PSCOOH-100 and PSNH2-100 (data not shown).

We concluded that, under the present experimental conditions, transport through the membranes was not driven by the functionalities of the beads, in other terms by physicochemical interactions. Figure 1: Retention of 500 nm polystyrene nanospheres after filtration on cellulose acetate membranes with various porosities. Polystyrene nanospheres were functionalized with carboxylic acid or amine or were not functionalized. RSD were given with n=6.

When 500 nm nanospheres were tested with membranes presenting greater pore sizes (1.6 to 5 times), there was anyway membrane retention (Figure 1). For example, membranes in CA or PVDF with a reported pore size of 5 µm retained 20 to 40% of the 500 nm nanospheres (Figure 2). With a reported pore size of 1 µm, the PTFE membrane retained approximately 90% of the 500 nm nanospheres (Figure 3 A ). It appeared that PTFE and PES presented higher retention rates than PC membranes (Figure 3). Testing 350 nm nanosphere with larger pore size membranes, we also observed important retention capacities. For example, 0.45 µm membranes in cellulose acetate retained 94% of the PSCOOH-350 (Figure SI5). Testing even larger membrane pore sizes, over 1 µm, the retention capacities for the PSCOOH-350 were above 20 to 25 %, all membrane type tested (Figure SI5). PSCOOH-100 was tested with various membranes type with pore sizes at 0.45 and 0.4 µm (Figure 4 ). Again, there were important differences depending on the polymerbased membrane. The 0.45 µm nylon membranes presented almost quantitative retention. The 0.45 µm PTFE membrane was the second membrane, retaining a substantial percentage of the particles (45%). On CA, with 0.7 to 0.8 µm pore size, bettween 15 to 32% of PSCOOH-100 was retained (Figure SI6). With the use of even larger pore size membranes (1 to 1.2 µm), the retention capacities were between 12 % (CA, Figure 2) and 30% (PC, Figure SI6). The retention capacities of the membranes appeared to be ruled both by the pore size but also by the nature of the polymer used. We thus considered a given membrane type with varying pore sizes using PSCOOH-100 (Figure 5 A). With CA, the smaller the porosity, the higher the proportion of particles trapped, ranging from 50% retention with 0.2 µm to 12% with 1.2 µm porosity.

With PC, the behavior was radically different because the percentage of retention was 30%, regardless of the pore size (from 0.2 to 1 µm). This difference was difficult to rationalize but microscopic imaging presented below helped to apprehend the very distinct structure of the polymer-based membranes tested.

A) B)

Figure 5: A) Retention of 100 nm PS nanospheres after filtration on cellulose acetate or polycarbonate membranes with increasing porosity. B) Retention of carboxylated polystyrene nanospheres after filtration on cellulose acetate membranes with various nanosphere sizes and membrane porosities. RSD were given with n=3.

Scanning electron micrographs (SEM) provided interesting information. This technic is nonetheless relevant when polystyrene nanospheres are used and certainly more difficult to interpret with real samples. SEM was informative about the morphologies of the membranes and how the beads could be retained on them. PC membranes with a porosity of 0.2 µm appear to have a relatively planar surface with holes approximately 0.2 µm wide. It appears that the 100 nm beads can be retained on the membrane surface (Figure 6). Enlargement (Figure 6, on the right) shows that several beads can enter the same pore and be retained inside it, thereby clogging the pore. This configuration could explain why the 0.2 µm PC membrane retained some PS-100; the retention capacities were of 30%. The CA membranes with the same porosity (0.2 µm) had a very distinct organization, with multiple layers (Figure 7A). The multiple layer Finally, PSCOOH-50, the smallest PSL tested, were filtered through 0.2 µm membranes (Figure SI8). The percentages of retention were between 6 and 14%, and the differences between membrane types were not significantly different.

Altogether, it appeared that the filtration process using polymer-based membranes was crucial in terms of PSL retention. We observed very important retention capacities, and there was always retention even if the porosity was up to 50 times larger than the beads diameter. This implies that for pre-filtration, in the view to NP quantification in a real sample, the recoveries would be insufficient and very difficult to rationalize because of the high particle heterogeneity [START_REF] Gigault | Nanoplastics are neither microplastics nor engineered nanoparticles[END_REF]. We then looked for an alternative to polymer-based membranes.

Grid filtration

Nylon single strand tissue and stainless-steel grids were tested (microscopic images given in Figure SI 9). These two products did not present a multiple layer organization and were promising in terms of high particles recoveries. The nylon single strand tissue with a cut off at 1 or 5 µm retained between 20 to 30 % of the PSL whatever the particle size tested (100, 350, 500, 750 and 1000 nm). The stainless-steel grids with cut off at 5 and 10 µm retained between 15 to 20 % with the same particles tested. As the stainless-steel grid can be calcined in order to remove any impurities and be re-used, we preferred its use to the nylon tissue.

Dialysis, ultrafiltration

In addition to gravimetric filtration, we also investigated PSL concentration changes upon ultrafiltration and dialysis. The dialysis of PSCOOH-100 on 1 kDa PES membranes led to important losses 50% after 48 hours and 70% after 72 hours. We hypothesized that the particles certainly interacted with the membrane and remained attached to it. We highly recommend controlling the particle concentrations after proceeding to dialysis.

Another option to extract NP is the use of ultrafiltration [START_REF] Ter Halle | Nanoplastic in the North Atlantic Subtropical Gyre[END_REF]. We investigated the impact of ultrafiltration using a 50 kDa PES membrane and we observed important losses: 40% with frontal filtration. Working with tangential ultrafiltration slightly diminished this value to 35%. This is in agreement with previous evaluations with 50% losses [START_REF] Mintenig | Closing the gap between small and smaller: towards a framework to analyse nano-and microplastics in aqueous environmental samples[END_REF]. As there are important PSL losses after ultrafiltration we recommend to evaluate these loses is selecting this technic for nanoplastic extraction.

NP collection on glass fiber filters

In order to analyze NP after the isolation step, it was proposed to deposit of a small volume of the dispersion in a pyrolysis tube [START_REF] Mintenig | Closing the gap between small and smaller: towards a framework to analyse nano-and microplastics in aqueous environmental samples[END_REF] or a pyrolysis cup [START_REF] Davranche | Nanoplastics on the coast exposed to the North Atlantic Gyre: Evidence and traceability[END_REF][START_REF] Wahl | Nanoplastic occurrence in a soil amended with plastic debris[END_REF]. But other options were described, like the trapping of the particles on a filter. Sullivan et al. used PTFE filters as a medium for particles introduction in the pyrolysis chamber [START_REF] Sullivan | Detection of trace sub-micron (nano) plastics in water samples using pyrolysis-gas chromatography time of flight mass spectrometry (PY-GCToF)[END_REF]. However, the use of PTFE limits pyrolysis to 500°C. As an alternative the use of fiberglass filters was proposed as a support to transfer plastic particles in the micrometric range [START_REF] Albignac | Determination of the microplastic content in Mediterranean benthic macrofauna by pyrolysis-gas chromatography-tandem mass spectrometry[END_REF][START_REF] Leslie | Discovery and quantification of plastic particle pollution in human blood[END_REF][START_REF] Okoffo | Identification and quantification of selected plastics in biosolids by pressurized liquid extraction combined with double-shot pyrolysis gas chromatography-mass spectrometry[END_REF][START_REF] Ribeiro | Quantitative Analysis of Selected Plastics in High-Commercial-Value Australian Seafood by Pyrolysis Gas Chromatography Mass Spectrometry[END_REF]. This is promising because fiberglass filters can be calcined to remove any plastic residues before use. Finally, it allows the use of higher pyrolysis temperatures compared to PTFE which allow a better response for some polymers [START_REF] Hermabessiere | Optimization, performance, and application of a pyrolysis-GC/MS method for the identification of microplastics[END_REF].

However, the retention capacity of the filters used to collect the NP was never estimated.

We measured that calcined 0.7 µm glass fiber filters (GF/F, Whatman®) retained over 95% of PS-500, PS-750 and PS-1000. They retained 77% of the PS-350 and below this size, the retention capacity was 20% (for PS-100 and PS-200; Figure 6 8). As a conclusion, in the objective to collect nanoplastic, the retention capacity of glass fiber filters was quantitative above 500 nm and below this size the retention capacity rapidly diminishes with the particle sizes.

Figure 8: Retention of PSL carboxylated polystyrene nanospheres after filtration on glass fiber filters (0.7 µm pore size). RSD were given with n=3.

Application to river sampling

As an illustration, 2 L of river water were processed with the two-step preparation consisting in pre-filtration with a stainless-steel grid (5 µm cut-off) and collection with a calcined glass fiber filter.

The colloidal content of the water was monitored by NTA. The pre-filtration of 2L (Figure 9) shows that there is no alteration of the colloidal size distribution in the range 200-500 nm. Below 200 nm, after pre-filtration, there was a slight increase of the particle abundance; this could be explained by shearing of the colloids and formation of smaller particles in this class range. Above 500 nm the colloids present in the river were not abundant enough to be monitored by NTA. We concluded that pre-filtration on 5 µm steel grid did not alter much the particle size distribution in the nanometric rangethis is a promising way to recover quantitatively the colloidal fraction of a natural sample.

Figure 9: NTA analysis of river water samples after pre-filtration on 5µm inox grid and filtration on glass fiber filter. RSD were given with n=3.

The second step consisted in filtration on glass fiber filters in order to trap NP. We observed that 85% of the particles present in the river sample between 400-500 were retained, 56% for 300-400 nm, and 25 % for 200-300 nm. Below 200 nm, it is more difficult to interpret the results because the particles were more numerous after filtration (Figure 9). Again, we assume that during filtration there is shearing of the colloids resulting in more numerous smaller particles. The results with river water are less clearcut than with PSL because colloids are polydisperse and are made of aggregated smaller particles that reorganize and rearrange easily under shear effect taking place during filtration but we can conclude that above 400 nm more than 85 % of the particles in the river sample were trapped in the glass fiber filter.

Conclusion

GF/F filtered water

As a conclusion, we insist on the fact that sample preparation for nanoplastics analysis

is not yet established. Filtration (frontal or tangential ultrafiltration) or dialysis evaluated here for nanoplastic retention capacities might be not the best option. These methods present drawbacks but assets are also various. Among them, it remains a universal method easily accessible to most research teams. It was important to demonstrate that it can be used for such challenging topic, as long as enough precautions are taken and yields of recoveries evaluated. In summary, this study demonstrates that there are important interactions between PSL and polymer-based membrane during filtration; important losses were recorded. Our recommendation would be to assess the retention capacity of any membrane used whenever model NP are handled during tests. From the perspective of quantifying NP in natural samples, the use of polymer-based membranes as a pre-filtration step would result in important losses. As an alternative we propose the use of stainless-steel grids because the losses were low with 20 % retention capacity regardless the bead sizes tested. Filtration on the grid allow to recover properly the colloidal fraction of the river water and alterations seemed minimal. In a second step, in order to collect NP, the use of fiberglass filters is promising because they present high retention capacities, and can be calcined to remove any plastic residues before use. But the retention capacities of the glass fiber filter, quantitative above 500 nm, rapidly decreased with the particle's diameter.

In conclusion, as very intense efforts are made for the technological development of performant detection systems to reach smaller and smaller detection limits for NP analysis [START_REF] Schwaferts | Methods for the analysis of submicrometer-and nanoplastic particles in the environment[END_REF], extraction recoveries were systematically overlooked. We recommend, in case of filtration before analysis that a well control should be developed, along with the careful clean on the membrane. In order to provide quantification, this step is absolutely necessary. The present paper proposes a method to estimate the recovery rates during sample preparation and gives an illustration with a river sample.

  Figure 2: Retention of 500 nm polystyrene nanospheres after filtration on membranes with 5 µm porosities either in cellulose acetate (CA) or polyvinylidene difluoride (PVDF). RSD were given with n=6.

Figure 4 :

 4 Figure 4 : Retention of 100 nm carboxylated polystyrene nanospheres after filtration on 0.45 µm membranes. Membranes of cellulose acetate (CA), polycarbonate (PC), polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE) and nylon were tested. The membrane marked with a star had a 0.4 µm porosity. RSD were given with n=3.

Figure 6 :Figure 7 :

 67 Figure 6: Scanning electron micrographs of a 0.2 µm polycarbonate membrane after filtration of 100 nm carboxylate polystyrene nanospheres. The image on the right (B)is an enlargement over a few pores, where several 100 nm beads can be observed as trapped in a single pore (approximately 200 nm wide).

Funding acquisition, editing. Funding: This work was supported by the French National Research Program for Environmental and Occupational Health (ANSES), 'EST/2017/1/2019'. This study was funded by the Agence de l'Eau Adour-Garonne (PLASTIGAR project) and by the Region Midi-Pyrenees. A part of this study was also funded by SETOM, dedicated society of Veolia for the public drinking water service of Toulouse Métropole operating under the brand Eau de Toulouse Métropole.


Charles H Wellman
email: c.wellman@sheffield.ac.uk

Borja Cascales-Miñana

Thomas Servais

Terrestrialization in the Ordovician

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Palaeontological research aimed at reconstructing life in the Ordovician has historically focussed on the marine environment and reconstructing the 'Great Ordovician Biodiversification Event' and effects of the 'End Ordovician Mass Extinction'. Studies on life in terrestrial environments has largely been neglected (but see reviews by [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Richardson | Lower and Middle Palaeozoic records of terrestrial palynomorphs In[END_REF][START_REF] Wellman | Ordovician land plants: evidence and interpretation[END_REF][START_REF] Retallack | Ordovician life on land and early Paleozoic global change In[END_REF][START_REF] Steemans | Miospore evolution from the Ordovician to the Silurian[END_REF][START_REF] Strother | Cryptospores: the origin and early evolution of the terrestrial flora In[END_REF][START_REF] Wellman | The microfossil record of early land plants[END_REF]Steemans & Wellman 2004;[START_REF] Wellman | Palaeophytogeography of Ordovician -Silurian land plants[END_REF][START_REF] Servais | Revisiting the Great Ordovician Diversification of land plants: recent data and perspectives[END_REF]. In part this is a consequence of the paucity of Ordovician non-marine sediments and hence a dismally poor fossil record of non-marine organisms (except for those that have been transported into marine settings). But it also reflects historical research agendas based on assumptions that the first land plants did not appear until the Silurian. Since the 1980s this latter view has been challenged and evidence has accumulated suggesting that land plants first appeared in the Ordovician and may have been accompanied by the first animals to emerge from the oceans (either directly or migrating into brackish-freshwater environments and finally venturing onto the land). More recently evidence emanating from molecular biology, including phylogenetic, molecular clock and evo-devo analyses, has added to this growing body of evidence for an Ordovician terrestrial biota. In this chapter we will discuss the evidence for this terrestrial biota and attempt to reconstruct it. We argue that the Ordovician was a critical period during the terrestrialization of planet Earth.

An Early Ordovican terrestrial microbial world

The earliest terrestrial ecosystems were essentially microbial comprising freshwater ecosystems, periodically inundated microbial mats adjacent to rivers and lakes, and microbial crusts developed on rudimentary soils (reviewed in Wellman & Strother 2015). Such ecosystems are well documented from billion-year-old Lagerstätten: The Torridonian of Scotland [START_REF] Strother | Earths earliest nonmarine eukaryotes[END_REF]) and the Nonesuch Formation of the USA (Strother & Wellman 2021). They have also been shown to have persisted into the Silurian after land plants (embryophytes) had evolved and begun to colonise the continents (Tomescu & Rothwell 2006;Tomescu et al. 2006[START_REF] Tomescu | Earliest fossil record of bacterial -cyanobacterial mat consortia: the Early Silurian Passage Creek biota (440 Ma, Virginia, USA)[END_REF][START_REF] Tomescu | A new genus and species of filamentous microfossil of cyanobacterial affinity from Early Silurian fluvial environments (lower Massanutten Sandstone, Virginia, USA)[END_REF][START_REF] Tomescu | Simulating fossilization to resolve the taxonomic affinities of thalloid fossils in Early Silurian (ca. 425 Ma) terrestrial assemblages[END_REF]. The aquatic biotas included primary producers such as photosynthetic cyanobacteria (including both benthic and planktonic forms) (Strother & Wellman 2015) and a variety of other prokaryotic and eukaryotic organisms (e.g. Strother et al. 2021). The microbial mats and microbial crusts were established around photosynthetic cyanobacteria. At present there is little evidence for a significant fungal or lichen component (discussed below). It seems likely that such microbial ecosystems dominated Ordovician terrestrial ecosystems until the origin of land plants in the Middle Ordovician. Fig. 1 illustrates an assemblage of microbial fossils recovered from an Early Ordovician terrestrial deposit. The components are essentially identical to those described from the billion-year-old terrestrial deposits of the Torridonian Group and Nonesuch Formation.

The fossil record of land plants

Historically research on terrestrialization in the Ordovician has relied heavily on the fossil record. Regarding land plants this includes plant megafossils, dispersed spores, and disarticulated fragments of the plants. The latter two fossil types are generally recovered by palynological acid maceration techniques. A major hindrance to research has been the paucity of non-marine deposits recorded from the Ordovician worldwide. Land plant megafossils can be transported into marine deposits but the expectation is that they will be much rarer than in terrestrial deposits due to their allochthonous nature. Also they are mixed with marine fossils and it can be difficult to distinguish between fossils deriving from marine and non-marine organisms. Dispersed microfossils (spores and phytodebris) are readily transported into marine environments where they may be abundant. Again, however, the problem is that the non-marine provenance of biologically ambiguous forms cannot be confirmed.

Plant megafossils

The oldest generally accepted plant megafossils are rhyniophytoid plants, preserved as coalified compressions, from the Silurian (Wenlock) [START_REF] Edwards | A late Wenlock flora from Co, Tipperary, Ireland[END_REF][START_REF] Libertin | Libertín Sporophytes of polysporangiate land plants from the early Silurian period may have been photosynthetically autonomous[END_REF]. These show evolutionary continuity with younger Silurian forms that possess unequivocal spore-containing sporangia, stomata and conducting tissues (in some cases true tracheids) [START_REF] Edwards | A vascular conducting strand in the early land plant Cooksonia[END_REF]. Approximately 25-or-so Silurian localities preserve such plant megafossil assemblages and by the Lower Devonian (Lochkovian) they are abundant with at least 30 assemblages known worldwide (most recently reviewed by [START_REF] Edwards | Embryophytes on land: The Ordovician to Lochkovian (Lower Devonian) Record[END_REF][START_REF] Wellman | Palaeophytogeography of Ordovician -Silurian land plants[END_REF]. The coalified compressions preserve little anatomical detail. However, the anatomy of these plants is now well known due to Lagerstätten yielding exquisitely preserved charcoalified remains: Ludford Lane of late Silurian (Pridoli) age and North Brown Clee Hill of Early Devonian (Lochkovian) age, both from the Anglo-Welsh basin of the UK (e.g. [START_REF] Morris | The timescale for early land plant evolution[END_REF]. The Lower Devonian (Pragian-?earliest Emsian) silicified plants from the Rhynie chert Lagerstätten are also important in preserving exquisite anatomical detail of early land plants (e.g. [START_REF] Edwards | History and contemporary significance of the Rhynie cherts-our earliest preserved terrestrial ecosystem[END_REF].

There are regular and numerous claims for pre-Silurian plant megafossils. However, these are discounted as they can be proven to be fragments of other organisms (e.g. [START_REF] Kenrick | Semblant land plants from the Middle Ordovician of the Prague Basin reinterpreted as an animal[END_REF] or they lack sufficient characters to be unequivocally assigned to land plants (e.g. [START_REF] Salamon | Putative Late Ordovician land plants[END_REF][START_REF] Naugolnykh | Plants of the first terrestrial ecosystems[END_REF][START_REF] Retallack | Ordovician land plants and fungi from Douglas Dam, Tennessee[END_REF]. Throughout the 1960s-1980s Banks, Chaloner and others thoroughly catalogued and discredited such claims (e.g. [START_REF] Chaloner | The Origin of Vascular Plants[END_REF][START_REF] Chaloner | The rise of the first land plants[END_REF]Banks 1975a,b). This procession continues to this day and it is still doubtful that any claimed pre-Silurian land plant possesses sufficient unequivocal plant characters to be confidently and unequivocally proven to be a land plant. As noted by Edwards et al. (2021a) "…when diagnostic features are absent, such fragmentary organic materials can be misinterpreted, leading to implausible attribution (e.g. [START_REF] Retallack | Ordovician land plants and fungi from Douglas Dam, Tennessee[END_REF].".

Occasionally spore masses or even partial sporangia have been recovered during palynological processing of Ordovician deposits [START_REF] Wellman | Fragments of the earliest land plants[END_REF][START_REF] Abuhmida | Palynology of the Middle Ordovician Hawaz Formation in the Murzuq Basin, south-west Libya[END_REF]. From the Late Ordovician (Katian) of Oman [START_REF] Wellman | Fragments of the earliest land plants[END_REF] report spore masses, including some enclosed within a homogenous covering, and partial sporangia with fragments of sporangial wall attached (Fig. 2). The enclosed spores can be identified among dispersed forms (see below). These plant remains provide a tantalizing insight into Ordovician land plants and, if nothing else, demonstrate that they produced vast numbers of spores within sporangia that were extremely small but clearly highly fecund.

Dispersed spores

Spores encased in a resistant sporopollenin wall are a synapomorphy of land plants and it seems likely that the invasion of the land by plants was coincident with development of a subaerial reproductive strategy that utilised such spores [START_REF] Blackmore | Embryophyte spore walls: origin, development and homologies[END_REF][START_REF] Wellman | Origin, function and development of the spore wall in early land plants[END_REF]). They have an excellent fossil record because they are produced in vast numbers, capable of dispersal over huge distances by wind and water and are relatively resistant to degradation. Research by the early palynological pioneers established by the 1970s a continual record of dispersed land plant trilete spores from the early Silurian (Llandovery) onwards (e.g. [START_REF] Richardson | Silurian and Devonian spore zones of the Old Red Sandstone Continent and adjacent regions[END_REF][START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF]. These spores are often identical to those reported in situ from the earliest rhyniophytoid-rhyniophyte land plant megafossils (see above). However, in a series of ground-breaking papers Jane Gray and colleagues reported on a new group of dispersed spores, extending back into the Ordovician, that were subsequently proven to be the spores of land plants [START_REF] Gray | Early Silurian spore tetrads from New York: earliest New World evidence for vascular plants?[END_REF][START_REF] Gray | Caradocian land plant microfossils from Libya[END_REF][START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Gray | Tetrahedraletes, Nodospora, and the 'cross' tetrad: an accretion of myth[END_REF] produced by eophyte plants (Edwards et al. 2021a,b,c). These were called cryptospores because they occur in unusual configurations (e.g. permanent dyads and tetrads) [START_REF] Richardson | Late Ordovician and Early Silurian cryptospores and miospores from northeast Libya in[END_REF]) (Fig. 2).

The oldest reported occurrence of cryptospores clearly related to land plants is currently considered to be in the Middle Ordovician (Dapingian or Darriwilian) suggesting that land plants had successfully invaded the land by this time. They have now been reported continuously from this time point, with records from most palaeocontinents, extending through time until the end Pragian after which they become extremely rare in the fossil record (see Table 1 andFigs. 3-6). Cryptospores include monads, dyads and tetrads that are either naked or enclosed within an envelope. The envelope may be laevigate or ornamented.

Cryptospore taxonomy utilises the following characters: the number of units (monad, dyad, tetrad), the nature of attachment of these units (fused or unfused), the presence/absence of an envelope, the infraornament/ornament of the wall and/or envelope. A number of Ordovician cryptospores (dispersed and in situ) have been examined ultrastructurally and their gross structure/wall ultrastructure evaluated with regard to evidence for biological affinities (see Table 2) (Fig. 2). More recently Ordovician cryptospores have also been examined using a synchrotron light source [START_REF] Guizar-Sicairos | Ptychographic nanotomography at the Swiss Light Source[END_REF].

Very similar cryptospore assemblages have been reported throughout the Ordovician (spatially and temporally). Biostratigraphic schemes have been proposed, but these tend to be rather coarse with respect to time resolution (e.g. [START_REF] Richardson | Late Ordovician and Early Silurian cryptospores and miospores from northeast Libya in[END_REF][START_REF] Steemans | Cryptospores and trilete spores from the Llandovery, Nuayyim Borehole, Saudi Arabia[END_REF]. This is a consequence of the morphological simplicity of cryptospores (and hence lack of characters compared to trilete spores) and their seemingly slow pace of acquisition of novel morphological characters. The palaeogeographical spread, consistency and relative stasis exhibited by cryptospore assemblages have been taken to indicate that the first flora to invade the land consisted of cosmopolitan generalists that occupied a wide range of environments and evolved relatively little throughout the course of the Ordovician [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Wellman | Cryptospores from the type area of the Caradoc Series in Southern Britain[END_REF].

Probable non-marine palynomorphs that occur as irregular clusters of dyads and other polyads (in packets) have been described from Early Ordovician (Tremadocian) and earlier Cambrian deposits [START_REF] Strother | A fossil record of land plant origins from charophyte algae[END_REF]. These have been included with the cryptospores by some authors but are excluded by others. This is largely a semantic debate as several very different definitions for the term cryptospore have been proposed (see discussion in [START_REF] Servais | Revisiting the Great Ordovician Diversification of land plants: recent data and perspectives[END_REF]. This debate centres on whether cryptospores are defined as the spores of land plants (our preferred interpretation) or represent palynomorphs produced by any non-marine organisms. The most recent interpretation for the enigmatic Cambrian-Early Ordovician palynomorphs is that they represent charophyte algal remains [START_REF] Strother | A fossil record of land plant origins from charophyte algae[END_REF]) and may thus represent intermediate organisms on the freshwater green algal to terrestrial land plant lineage.

The first trilete spores, similar to those produced by Silurian rhyniophytoid plants, first appear in low numbers in the Ordovician [START_REF] Steemans | Origin and radiation of the earliest vascular land plants[END_REF]). They are difficult to identify because spores physically removed from cryptospore tetrads often bear a false trilete mark resulting from physical tearing as the polyads are broken apart. Similar to trilete spores, hilate cryptospores are produced by the natural dissociation of a polyad, but in this case a dyad [START_REF] Richardson | Late Ordovician and Early Silurian cryptospores and miospores from northeast Libya in[END_REF]). On their proximal surface they bear a circular contact area (hilum) formed where they were in contact with the other spore in the dyad. Like trilete spores, these naturally dissociated spores are common in the Silurian. They occur rarely in the Ordovician and again can be easily mistaken for spores physically separated from permanent dyads [START_REF] Steemans | Cryptospores and trilete spores from the Llandovery, Nuayyim Borehole, Saudi Arabia[END_REF].

Trilete spores only become common in dispersed spore assemblages in the Late Silurian whereupon they rapidly increase in abundance and diversity and have stratigraphical continuity with living trilete spore-producing plants. It has been proposed that this pattern reflects the origin and diversification of vascular plants (tracheophytes), because most Silurian-Devonian vascular plants have been shown to produce trilete spores (as do many extant basal groups of vascular plants) (e.g. [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Wellman | The microfossil record of early land plants[END_REF][START_REF] Wellman | Palaeophytogeography of Ordovician -Silurian land plants[END_REF]. The rare reports of trilete spores from the Ordovician-early Silurian may have derived from various non-vascular plants as trilete spores are known to be produced by some extant non-vascular plants (see [START_REF] Steemans | Origin and radiation of the earliest vascular land plants[END_REF]) and various plant groups are likely to have evolved this basic character independently.

We can only speculate as to where the first land plants appeared and the course of their spread across the continents. The earliest generally accepted reports of cryptospores are all currently from Gondwana which has led to speculation that they may have evolved on this continent before rapidly spreading out across the other continents of the planet (Steemans et al. 2010;[START_REF] Wellman | The invasion of the land by plants: when and where?[END_REF].

Phytodebris

Plants shed organs naturally during their lifetime and on death begin to rot and disassociate.

These processes produce recalcitrant fragmentary remains that are dispersed by gravity, wind and water and are ultimately incorporated into sediment from which they can be recovered by palynological processing. Such fragmentary plant remains, composed of recalcitrant biomacromolecules that survive as fossils, are termed phytodebris. They include plant cuticles composed of cutin, lignified tissues such as tracheids and reproductive propagules composed of sporopollenin (spores, megaspores, ovules and seeds), in addition to a number of more enigmatic remains. The fossil record and interpretation of phytodebris produced by early land plants was recently reviewed by [START_REF] Wellman | Applications of Non-Pollen Palynomorphs: from palaeooenvironmental reconstructions to biostratigraphy[END_REF].

There are surprisingly few reports of Ordovician phytodebris, perhaps reflecting the dearth of non-marine deposits examined (Fig. 2). Silurian non-marine deposits usually yield a diverse array of phytodebris. These include cuticles (sometimes with stomata) and tracheids that clearly derive from land plants. More enigmatic are cuticle-like sheets and tubular structures (including banded tubes) that are now known to derive from nematophytes.

Nematophytes were recently demonstrated to have fungal and possibly also lichen affinities [START_REF] Edwards | Evidence for a fungal affinity for Nematosketum, a close ally of Prototaxites[END_REF][START_REF] Edwards | Contributions to the diversity in cryptogamic covers in the mid-Palaeozoic; Nematothallus revisited[END_REF][START_REF] Edwards | Anatomically preserved Silurian 'nematophytes' from the Welsh Borderland (UK)[END_REF][START_REF] Honegger | The earliest records on internally stratified cyanobacterial and algal lichens from the Lower Devonian of the Welsh Borderland[END_REF][START_REF] Honegger | Fertile Prototaxites taiti: a basal ascomycete with inoperculate, polysporous asci lacking croziers[END_REF]. Related nematophyte remains appear not to extend back into the Ordovician. Rare cuticle-like sheets have been reported [START_REF] Gray | Caradocian land plant microfossils from Libya[END_REF][START_REF] Strother | New evidence for land plants from the lower Middle Ordovician of Saudi Arabia[END_REF]) (see Fig. 2). Ordovician tubular structures are usually smooth walled forms [START_REF] Burgess | Classification of uppermost Ordovician to Lower Devonian tubular and filamentous macerals from the Anglo-Welsh Basin[END_REF][START_REF] Strother | New evidence for land plants from the lower Middle Ordovician of Saudi Arabia[END_REF] with the reported banded tubes unconvincing (reviewed in [START_REF] Taylor | Ultrastructure of enigmatic phytoclasts (banded tubes) from the Silurian-Lower Devonian: Evidence for affinities and role in early terrestrial ecosystems[END_REF]. Because all of the reported Ordovician phytodebris are from marine deposits it is difficult to prove if they derive from terrestrial organisms rather than any variety of marine organism.

Regarding the biological affinities of the Ordovician phytodebris, the cuticle-like sheets may possibly derive from early nematophytes, although these have not been convincingly reported from the Ordovician. They may derive from early land plants, but in the absence of a megafossil record it is difficult to make comparisons. The smooth-walled tubular structures may derive from nematophytes or land plants. It is also possible that they represent cyanobacterial sheathes such as those described from the Silurian by Tomescu and colleagues (e.g. Tomescu & Rothwell 2006).

Geochemical biomarkers

In recent years reports on the first searches for early land plant biomarkers have begun to appear. For example, [START_REF] Romero-Sarmiento | Aliphatic and aromatic biomarkers from Gondwanan sediments of Late Ordovician to earlty Devonian age: an early terrestrialisation approach[END_REF] analysed Gondwanan Late Ordovican-Early Devonian sediments for the presence of aliphatic and aromatic biomarkers indicative of the presence of land plants [START_REF] Versteegh | The terrestrialization process: Modelling complex interactions at the biosphere-geosphere interface[END_REF]. [START_REF] Spaak | Environmental conditions and microbial community structure during the Great Ordovician Biodiversification Event; a multi-disciplinary study from the Canning Basin, Western Australia[END_REF] reported on the presence of benzonaphthofurans and delta δ 13 C-depleted mid-chain n-alkanes that they interpreted as indicative of the presence of bryophyte-like early land plants. This may be a fruitful area for future research as analytical techniques continue to improve in precision and we gain a better understanding of the biological affinities of the earliest land plants and their likely biomarker trails.

Evidence from extant land plants

Phylogenetic analysis

In the early 1980s the first cladistic analyses of land plants began to appear in publications, more-or-less coincident with the recognition of an Ordovician flora [START_REF] Gray | Caradocian land plant microfossils from Libya[END_REF]. These pioneering cladistic analyses considered only extant plants and utilised morphological/anatomical characters (e.g. [START_REF] Mishler | Transition to a land flora: phylogenetic relationships of the green algae and bryophytes[END_REF]. The majority of these:

(i) recovered a member of the charophycean green alga as sister group to the monophyletic embryophytes (land plants); (ii) indicated that the bryophytes were paraphyletic with the liverworts basal and a sister group relationship between vascular plants and either hornworts or mosses. For several decades these analyses were hugely influential providing a model for the physiological/anatomical aquatic algal-subaerial land plant transition (e.g. [START_REF] Graham | Origin of land plants[END_REF][START_REF] Graham | The origin, morphology, and ecophysiology of early embryophytes: neontological and paleontological perspectives in[END_REF]) and also the nature of the earliest land plants [START_REF] Gray | Ordovician-Silurian land plants: the interdependence of ecology and evolution[END_REF][START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Gray | Tetrahedraletes, Nodospora, and the 'cross' tetrad: an accretion of myth[END_REF].

The charophycean green algae-liverwort transition was seen to mirror the origin of subaerial land plants from freshwater aquatic green algal ancestors (e.g. [START_REF] Graham | Origin of land plants[END_REF]). The liverworts were regarded as the most basal extant land plants and used as a broad model for the morphology/anatomy, physiology and ecology of the first land plants (e.g. [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF].

The many subsequent morphology-based analyses that followed often mirrored these findings, although just about every possible relationship among the embryophytes (liverworts, hornworts, mosses, vascular plants) was been proposed at some point (reviewed in [START_REF] Kenrick | The origin and early diversification of land plants: a cladistics study[END_REF]. A significant advance was the inclusion of fossil data in cladistic analyses. Fossil data were important in recognising a group of fossil plants (Protracheophytes) that fell between the paraphyletic 'bryophytes' and vascular plants [START_REF] Kenrick | The origin and early diversification of land plants: a cladistics study[END_REF]. These included various Rhynie chert plants that preserve exquisite anatomical detail including some rather unusual character combinations, such as more-or-less isomorphic gametophyte and sporophyte generations, tracheids and stomata present in both generations, etc. [START_REF] Edwards | History and contemporary significance of the Rhynie cherts-our earliest preserved terrestrial ecosystem[END_REF]. Nevertheless, liverworts remained the model of choice for the earliest land plants.

As the molecular revolution dawned, the use of sequence data in cladistic analyses became possible. Initial analyses seemed to support the existing favoured topology. However, as more sequence data became available and ever-refined analytical techniques emerged, the tree topologies generated began to diverge from this model (recently reviewed by [START_REF] Wickett | Phylotranscriptomic analysis of the origin and early diversification of land plants[END_REF][START_REF] Puttick | The interrelationships of land plants and the nature of the ancestral embryophyte[END_REF][START_REF] Otpti | One thousand plant transcriptomes and the phylogenomics of green plants[END_REF]. Firstly, it began to appear that the zygnematophycean green algae were the most likely sister group to the embryophytes.

Secondly, embryophyte tree topologies changed significantly with monophyletic bryophytes and tracheophytes emerging as sister groups. Within the bryophytes the hornworts appeared as most basal and sister to a 'setaphyte clade' consisting of the liverworts and mosses.

The newly accepted topologies have important implications. Regarding the algal sister group, it suggests that many of the extant zygnematophycean algae, some of which are unicellular, are highly reduced [START_REF] Puttick | The interrelationships of land plants and the nature of the ancestral embryophyte[END_REF][START_REF] Cheng | Genomes of subaerial Zygnematophyceae provide insights into land plant evolution[END_REF][START_REF] Jiao | The Penium margaritaceum genome: hallmarks of the origins of land plants[END_REF][START_REF] Donoghue | Plant evolution: assembling land plants[END_REF][START_REF] Rensing | How plants conquered the land[END_REF]. This makes modelling of the algal-plant transition problematic based solely on consideration of the living zygnematophycean algae. In terms of the basal embryophytes, it means that the most basal of the extant vascular plants (lycopsids) are as closely related to the stem group land plants as the earliest diverging extant bryophytes (hornworts) [START_REF] Puttick | The interrelationships of land plants and the nature of the ancestral embryophyte[END_REF]. It also suggests that the bryophyte groups are reduced and have lost certain characters (e.g. liverwort stomata) or are secondarily simplified (e.g. possibly bryophyte conducting tissues). A critical next step will be the inclusion of fossil data into the new phylogenetic schemes. It is important to assess the position of the Rhynie chert protracheophytes. For example, if Aglaophyton can be shown to be sister group to the [bryophytes + tracheophytes] (i.e. a stem group embryophyte) it would suggest that stem group embryophytes may have been more complex than previously anticipated, possessing a more-or-less isomorphic gametophyte and sporophyte, with both generations possessing stomata and containing conducting tissues (possibly of Aglaophyton-type).

Recently Edwards et al. (2021a,b,c) recognised a new group of plants, called eophytes, among their charcoalified late Silurian-Early Devonian plant Lagerstätte (see above). These diminutive plants exhibit a primitive anatomy based on sporophytes with food conducting cells that yield in situ cryptospores. It is highly likely that these were parasitic on gametophytes characterised by transfer cells. Both stages of the lifecycle were likely poikilohydric, and thus able to desiccate and rehydrate, bearing ecological/physiological similarities to extant bryophytes as opposed to most vascular plants. Edwards et al. (2021a, fig. 8) place the eophytes as stem group polysporangiates, although we consider that they may represent stem group embryophytes that have a sister group relationship with a clade comprising both the bryophytes and vascular plants. Again their relative position in the phylogeny with respect to Rhynie chert plants such as Aglaophyton is critical in influencing our perception of the earliest stem embryophytes. It seems likely that they are more basal than Aglaophyton and provide the best current model for the earliest stem group embryophytes.

Molecular clock analyses

In the early 2000s the first molecular clock analyses began to appear concerning the dating of the origin of land plants and the major land plant groupings (reviewed in [START_REF] Morris | The timescale for early land plant evolution[END_REF].

Initial results indicated land plant origins far earlier than that suggested by the fossil record (as described above). Subsequently molecular clock techniques have rapidly evolved, and different strategies have been employed and a variety of palaeontological calibration systems experimented with. These analyses have provided highly variable results (reviewed in Table 3), and nearly all are incongruent with the fossil record, although the discrepancy is decreasing in some of the most recent analyses. An early origin of land plants as suggested by some molecular clock analyses would require that these plants left no fossil record for a condiderable period of Earth history, which seems unlikely if they reproduced by sporopollenin-walled spores, unless they were only present in very restricted environments and/or a very confined palaeogeographical area.

Fungi and lichens (including nematophytes)

Phylogenetic analyses and molecular clock evidence indicate that fungi originated in the Mesoproterozoic (e.g. [START_REF] Parfrey | Estimating the timing of early eukaryotic diversification with multigene molecular clocks[END_REF]), but it is not clear when they first appeared in terrestrial settings (aquatic or subaerial). Fungal remains are well known from the Silurian where they occur in palynological preparations as dispersed fungal spores and hyphae (e.g. [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF]. Such remains have rarely been reported from the Ordovician, but this may reflect the paucity of non-marine deposits available for analysis. [START_REF] Thusu | Latest Ordovician-Earliest Silurian acritarchs and chitinozoans from subsurface samples in Jebel Asba, Kufra Basin, SE Libya[END_REF] report the enigmatic Tortotubulus protuberans from the Late Ordovician. This tubular structure has been interpreted as a fungus [START_REF] Smith | Cord-forming Palaeozoic fungi in terrestrial assemblages[END_REF], although more precise affinities are not possible [START_REF] Auxier | No place among the living: phylogenetic considerations place the Palaeozoic fossil T. protuberans in Fungi but not in Dikarya. A comment on M. Smith[END_REF]. Most of the other reported fungal remains from pre-Silurian strata are more contentious (reviewed in [START_REF] Taylor | Fossil fungi[END_REF][START_REF] Berbee | Genomic and fossil windows into the secret lives of the most ancient fungi[END_REF][START_REF] Wellman | Applications of Non-Pollen Palynomorphs: from palaeooenvironmental reconstructions to biostratigraphy[END_REF]. However, it is worth noting that recent molecular development research on extant charophycean algae and land plants suggests that fungal symbioses may have been crucial to the colonisation of the land by plants (Berbee et al. 2011).

Sherwood-Pike

Lichenization has evolved numerous times involving different combinations of fungi and algal/cyanobacterial host [START_REF] Lűcking | Ediacarans, protolichens, and lichen-derived penicillium: A critical reassessment of the evolution of lichenization in fungi In[END_REF]. However, recent phylogenetic analysis suggests that lichens may not have evolved until after the evolution of vascular plants (Nelson et al. 2020).

The enigmatic Silurian-Devonian nematophytes have recently been demonstrated to have fungal, and possibly also lichen, affinities [START_REF] Edwards | Evidence for a fungal affinity for Nematosketum, a close ally of Prototaxites[END_REF][START_REF] Edwards | Contributions to the diversity in cryptogamic covers in the mid-Palaeozoic; Nematothallus revisited[END_REF][START_REF] Edwards | Anatomically preserved Silurian 'nematophytes' from the Welsh Borderland (UK)[END_REF][START_REF] Honegger | The earliest records on internally stratified cyanobacterial and algal lichens from the Lower Devonian of the Welsh Borderland[END_REF][START_REF] Honegger | Fertile Prototaxites taiti: a basal ascomycete with inoperculate, polysporous asci lacking croziers[END_REF]. These occur as megafossils [START_REF] Lang | On the plant-remains from the Downtonian of England and Wales[END_REF][START_REF] Strother | New species of Nematothallus from the Silurian Bloomsburg Formation of Pennsylvania[END_REF] and dispersed microfossils in the form of 'cuticle-like sheets' and tubular structures, including 'banded tubes' (recently reviewed by [START_REF] Wellman | Applications of Non-Pollen Palynomorphs: from palaeooenvironmental reconstructions to biostratigraphy[END_REF]. However, no convincing nematophyte remains have been reported from the Ordovician thus far.

Land animals in the Ordovician

Silurian continental deposits have yielded a diverse array of fossil evidence for land animals indicative of diverse and complex ecosystems developed in both freshwater aquatic and fully terrestrial settings. These fossils include whole organisms such as fish (e.g. [START_REF] Blom | Birkeniid anaspids from the northern hemisphere[END_REF] and arthropods (e.g. [START_REF] Jeram | Land animals in the Silurian: Arachnids and Myriapods from Shropshire, England[END_REF], dispersed arthropod cuticles recovered using palynological techniques (e.g. [START_REF] Gray | Early Silurian nonmarine animal remains and the nature of the early continental ecosystem[END_REF], coprolites (e.g. [START_REF] Edwards | Coprolites as evidence for plant-animal interaction in Siluro-Devonian terrestrial ecosystems[END_REF] and a variety of trace fossil evidence (e.g. [START_REF] Mccoy | A possible tracemaker of Arthrophycus alleghaniensis[END_REF].

Similar evidence for Ordovician terrestrial organisms is much rarer, almost certainly reflecting the paucity of Ordovician non-marine deposits available for study. To date all reported Ordovician fish remains are considered to be from fish inhabiting nearshore shallow marine environments [START_REF] Davies | Ordovician vertebrate habitats: a Gondwanan perspective[END_REF]. There are rare reports of potential nonmarine arthropods, but these are all controversial with questions remaining regarding either their age or habitat (e.g. [START_REF] Mcnamara | A euthycarcinoid arthropod from the Silurian of Western Australia[END_REF]. There are several reports of potential non-marine trace fossil assemblages. However, these are all from terrestrial deposits that are either doubtful or closely associated with nearshore marine deposits, making judgements on the habitat and mode of life of their makers doubtful. For example, it has been suggested that purported millipede burrows in Ordovician palaeosols described by [START_REF] Retallack | Trace fossil evidence for Late Ordovician animals on land[END_REF] are possibly of marine origin (Davies et al. 2010). Ordovician non-marine arthropod traces described by [START_REF] Johnson | Nonmarine arthropod traces from the subaerial Ordovician Borrowdale Volcanic Group, English Lake District[END_REF] have also been re-interpreted as marine in origin [START_REF] Shillito | Death on the shoreline, not life on land: Ordovician arthropod trackways in the Borrowdale Volcanic Group, UK[END_REF], and the trackways in Cambrian-Ordovician aeolian deposits described by [START_REF] Macnaughton | First steps on land: arthropod trackways in Cambrian-Ordovician eolian sandstone, southeast Ontario, Canada[END_REF] are from a marginal marine setting. It has recently been suggested that the exquisite trace fossils from the Tumblagooda Sandstone of Australia [START_REF] Trewin | Arthropods invade the land: trace fossils and palaeoenvironments of the Tumblagooda Sandstone (?Late Silurian) of Kalbarri, Western Australia[END_REF] were deposited in a littoral setting and are possibly of Silurian age [START_REF] Shillito | The Tumblegooda Sandstone revisited: Exceptionally abundant trace fossils and geological outcrop provide a window into Palaeozoic littoral habitats before invertebrate terrestrialization[END_REF].

Molecular clock studies have also been employed to ascertain when the various terrestrial arthropod groups appeared. To date most of these studies suggest that terrestrial arthropod groups invaded the land much earlier than the fossil record would indicate and by at least the Cambrian [START_REF] Lozano-Fernandez | A molecular palaeobiological exploration of arthropod terrestrialization[END_REF]. Terrestrial trace fossil assemblages from the Cambrian and earlier are all considered doubtful (reviewed by [START_REF] Minter | The trace fossil record of major evolutionary events[END_REF].

Ordovician non-marine sediments, soils and terrestrial environment

As emphasised throughout this review globally Ordovician non-marine deposits are extremely rare. The reasons for this are not clear (Davies & Gibling 2010) but is usually considered to be a consequence of high sea levels and the difficulties involved in identifying Ordovician non-marine evidence that relies largely on absence of evidence. Davies & Gibling (2010) summarise the sedimentology of the best known Ordovician non-marine deposits. It is clear that many of these are actually very near shore and are often interdigitated with marine deposits making it difficult to ascertain the degree of marine influence. None-the-less, some Ordovician palaeosols are reported (summarised by [START_REF] Retallack | Ordovician life on land and early Paleozoic global change In[END_REF].

There has been considerable debate regarding the influence of Ordovician terrestrial life, which transitioned from microbial mat communities to those including the earliest land plants (embryophytes), on the nature of sedimentation and soil formation (Davies & Gibling 2010;[START_REF] Gibling | Palaeozoic landscapes shaped by plant evolution[END_REF][START_REF] Santos | Tectonic and environmental controls on Palaeozoic fluvial environments:reassessing the impacts of early land plants on sedimentation[END_REF][START_REF] Davies | Discussion on 'Tectonic and environmental controls on Palaeozoic fluvial environments: reassessing the impacts of early land plants on sedimentation[END_REF]. This avenue of research has considered changes to weathering rate (e.g. D 'Antonio et al. 2019), sediment stabilising properties (e.g. [START_REF] Davies | Land plant evolution and global erosion rates[END_REF], patterns of sedimentation as geomorphological agents such as rivers change form (e.g. [START_REF] Ganti | Low gradient, singlethreaded rivers prior to greening of the continents[END_REF], consideration of how land plants promoted terrestrial mud deposition (e.g. [START_REF] Mcmahon | Evolution of alluvial mudrock forced by early land plants[END_REF][START_REF] Zeichner | Early plant organics increased global terrestrial mud deposition through enhanced flocculation[END_REF], and much more. This debate has also extended to biogeochemical effects [START_REF] Lenton | Matworld -the biogeochemical effects of early life on land[END_REF] and how these impacted atmospheric composition (e.g. [START_REF] Adiatma | Did early land plants produce a stepwise change in atmospheric oxygen during the Late Ordovician (Sandbian ~458 Ma)? Palaeogeography[END_REF], climate (e.g. [START_REF] Lenton | First plants cooled the Ordovician[END_REF], and so on. One avenue of research that has also recently received attention is the effects of increasing land plant cover on terrestrial biomass and thus patterns of carbon isotopes in the Ordovician sedimentary record [START_REF] Tomescu | A new genus and species of filamentous microfossil of cyanobacterial affinity from Early Silurian fluvial environments (lower Massanutten Sandstone, Virginia, USA)[END_REF][START_REF] Quinton | Sequence stratigraphy and carbon isotopes from the Trenton and Black River Groups near Union Furnace, PA: constraining the role of land plants in the Ordovician world[END_REF].

It should be noted that much of the above debate was focussed on the assumption that Ordovician terrestrial floras comprised bryophyte-like (more specifically liverwort-like plants). This assumption was based on prevailing land plant phylogenies (see above). Thus much of the debate utilised evidence from experiments on extant bryophytes (particularly liverworts) (e.g. [START_REF] Quirk | Constraining the role of early land plants in Palaeozoic weathering and global cooling[END_REF]. As discussed above recent phylogenetic analyses and the recognition of eophyte plants has altered our understanding and perception of the nature of the earliest stem group land plants. However, it should be noted that workers such as Jane Gray always stressed that the earliest land plants may not be directly related to extant bryophytes/liverworts, but that these plants were probably 'bryophyte-like' / 'liverwort-like' in their physiology and ecology [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Gray | Tetrahedraletes, Nodospora, and the 'cross' tetrad: an accretion of myth[END_REF], and they may thus still provide a reasonable analogue (if not 'nearest living relative') for the earliest land plants.

Terrestrial life and the End Ordovician glaciation and mass extinction

The dispersed spore fossil record exhibits no appreciable change from the Late Ordovician into the Early Silurian (reviewed by [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Richardson | Lower and Middle Palaeozoic records of terrestrial palynomorphs In[END_REF][START_REF] Strother | Cryptospores: the origin and early evolution of the terrestrial flora In[END_REF][START_REF] Steemans | Miospore evolution from the Ordovician to the Silurian[END_REF][START_REF] Wellman | Palaeophytogeography of Ordovician -Silurian land plants[END_REF]) with a continuous increase in diversity not interrupted by the Late Ordovician extinction interval evident among marine invertebrates. Identical dispersed spore assemblages have been reported worldwide before, during and after the Hirnantian glaciation and End Ordovician mass extinction. This has been taken to suggest that terrestrial floras were relatively unaffected by these interconnected events, with speculation that the earliest land plants were immune because they were cosmopolitan generalists that flourished in a wide range of environments [START_REF] Gray | The microfossil record of early land plants: advanced in understanding of early terrestrialisation, 1970-1984[END_REF][START_REF] Wellman | Cryptospores from the type area of the Caradoc Series in Southern Britain[END_REF]. Indeed dispersed spore assemblages are well known from cold, high latitude localities where they are often associated with glacial deposits (e.g. [START_REF] Gray | Age of the Cedarberg Formation, South Africa and early land plant evolution[END_REF]).

Conclusions

It is evident from the above discussion that the Ordovician represents a critical period in Earth's terrestrialisation. It seems likely that is witnessed the transition from a terrestrial, microbial soil-vegetation system to one that included the first land plants. At the same time non-marine aquatic biotas began to increase in diversity, although it seems that the only animals on the land were probably temporary visitors that were not obligate subaerial dwellers. Our understanding of Ordovician terrestrial life will continue to improve as refined techniques in phylogenetic analysis, molecular clock studies, Evo-Devo research and Earth systems modelling become available. But ultimately it seems likely that newly discovered fossils will shed most light on this subject area -particularly if convincing Ordovician plant remains can be recovered. Table 1: Dispersed spore assemblages reported from the Ordovician. C = conodonts; Ch = chitinozoans; G = graptolites, Inv = invertebrates .

FiguresFig. 1 .

 1 Figures

  (o) Tube with expanded tip. Scale bar: (a-j) = 30 μm; (k-l) = 40 μm; (m) = 30 μm; (n) = 100 μm; (o) = 30 μm.

Fig. 2 .

 2 Fig. 2. Land plant remains recovered by palynological processing of Ordovician (Katian) continental deposits from the Hasirah Formation of Oman. (a-e) Light microscope images of dispersed spores (cryptospores). (a) Naked permanent fused dyad (pseudodyad). (b) Permanent unfused dyad (true dyad) enclosed in a laevigate envelope. (c) Naked permanent unfused tetrad. (d) Permanent unfused tetrad enclosed in a laevigate envelope. (e) Permanent fused tetrad enclosed in an ornamented envelope. (f-l) Scanning Electron Microscope images of dispersed spores (cryptospores). (f) Naked permanent unfused dyad (true dyad). (g) Naked permanent unfused tetrad. (h) Permanent unfused tetrad enclosed in a laevigate envelope. (i) Naked permanent unfused tetrad. (j) Naked permanent fused dyad (pseudodyad) with microgranulate ornament. (k) Naked permanent unfused tetrad. (l) naked permanent unfused tetrad. (m-n) Transmission Electron Microscope images of a section cut from an individual dispersed spore (Naked permanent unfused dyad). (m) Entire dyad. (n) Close up of left hand part of dyad. (o-p) Scanning Electron Microscope images of a spore mass. (o) Entire spore mass. (p) Close up of part of the spore mass illustrating the nature of the spores. (q-r) Fragments of dispersed cuticle-like sheets. (q) (r) (s-t) Transmission Electron Microscope images of sectioned sporangia/spore masses. (s) Sporangium containing spores with wall ultrastructure lamellate. (t) Spore mass containing spores with wall ultrastructure homogeneous. Scale bar: (a-e) = 25 μm; (f-l) = 25 μm; (m) = 7.25 μm; (n) = 4.6 μm; (o) = 75 μm; (p) = 25 μm; (q) = 85 μm; (r) = 115 μm; (s) = 1.8 μm; (t) = 2.5 μm.

Fig. 3 :

 3 Fig. 3: Palaeogeographic map of the Dapingian and Darriwilian showing the location of dispersed spore assemblages. Locality codes refer to those used in Table 1. Modified from Map 80 (CR Scotese, Paleomap Project).

Fig. 4 :

 4 Fig. 4: Palaeogeographic map of the Sandbian showing the location of dispersed spore assemblages. Locality codes refer to those used in Table 1. Modified from Map 79 (CR Scotese, Paleomap Project).

Fig. 5 :

 5 Fig. 5: Palaeogeographic map of the Katian showing the location of dispersed spore assemblages. Locality codes refer to those used in Table 1. Modified from Map 78 (CR Scotese, Paleomap Project).

Fig. 6 :

 6 Fig. 6: Palaeogeographic map of the Hirnantian showing the location of dispersed spore assemblages. Locality codes refer to those used in Table 1. Modified from Map 77 (CR Scotese, Paleomap Project).Tables

Table 2 :

 2 Cryptospores examined ultrastructurally

	SPORE TAXON	MORPHOLOGY	LOCALITY	AGE	REFERENCE
	Tetrahedraletes	Naked permanent unfused tetrad	Ohio, USA	Katian	Taylor (1995)
	medinensis
	Pseudodyadospora sp.	Naked permanent fused dyad	Ohio, USA	Katian	Taylor (1996)
	Segestrespora	Envelope-enclosed permanent unfused	Ohio, USA	Katian	Taylor (1996)
	membranifera	dyad
	Dyadospora	Naked permanent unfused dyad	Ohio, USA	Katian	Taylor (1997)
	murusattenuata
	Tetrahedraletes spp.	Naked permanent unfused tetrad	Oman	Katian	Wellman et al. (2003)
	Dyadospora spp.	Naked permanent unfused dyad	Oman	Katian	Wellman et al. (2003)
	Cryptotetras erugata	Naked permanent unfused	Saudi Arabia	Darriwilian	Taylor et al. (2017)
	Pseudodyadospora sp. cf.	Naked permanent fused dyad	Saudi Arabia	Darriwilian	Taylor et al. (2017)
	P. laevigata
	Monad	Naked monad	Saudi Arabia	Darriwilian	Taylor et al. (2017)

Acknowledgements

This research was funded by various NERC grants to CHW including NE/R001324/1 and NE/V001639/1. B.C.M. and T.S. were supported by the EARTHGREEN project (ANR-20-CE01-0002-01). We would like to thank Claudia Rubinstein for alerting us to some cryptospore occurrences we were not aware of and Neil Davies for advice on Ordovician terrestrial palaeoenvironments and ichnology.