Azucena Molina-Solís

Christopher J Cleal

Eliott Capel

Borja Cascales- Miñana
email: borja.cascales-minana@cnrs.fr

Vegetation dynamics in the Early-Middle Pennsylvanian coal swamps of the Nord-Pas-de-Calais Coalfield, France

Keywords: Namurian, Westphalian, biodiversity, palaeobotany, Variscan Foreland

come

Introduction

The foreland that developed north of the Variscide orogen in tropical Euramerica during the late Carboniferous [START_REF] Opluštil | A comparative analysis of some Late Carboniferous basins of Variscan Europe[END_REF]Cleal, 2008aCleal, , 2008b;;[START_REF] Opluštil | A review of the Middle-Late Pennsylvanian west European regional substages and floral biozones, and 19. their correlation to the Geological Time Scale based on new U-Pb ages[END_REF][START_REF] Knight | The challenge of relating the Kasimovian to West European chronostratigraphy -a critical review of the Cantabrian and Barruelian substages of the Stephanian Stage[END_REF][START_REF] Opluštil | Middle-Late Pennsylvanian tectonosedimentary, climatic and biotic records in basins of Europe, NW Turkey and North Africa: an overview[END_REF] was covered by about 8 × 10 5 km 2 of wetlands, often referred to as coal swamps [START_REF] Cleal | Palaeozoic tropical rainforests and their effect on global climates: is the past the key to the present?[END_REF][START_REF] Cleal | The Carboniferous coal swamp floras of England: a window on an ancient tropical ecosystem[END_REF]. The result has been a belt of coal-bearing strata between the Canadian Maritimes and northern Turkey, containing a 10 Myr fossil record of this wetland biome (Cleal et al., 2009, fig. 1;[START_REF] Opluštil | Middle-Late Pennsylvanian tectonosedimentary, climatic and biotic records in basins of Europe, NW Turkey and North Africa: an overview[END_REF]. Numerous isochronous beds, notably eustatic marine bands (e.g., [START_REF] Bless | Palaeogeography of Upper Westphalian deposits in NW Europe with reference to the Westphalian C north of the mobile Variscan belt[END_REF] and volcanic ash beds ("tonsteins" -e.g., [START_REF] Bouroz | Corrélation des tonsteins d'origine volcanique entre les bassins houillers de Sarre-Lorraine et du Nord-Pas-de-Calais[END_REF], allow detailed correlations of the successions in and between the coalfields, making it possible to document the changes in the biotas across this substantial area. Evidence from modern-day vegetation has shown that species richness is a sensitive indicator of extrinsic environmental change (e.g., [START_REF] Magurran | Measuring Biological Diversity[END_REF]. The objective of this paper is to investigate whether there is a correlation between ecological factors and vegetation diversity during the Carboniferous period. Specifically, we aim to explore the impact of climatic cooling associated with the Late Palaeozoic Ice Age (e.g. [START_REF] Fielding | Stratigraphic imprint of the Late Palaeozoic Ice Age in eastern Australia: a record of alternating glacial and nonglacial climate regime[END_REF][START_REF] Montañez | The Late Paleozoic Ice Age: An evolving paradigm[END_REF][START_REF] Montañez | The Late Paleozoic Ice Age: An evolving paradigm[END_REF] and landscape disturbance caused by the Variscan Orogeny on vegetation diversity. (e.g., [START_REF] Besly | Sedimentation in a Synorogenic Basin Complex: The Upper Carboniferous of Northwest Europe[END_REF].

The Nord-Pas-de-Calais Coalfield (Fig. 1) lies near the centre of the belt of Variscan Foreland coalfields [START_REF] Cleal | Late Moscovian terrestrial biotas and palaeoenvironments of Variscan Euramerica[END_REF][START_REF] Cleal | Pennsylvanian vegetation and climate in tropical Variscan Euramerica[END_REF]Cleal and Cascales-Miñana, 2019, fig. 1). It has an almost complete succession through much of the Namurian and 3.

Westphalian [START_REF] Becq-Giraudon | Synthèse structurale et paléogéographique du basin houiller du Nord et du Pas-de-Calais[END_REF]Laveine, 1987;[START_REF] Guéguen | Monitoring residual mining subsidence of Nord/Pas-de-Calais coal basin from differential and Persistent Scatterer Interferometry (Northern France)[END_REF]Fig. 2).

These strata yield abundant adpression floras (macrofloras) that have been studied over the last century and a half (e.g., [START_REF] Brongniart | Prodrome d'une Histoire des végétaux fossiles[END_REF][START_REF] Boulay | Le terrain houiller du Nord de la France et ses végétaux fossiles[END_REF]Zeiller, 1888;[START_REF] Carpentier | Contribution à l'Étude du Carbonifère du Nord de la France[END_REF]Danzé-Corsin, 1953;Danzé, 1956;Buisine, 1961;Laveine, 1967Laveine, , 1987;;Laveine et al., 1978;Laveine and Goubet, 1995;[START_REF] Laveine | Frond architecture of the seed-fern Macroneuropteris scheuchzeri, based on Pennsylvanian specimens from the Northern France coal field[END_REF]. In this paper, we collated the distribution of meaningful fossil-species through the Nord-Pas-de-Calais succession and then used this to estimate patterns of vegetation diversity in this part of the coal swamps. We then compared the results of similar studies undertaken in other Variscan coalfields [START_REF] Cleal | The Westphalian macrofloral record from the cratonic central Pennines Basin, UK[END_REF][START_REF] Cleal | The Westphalian-Stephanian macrofloral record from the South Wales Coalfield, UK[END_REF](Cleal, , 2008c;;[START_REF] Cleal | Late Westphalian-Early Stephanian vegetational changes across the Variscan Foreland[END_REF][START_REF] Cleal | Late Moscovian terrestrial biotas and palaeoenvironments of Variscan Euramerica[END_REF][START_REF] Uhl | Late Carboniferous vegetation change in lowland and intramontane basins in Germany[END_REF][START_REF] Opluštil | A 25 million year macrofloral record (Carboniferous-Permian) in the Czech part of the Intra-Sudetic Basin; biostratigraphy, plant diversity and vegetation patterns[END_REF].

Geological background

Throughout this paper we used the West European Regional Chronostratigraphy as outlined by [START_REF] Wagner | The chronostratigraphic units of the Upper Carboniferous in Europe[END_REF][START_REF] Wagner | The 'global' scheme of Pennsylvanian chronostratigraphic units contrasted with the West European and North American regional classifications: discussion of paleogeographic zones/regions and problems of correlation[END_REF] and [START_REF] Wagner | History and current status of the Pennsylvanian chronostratigraphic units: problems of definition and interregional correlation[END_REF], as it expresses the stratigraphical relationships of these terrestrial, coal-bearing sequences better than the Global IUGS Chronostratigraphy [START_REF] Cohen | The ICS International Chronostratigraphic Chart[END_REF], which is based on marine sequences.

The Nord-Pas-de-Calais Coalfield has approximately 1150 km 2 of formerly mined concessions, over an area of about 105 km by 8 km (Fig. 1). The basin contains a paralic sequence (Bouroz, 1969) deposited in the rapidly subsiding foredeep near the Variscan Front [START_REF] Laurent | 3-D Structure of the Variscan Thrust front in northern France: New insights from seismic reflection profiles[END_REF]. The sequence is divided into five formations and twelve members (in French, termed Assise and Faisceau, respectively) ranging from the Arnsbergian (Namurian) to the lower Asturian (Westphalian) (Fig. 3). These lithostratigraphic units are delimited by coalfield-wide marine bands and tonsteins (Bouroz, 1960a(Bouroz, , 1969;;[START_REF] Becq-Giraudon | Synthèse structurale et paléogéographique du basin houiller du Nord et du Pas-de-Calais[END_REF]. The Namurian deposits are predominantly marine shales, sandstones, and limestones, with generally low diversity, allochthonous macrofloras [START_REF] Chalard | Contribution à l'étude du Namurien du bassin houiller du Nord de la France[END_REF]Fig. 4). However, continental influence increases from the upper St. Georges Member and Marie Member, with a declining number of marine bands and increasing macrofloras (Fig. 4). The Westphalian begins at the base of the Olympe Member of the Flines Formation (Fig. 5). This latter member is thin (15 to 90 m from west to east) and has numerous marine 4. bands but relatively few coals. The overlying Vicoigne Formation, whose base is marked by the Laure Marine Band, has major coals, up to 12 m thick, and has yielded abundant macrofloras that span much of the Langsettian Substage (Laveine, 1987).

Two members (Chandeleure and Modeste) are recognised, separated at the Graziella Tonstein but there is no significant change in the macrofloras at this level (Fig. 5).

The boundary with the overlying Anzin Formation is placed at the Poissonnière Marine Band, which is equivalent to the Amman Marine Band in the Westphalian stratotype in Britain [START_REF] Owens | Boundary stratotypes and new stage names for the lower and middle Westphalian sequences in Britain[END_REF] and defines the boundary between the Langsettian and Duckmantian Substages. The Anzin Formation continues to have thick coals and abundant macrofloras. It is divided into two members (Meunière and Pouilleuse) at the Wingles Marine Band (Bouroz, 1960b), with the lower member being characterised by numerous freshwater faunal horizons, and the upper member by having fewer freshwater horizons and thinner coals (Fig. 5).

The base of the overlying Bruay Formation is placed at the distinctive Rimbert Marine Band, whose thickness can exceed 30 m and is remarkably rich in fauna (Fig. 5). It correlates with the Aegiranum Marine Band in the Westphalian stratotype in Britain and marks the base of the Bolsovian Substage [START_REF] Owens | Boundary stratotypes and new stage names for the lower and middle Westphalian sequences in Britain[END_REF]. Four members are recognised in the Bruay Formation. The Six-Sillons Member is very similar to the underlying Pouilleuse Member in terms of sedimentology, flora, and fluvial fauna, but has a greater number of tonsteins (Fig. 5). The overlying Ernestine is characterized by numerous, thick, extensively-exploited coal seams; also by a distinctive feldspar-rich arkosic sandstone called the Sainte-Croix sandstone [START_REF] Dollé | Étude préliminaire sur les grès du bassin houiller du Nord et du Pasde-Calais[END_REF]. The Dusouich Member also contains numerous, economically-important coals, but is distinguished from the Ernestine Member by the presence of abundant Neuropteris ovata Hoffmann, regarded as an important index for the Asturian Substage [START_REF] Laveine | Report on the Westphalian D[END_REF]; the base of the Dusouich Member is placed at the Talence Tonstein (Fig. 5). The fourth and highest member (Edouard) is distinguished by a distinctive conglomerate at its base (the so-called poudingue d'Edouard), probably the result of uplift of the Variscan Orogen to the south; immediately overlying this conglomerate is Tonstein Valence (Fig. 5), which has been correlated with the lower Asturian Tonstein 1 of the Saar-Lorraine basin [START_REF] Bouroz | Corrélation des tonsteins d'origine volcanique entre les bassins houillers de Sarre-Lorraine et du Nord-Pas-de-Calais[END_REF]. The upper limit of the 5.

Edouard Member is marked by the overburden of Late Cretaceous age, separated by a major erosional discordance [START_REF] Becq-Giraudon | Synthèse structurale et paléogéographique du basin houiller du Nord et du Pas-de-Calais[END_REF].

Methods

Late Carboniferous coal swamp vegetation can be divided into eight broad, partly informal plant groups: (1) lycopsids, (2) calamite sphenopsids, (3) sphenophyll sphenopsids, (4) non-marattialean ferns, (5) marattialean ferns, (6) lyginopteridaleans, (7) medullosaleans, and ( 8) cordaitales (e.g., [START_REF] Cleal | The Carboniferous coal swamp floras of England: a window on an ancient tropical ecosystem[END_REF]. Since there was a strong ecological segregation of these plant groups between habitats within the coal swamps (e.g., [START_REF] Dimichele | Ecological sorting of vascular plant classes during the Paleozoic evolutionary radiation[END_REF][START_REF] Dimichele | Wetland-dryland vegetational dynamics in the Pennsylvanian Ice Age tropics[END_REF], it makes sense to look separately at the diversity dynamics of each of these plant groups.

Two approaches were used with the aim of determining the pattern of changing vegetation diversity from the macrofloral record. First, we looked at the changing species-richness for the selected plant parts for each of the major plant groups (as discussed in Section 4) through the stratigraphical succession. We used the rangethrough method, where a species is counted as being present at a stratigraphical level if it has been documented both above and below that level [START_REF] Boltovskoy | The range-through method and first-last appearance data in paleontological surveys[END_REF]. This approach avoids the problems of uneven sampling through the succession and, although it can obscure fine-scale variations in species diversity, it still allows the broad vegetation trends to be recorded. Separate diversity curves were plotted for the eight major plant groups and then plotted as stacked charts to reveal the pattern of total species richness. Importantly, this facilitates comparisons with previous studies on Variscan coalfield macrofloras (Central and South Pennines, South Wales and Ruhr basins), which used the same approach [START_REF] Cleal | The Westphalian macrofloral record from the cratonic central Pennines Basin, UK[END_REF][START_REF] Cleal | The Westphalian-Stephanian macrofloral record from the South Wales Coalfield, UK[END_REF](Cleal, , 2008c;;[START_REF] Uhl | Late Carboniferous vegetation change in lowland and intramontane basins in Germany[END_REF].

Second, a set of evolutionary metrics were evaluated. We used the boundarycrosser method to calculate per-capita origination and extinction rates (e.g., [START_REF] Warnock | Assessing the impact of incomplete species sampling on estimates of speciation and extinction rates[END_REF][START_REF] Capel | The Silurian-Devonian terrestrial revolution: Diversity patterns and sampling bias of the vascular plant macrofossil record[END_REF]. Per-capita rates are singleton-free metrics based on the proportions of range-through and boundary-crossing taxa observed in a given interval (see Foote, 2000, table 1, for implementation). These rates (1) summarize the changing pattern underlying the diversity distribution, (2) derive directly from branching theory instead of a normalization of the number of taxa per interval, and (3) assume that the number of lineages depends only on the observed taxonomic rates [START_REF] Foote | Origination and extinction components of taxonomic diversity: General problems[END_REF]. It provides a rate regardless of the origination-extinction magnitude frequently used as a descriptor of diversity changes. Other metrics, such as Sepkoski or Van Valen rates (Cascales-Miñana et al., 2013, table 1) were also considered.

However, since the proportion of single-appearance data is almost negligible, no significant differences were observed.

Macrofloral data

Primary data were extracted from the classic monographs on the Nord-Pas-de-Calais medullosaleans, lyginopteridaleans and ferns (Danzé-Corsin, 1953;Danzé, 1956;Dalinval, 1960;Buisine, 1961;Laveine, 1967). These were supplemented by taxonomic lists that have given data for the lycopsids, sphenopsids, and cordaites (Zeiller, 1888;[START_REF] Carpentier | Contribution à l'Étude du Carbonifère du Nord de la France[END_REF]Bertrand, 1914[START_REF] Bertrand | Les zones végétales du terrain houiller du Nord de la France[END_REF]Corsin andCorsin, 1970, 1971;[START_REF] Laveine | Report on the Westphalian D[END_REF]Laveine, , 1987)). Together, these sources provide a full overview of the species dynamics of the Nord-Pas-de-Calais Coalfield macrofloras (Fig. 6).

Since this analysis is aimed to determine underlying patterns of vegetation taxonomic diversity, not all fossil-taxa were collated. Following the approach summarised by [START_REF] Cleal | Plant biodiversity changes in Carboniferous tropical wetlands[END_REF], the fossil-species of just one plant part per plant group were collated: for lycopsids, the bark/periderm species; for the cordaitales, the seed and pre-pollen cone species, and for the other groups, the foliage species.

Although this may not be a perfect solution to the problem of fragmentation of the plant remains during preservation and the modular evolution where different plant parts have changed at different rates, [START_REF] Cleal | Plant biodiversity changes in Carboniferous tropical wetlands[END_REF] argued that it is the most robust way of determining the broad pattern of vegetation diversity dynamics from the upper Carboniferous macrofloral record.

The fossil-species were collated against the formations and members in the currently-used lithostratigraphical classification for Nord-Pas-de-Calais (e.g., Bouroz, 1969;[START_REF] Becq-Giraudon | Synthèse structurale et paléogéographique du basin houiller du Nord et du Pas-de-Calais[END_REF]Figs. 4, 5). Following the approach used in the aforementioned palaeobotanical monographs, each member has been divided into three informal subunits (lower, middle, upper). In total, we traced the stratigraphical 7.

distribution of 255 plant macrofossil species. The resulting dataset is available from supplementary files (Table S1).

Results

The changing pattern in species richness is shown in Fig. 6. The Namurian represents a generally low diversity interval (0-20 species), with approximately even representation of lycopsids, sphenopsids, lyginopteridaleans and medullosaleans.

However, there are small diversity pulses at the top of the Stérile Member and the base of the Marie Member, driven by medullosalean and lyginopteridalean pteridosperms, respectively. Also notable is a significant increase in lycopsid diversity towards the top-Namurian.

Species richness increases exponentially through the Olympe Member (lowermost Westphalian) to reach ca. 60 species by the base of the Vicoigne Formation, mainly driven by increases among non-marattialean ferns and pteridosperms (Fig. 6). Species diversity then continues to grow more gradually through the Vicoigne and Anzin formations (Langsettian-Duckmantian) eventually reaching about 100 species by the upper Duckmantian Pouilleuse Member. A further increase is then recorded through the lower Bruay Formation (Bolsovian), reaching a peak in the upper Six-Sillons Member of > 110 species, which includes a notably high diversity of non-marattialean ferns and medullosalean pteridosperms (Fig. 6).

A significant reduction in diversity then occurs through the two upper (Asturian) members of the Bruay Formation (Asturian) declining to ca. 40 species at the top of the succession. This depletion of species sees more than a half of the Bolsovian flora disappearing, affecting all groups of ferns and pteridosperms, resulting in a more lycopsid-dominated flora.

The per-capita origination-extinction rate values of overall plant diversity are plotted in Fig. 7A. As is to be expected, there are high origination values in the lower part of the succession (Namurian-lowermost Langsettian) and high extinction (extirpation) values in the upper (Asturian) part of the succession. In the rest of the succession, total origination levels tend to be higher than extinction, explaining the overall increase in diversity.

However, some differences are observed between plant groups. For instance, the highest origination levels for lycopsids are observed in the lowermost and uppermost Langsettian (Fig. 7B), while the most significant sphenopsid expansions are observed in the uppermost part of Namurian and the Duckmantian (Fig. 7C). Results further show high origination levels in the upper Namurian, lowermost Duckmantian and Bolsovian among ferns (Fig. 7D), while lyginopteridalean and medullosalean pteridosperms mainly diversify during the lower Westphalian. Moreover, the main lycopsid and sphenopsid disappearances occur at the top Duckmantian and lower Bolsovian, respectively (Fig. 7B,C). The main fern and pteridosperm extinctions are concentrated in the Asturian, although some pulses of diversity loss are also observed at the end Bolsovian and middle Duckmantian, respectively (Fig. 7D,E).

Discussion

The Nord-Pas-de-Calais Coalfield macrofloras show an essentially skewedunimodal pattern in species richness. This pattern is characterised by a sudden increase in the lowermost Westphalian, followed by a gradual growth during the Langsettian, peak values in the Duckmantian, but especially in Bolsovian, and a sharp decline in the Asturian (Fig. 6). Broadly similar diversity patterns have also been found in the macrofloras of the Westphalian coalfields in Britain and northern Germany [START_REF] Cleal | The Westphalian macrofloral record from the cratonic central Pennines Basin, UK[END_REF][START_REF] Cleal | The Westphalian-Stephanian macrofloral record from the South Wales Coalfield, UK[END_REF](Cleal, , 2008c;;[START_REF] Uhl | Late Carboniferous vegetation change in lowland and intramontane basins in Germany[END_REF]Fig. 8), but there are some differences, which throw light on the factors that were affecting the coal swamp vegetation at this time.

Namurian macrofloras in Nord-Pas-de-Calais are more diverse than those found in Britain (e.g., [START_REF] Lacey | Additions to the Millstone Grit flora of Lancashire[END_REF], but less than those of the Ruhr (e.g., [START_REF] Josten | Die fossilen floren im Namur des Ruhrkarbons[END_REF][START_REF] Uhl | Late Carboniferous vegetation change in lowland and intramontane basins in Germany[END_REF], probably reflecting the westwards retreat of marine influence across the Variscan Foreland. Most of the Variscan coalfields then show a similar, progressive increase in diversity through the Langsettian reaching about 60-80 species by the top of the substage. If the chronology of [START_REF] Davydov | High-precision U-Pb zircon age calibration of the global Carboniferous time scale and Milankovitch band cyclicity in the Donets Basin, eastern Ukraine[END_REF] and [START_REF] Waters | Nature and timing of Late Mississippian to Mid-Pennsylvanian glacio-eustatic sea-level changes of the Pennine Basin, UK[END_REF] of ca. 1.5 Myr for the Langsettian is accepted, we are seeing a new species appear every ca. 20-25 kyr. However, is this reflecting adaptive speciation, or migration of pre-existing and pre-adapted species into the new swamp 9.

habitats? There were certainly areas of very comparable swamp vegetation in existence in early Namurian times, most notably in Upper Silesia (Poland and Czech Republic) and the Lower Loire (north-western France), although many of the species were different to those found in the lower Westphalian coal swamps (e.g., [START_REF] Bureau | Bassin houiller de la Basse Loire. Fascicule II. Description des flores fossiles[END_REF][START_REF] Purkyňová | Die Unternamurflora des Beckens von Horní Slezsko (CSSR)[END_REF][START_REF] Strullu-Derrien | A rare late Mississippian flora from Northwestern Europe (Maineet-Loire Coalfield, Pays de la Loire, France)[END_REF]. There are also macrofloras from the upper Namurian of Upper Silesia and the Ruhr, which include species that later appear in the Westphalian Nord-Pas-de-Calais macrofloras [START_REF] Stopa | Rośliny paprociolistne (Pteridophylla) górnegu namuru i najniźszego westfalu na Górnym Sla̧sku[END_REF][START_REF] Josten | Die fossilen floren im Namur des Ruhrkarbons[END_REF], but there are other species (e.g., Calymmotheca hoeninghausii or Neuropteris obliqua) that do not seem to occur in these upper Namurian floras. This fossil record of the transition from late Namurian (proto coal swamp) to early Westphalian (full coal swamp) vegetation needs further investigation.

There are, however, differences in the dynamics of this Langsettian diversification of coal swamp vegetation between the different Variscan coalfields.

For instance, in the Nord-Pas-de-Calais and Ruhr, species diversification is rapid, reaching over 60 species by the lower Modeste Member and lower Bochum Formation, respectively, and then slows down through the rest of the Langsettian (Fig. 8A,B). In the British coalfields, in contrast, diversification was initially slower, reaching levels of only ca. 20 species until the middle Langsettian, and then speeding up in the upper Langsettian (Fig. 8C,D). This may again be the result of delayed withdrawal of marine influence in the more western parts of the Variscan Foreland in Britain, reflected in the more-frequent marine bands followed by lacustrine deposits in the British lower and middle Langsettian (e.g., [START_REF] Evans | The Westphalian succession of the Glyn-neath area, South Wales: an internationally important geological resource[END_REF] and this delayed the full development of the coal swamp biome here. Across all the Variscan Foreland coalfields, macrofloral diversity tends to plateau in the Duckmantian, reaching values of 80-100 species in Nord-Pas-de-Calais and the Ruhr, but only about 60 species in the British coalfields (Fig. 8C-E). This lower diversity in the British coalfields is unlikely to be purely a sampling artefact: rarefaction analysis in South Wales showed that the samples were sufficiently large for this not to be an issue [START_REF] Cleal | The Westphalian-Stephanian macrofloral record from the South Wales Coalfield, UK[END_REF][START_REF] Cleal | Plant biodiversity changes in Carboniferous tropical wetlands[END_REF]. It was also shown that a single horizon in the Central Pennine Basin (the Barnsley Main seam) showed a sharp peak in diversity that resembles the values seen in the Anzin Formation of Nord-Pas-de-Calais and the Essen Formation of the Ruhr (Fig. 8A-C). This suggests that the coal swamp vegetation was more fully developed in the Nord-Pas-de-Calais and Ruhr compared with the one in Britain (other than the Barnsley Main seams floras). This discrepancy clearly needs a more detailed comparative taxonomic analysis.

In most of the Variscan coalfields, macrofloral diversity remains high in the lower Bolsovian (Fig. 7), The main exception is in the British Pennine Coalfields, where there is a major reduction in species numbers [START_REF] Cleal | The Westphalian macrofloral record from the cratonic central Pennines Basin, UK[END_REF](Cleal, , 2008c;;[START_REF] Cleal | Plant biodiversity changes in Carboniferous tropical wetlands[END_REF] coinciding with a significant drying of substrates here and the development of red-beds known as the Etruria Formation [START_REF] Besly | Origin of red beds in a moist tropical climate (Etruria Formation, Upper Carboniferous, UK)[END_REF][START_REF] Turner | Remanence acquisition in red beds from the Coal Measures of Central England[END_REF][START_REF] Besly | Palaeosols in Westphalian coal-bearing and redbed sequences, central and northern England[END_REF][START_REF] Besly | Upper Carboniferous stratigraphy of the West Midlands (UK) revised in the light of borehole geophysical logs and detrital compositional suites[END_REF]. This drying of the substrates is unlikely to be due to global climatic change (full swamp conditions were prevailing further south in the Variscan foredeep such as in Nord-Pas-de-Calais) or to the northwards drift of Euramerica [START_REF] Besly | Sedimentological evidence for Carboniferous and Early Permian palaeoclimates of Europe[END_REF]. Hence, we are likely seeing the results of a combination of reduced subsidence but continued sediment influx in these more northern cratonic areas. Whatever the causes, however, the result was a collapse of the coal swamp biome in the more northern cratonic areas, whilst it continued to flourish in the Variscan foredeep.

In Nord-Pas-de-Calais, in contrast, macrofloral diversity increases in the Bolsovian, mainly through larger numbers of non-marattialean fern taxa (Fig. 6), but no similar diversification is seen in Ruhr or South Wales (Fig. 8B,E). This diversification of ferns in the Six-Sillons Member (Fig. 7D) is associated with the development of numerous thick coal seams suggesting the coal swamp biome was especially well-developed at this time, but why this affected the ferns more than the other plant groups is unclear.

The Nord-Pas-de-Calais macrofloras undergo a substantial decline in diversity in the Asturian (Fig. 6). This cannot be explained as an edge-effect that often characterises diversity patterns at the top of stratigraphical sequences, and is merely a sampling artefact [START_REF] Foote | Origination and extinction components of taxonomic diversity: General problems[END_REF]; the Dusouich Member in particular has numerous coal seams (Fig. 5) and the macrofloras were well-sampled as part of the work to delineate the Asturian Substage here (e.g., [START_REF] Laveine | Report on the Westphalian D[END_REF]. A similar reduction in diversity also occurs in the lower Pennant Formation of South Wales [START_REF] Cleal | The Westphalian-Stephanian macrofloral record from the South Wales Coalfield, UK[END_REF], and the Lembeck Formation in the Ruhr [START_REF] Uhl | Late Carboniferous vegetation change in lowland and intramontane basins in Germany[END_REF], associated with an increase in coarse clastic sediment resulting from hinterland uplift caused by the northwards migration of the Variscan Front to the south [START_REF] Jones | The influence of contemporaneous tectonic activity on Westphalian sedimentation in the South Wales Coalfield[END_REF][START_REF] Gayer | Comparative evolution of coal-bearing foreland basins along the Variscan northern margin in Europe[END_REF][START_REF] Wrede | Das Oberkarbon (Silesium) am Nordrand des rechtsrheinischen Schiefergebirges (Ruhrkarbon)[END_REF][START_REF] Wood | Rivers of the Variscan Foreland: fluvial morphodynamics in the Pennant Formation of South Wales, UK[END_REF]. However, there is not an exact correlation between this diversity decline and the influx of coarse clastic sediment in the different areas; in Nord-Pas-de-Calais and the Ruhr the diversity change occurs before the change in sedimentation, whereas in South Wales the coarse-grained clastics appear first. This reduction in macrofloral diversity evidently needs further investigation into the distribution and taxonomy of the macrofloras in these different coalfields.

Conclusions

Macrofloral evidence indicates that the coal swamp vegetation in the Nord-Pasde-Calais area followed a broadly similar diversity pattern during Namurian-Westphalian times to that seen in many other parts of the Variscan Foreland. The upland intramontane basins such as Saar-Lorraine and the Bohemia Massif, as this relationship seems to have played a key role in the overall evolution of the coal swamp vegetation during Westphalian times (e.g., see discussion in [START_REF] Thomas | Distinguishing Pennsylvanian-age lowland, extrabasinal and upland vegetation[END_REF][START_REF] Cleal | The floristic relationship between the upland and lowland Carboniferous wetlands of Variscan Euramerica -Evidence from some medullosalean pteridosperm fronds[END_REF][START_REF] Šimůnek | A synopsis of Westphalian-earliest Stephanian medullosalean and allied plant fossils from the Central and Western Bohemian basins, Czech Republic[END_REF].

Nevertheless, the new data presented here seem to confirm other studies in Europe (e.g., [START_REF] Cleal | Late Moscovian terrestrial biotas and palaeoenvironments of Variscan Euramerica[END_REF][START_REF] Cleal | Pennsylvanian vegetation and climate in tropical Variscan Euramerica[END_REF] that climate change was at most only a subsidiary factor in the collapse of the coal swamp biome. Bouroz, 1969, fig. 6 andGuéguen et al., 2009, fig. 2); lithological data according to [START_REF] Bouroz | Carte des zones stratigraphique à la côte stratigraphique -300 m du Bassin Houiller du Nord et du Pas de Calais[END_REF]. Mining concessions: (1) Fléchinelle, (2) Auchy-au-Bois, (3) Ferfay, (4) Marles, (5) Cauchy-à-la-Tour, (6) Camblain-Chatelain, (7) Bruay, (8) Beugin, (9) Vendin, (10) Noeux, (11) Fresnicourt, (12) Grenay, ( 13) Gouy-Servins, ( 14) Douvrin, ( 15) Ablain-Saint-Nazaire, [START_REF] Corsin | Un siècle et demi de Paléobotanique dans le Nord de la France[END_REF] Liévin, ( 17 (Bouroz, 1960a(Bouroz, , 1969)). S1.

Abbreviations: Arns., Arnsbergian; Chock., Chokierian. S1. Species diversity of plant macrofossils from the Nord-Pas-de-Calais Coalfield. For each taxon, the plant group to which it belongs, as well as its first and last appearance (i.e., FAD and LAD, respectively) in the basin are provided. Compiled fossil sites, when available, are justificative of exposed range only. NA: Not available. Laveine (1967, pp. 196-197) Neuropteris heterophylla (Brongniart) Liévin, pit N° 3, Alfred Danzé (1956, pp. 198-199) Aniche, pit Dechy, Eloi Danzé (1956, p. 480)

  vegetation of this biome diversified more rapidly in Nord-Pas-de-Calais and the Ruhr compared with the British parts of the foreland, probably because marine incursions and lacustrine conditions delayed the full development of this biome in the latter areas until middle-late Langsettian times. The biome then flourished during Duckmantian times over much of the Variscan Foreland but went into decline during Bolsovian and Asturian times. This decline of the coal swamp biome started in Nord-Pas-de-Calais during Asturian times but elsewhere occurred earlier, in the early Bolsovian in the Pennine Basin, in the middle Bolsovian in the Ruhr, and in the late Bolsovian in South Wales. The evidence suggests that the collapse in plant diversity in the coal swamp biome was probably caused by changing substrate conditions due to increased sediment influx and reduced subsidence, resulting from the northwards migration of the Variscan Mountains. Further work is clearly needed on the detailed dynamics of the different macrofloral fossil-species in order to determine exactly how the various plant species of the coal swamp vegetation were responding to these ecological pressures. It will also be important to investigate further the relationship between the macrofloras of the lowland paralic basins such as Nord-Pas-de-Calais and of the 12.

Fig. 1 .

 1 Fig. 1. Geographic position of the Nord-Pas-de-Calais Coalfield. (A) Map of North France showing distribution of the Upper Carboniferous (Namurian-Westphalian) outcrops (modified from Bouroz, 1969, fig. 1); dotted line highlights the study area. (B) Map of Nord-Pas-de-Calais Coalfield showing distribution of stratigraphical zones (modified fromBouroz, 1969, fig. 6 and Guéguen et al., 2009, fig. 2);

  ) Lens, (18) Meurchin, (19) Vimy-Fresnoy, (20) Drocourt, (21) Courrières, (22) Carvin, (23) Dourges, (24) Ostricourt, (25) Courcelles-lez-Lens, (26) Escarpelle, (27) Aniche, (28) Flines-lez-Raches, (29) Azincourt, (30) Anzin, (31) Douchy, (32) Denain, (33) Hasnon, (34) Vicoigne, (35) Raismes, (36) Marly, (37) Saint-Saulve, (38) Crespin, (39) Thivencelle, (40) Escaupont, (41) Fresnes, (42) Odomez, (43) Bruille, (44) Château l'Abbaye, (45) Vieux-Condé, (46) Saint-Aybert.

Fig. 2 .

 2 Fig. 2. Namurian-Westphalian stratigraphy of the Nord-Pas-de-Calais Coalfield, based on successions in Marles, Béthune, Douai and Valenciennes areas, including massif of Douvrin, Bruay-Len and Dorignies-Denain. Based on data in Becq-Giraudon (1983, fig. 7).

Fig. 3 .

 3 Fig. 3. Stratigraphic divisions of the Nord-Pas-de-Calais Coalfield(Bouroz, 1960a,

Fig. 4 .

 4 Fig. 4. Synthetic column of Namurian lithostratigraphy of the Nord-Pas-de-Calais Coalfield. Raw data based on successions in Douai and Valenciennes areas. Modified fromBouroz (1969, fig. 4).

Fig. 5 .

 5 Fig. 5. Synthetic column of Westphalian lithostratigraphy of the Nord-Pas-de-Calais Coalfield. Legend as same in Fig. 4.

Fig. 7 .

 7 Fig. 7. Per-capita origination-extinction rate values (Lmy) observed in the Nord-Pasde-Calais Coalfield macrofloras. Legend as same in Fig. 6.

Table S1 .

 S1 Continued.

	Taxon Taxon	Plant group Plant group	FAD FAD	Temporal distribution Fossil site LAD Temporal distribution Fossil site LAD	Fossil site Fossil site	References References
	Sphenophyllum majus (Bronn) Bronn Sphenophyllales Sphenopteris hansae Gothan Non-marattialean	Pouilleuse Member Modeste Member	Undetermined Aniche, pit Gayant,	Edouard Member (Upper) Undetermined NA NA	Laveine (1987, p. 89) Danzé (1956, p. 425); Bouroz (1969, p. 52)
		ferns	(Middle) (Middle)	Cécile
	Sphenophyllum myriophyllum Crépin Sphenophyllales Sphenopteris kayi Arber Non-marattialean	Olympe Member Ernestine Member	Undetermined Dourges, pit N° 2, St.	Six-Sillons Member NA	Undetermined NA	Laveine (1987, p. 89) Danzé (1956, p. 495)
		ferns	(Upper) (Upper)	Alfred	(Lower)
	Sphenophyllum tenerrinum Sphenopteris kilimli Kidston	Sphenophyllales Non-marattialean	Stérile Member (Upper) Undetermined Dusouich Member Béthune, pit N° 4,	Marie Member (Upper) NA	Undetermined NA	Laveine (1987, p. 89) Danzé (1956, p. 504)
	Ettinghausen	ferns	(Lower)	Marie-Antoinette
	Sphenopteris andraeana von Roehl Sphenopteris latinervis Danzé	Lyginopteridales Non-marattialean	Meunière Member Pouilleuse Member	Anzin, pit Roeulx, Aniche, pit Notre-	Six-Sillons Member Ernestine Member (Upper) Liévin, pit N° 3, Céline Danzé (1956, pp. 414-415) Anzin, pit Crespin, Danzé-Corsin (1953, p. 243)
		ferns	(Lower) (Lower)	coal seam Dame, coal seam N°	(Lower)	level 745
				undetermined 5
	Sphenopteris cf. arberi Kidston Sphenopteris microcyclos Gothan	Non-marattialean Non-marattialean	Six-Sillons Member Six-Sillons Member	Undetermined Béthune, pit N° 9,	Six-Sillons Member Dusouich Member	Undetermined Lens, pit	Danzé (1956, p. 491) Danzé (1956, p. 345, 458); Loboziak (1971,
		ferns ferns	(Lower) (Lower)	coal seam D	(Upper) (Middle)	undetermined,	p. 92-93)
	Sphenopteris cf. aschenborni Stur	Non-marattialean	Six-Sillons Member	Undetermined	Six-Sillons Member	Undetermined Dusouich	Danzé (1956, p. 421)
	Sphenopteris microscopica Crépin	ferns Non-marattialean	(Lower) Six-Sillons Member	Undetermined	(Upper) Six-Sillons Member	Undetermined	Danzé (1956, p. 465)
	Sphenopteris aureli Danzé	Non-marattialean ferns	Meunière Member (Lower)	Anzin, pit Thiers,	Dusouich Member (Upper)	; Loboziak Liévin, pit N° 3, Alfred Danzé (1956, pp. 393-397) ; Loboziak
	Sphenopteris mixta (Schimper)	ferns Non-marattialean	(Upper) Six-Sillons Member	Boulangère Undetermined	(Middle) Ernestine Member (Upper) Undetermined	(1971, p. 92-93) (1971, p. 92-93); Laveine et al. (1977, p. Danzé (1956, p. 435)
	Renaultia crepinii (Stur) Gothan Zeiller	Non-marattialean ferns	Meunière Member (Lower)	Douai, pit St. René,	NA	NA	Danzé (1956, p. 210); Bouroz (1969, p. 52) 785)
	Sphenopteris cf. cantiana Kidston Sphenopteris cf. mixta (Schimper)	ferns Non-marattialean Non-marattialean	(Middle) Six-Sillons Member Ernestine Member	Déjardin Bruay, pit N° 5, coal Courrières, pit N° 7,	NA NA	NA NA	Danzé (1956, p. 411); Loboziak (1971, p. Danzé (1956, p. 437); Bouroz (1969, p. 52)
	Renaultia footneri (Marrat) Zeiller	Non-marattialean ferns ferns	Modeste Member (Upper) (Upper)	Aniche, pit Gayant, seam N° 18 St. Nicolas	Chandeleur Member	Aniche, pit Vuillemin,	Danzé (1956, p. 448); Bouroz (1969, p. 52) 92-93)
	Brousmiche Sphenopteris corifolia Kidston Sphenopteris obtusedentata Gothan Non-marattialean ferns Non-marattialean	(Middle) Six-Sillons Member Six-Sillons Member	Cécile Noeaux, pit N° 2, Undetermined	(Lower) Ernestine Member (Upper) Bruay, pit N° 3, coal Vieille Marie Ernestine Member Liévin, pit N° 15,	Danzé (1956, pp. 512-513); Loboziak Danzé (1956, pp. 453-454)
	Renaultia cf. footneri (Marrat)	Non-marattialean ferns ferns	Ernestine Member (Lower) (Lower)	Ferfay, pit N° 4, Ste Espérance	Ernestine Member (Upper) Liévin, pit N° 6, seam N° 8 (Middle) Omérine	Danzé (1956, p. 450) (1971, p. 92-93)
	Brousmiche Sphenopteris delavalii Zeiller Sphenopteris parviloba Danzé	ferns Non-marattialean Non-marattialean	(Lower) Modeste Member Dusouich Member	Marie Aniche, pit Lens, pit	NA NA	Delphine NA NA	Danzé (1956, p. 508); Bouroz (1969, p. 52) Danzé (1956, p. 516)
	Renaultia gracilis Brongniart	Non-marattialean ferns ferns	Modeste Member (Middle) (Middle)	Undetermined undetermined, Cécile undetermined,	Dusouich Member (Upper) Aniche, pit Déjardin,	Danzé (1956, pp. 216, 221)
	Sphenopteris cf. delavalii Zeiller	ferns Non-marattialean	(Lower) Six-Sillons Member	Undetermined Dusouich	Six-Sillons Member	August Undetermined	Danzé (1956, p. 509)
	Renaultia rotundifolia Andrae Sphenopteris pecopteroides Kidston Non-marattialean Non-marattialean ferns	Pouilleuse Member (Lower) Pouilleuse Member	Aniche, pit Dechy, Douai, pit Notre-	Ernestine Member (Upper) Béthune, pit N° 4, coal (Upper) Ernestine Member (Upper) Dourges, pit N° 7,	Danzé (1956, p. 206) Danzé (1956, p. 521)
	Sphenopteris dentaefolia Danzé	ferns Non-marattialean ferns	(Lower) Meunière Member (Lower)	coal seam N° 2 Auchel, pit N° 3, Dame, coal seam	Dusouich Member	seam undetermined Lens, pit N° 2, coal Elisabeth	Danzé (1956, p. 473)
	Renaultia schatzlarensis Stur	Non-marattialean ferns	Chandeleur Member (Lower)	Lens, pit N° 18, coal seam undetermined	Chandeleur Member (Lower)	Douai, pit Sessevalle, seam undetermined	Danzé (1956, p. 180); Bouroz (1969, p. 52)
	Sphenopteris cf. pecopteroides	ferns Non-marattialean	(Lower) Ernestine Member	Désirée undetermined Undetermined	(Upper) Ernestine Member (Upper) Undetermined Ernest	Danzé (1956, p. 523)
	Renaultia schwerini Stur Sphenopteris cf. devians Gothan Kidston	Non-marattialean Non-marattialean ferns	Modeste Member Meunière Member (Lower)	Aniche, pit St. René, Undetermined	Ernestine Member (Upper) Ferfay, pit N° 4, Sainte NA NA	Danzé (1956, p. 193); Bouroz (1969, p. 52) Danzé (1956, p. 389)
	Sphenopteris pseudomarrati Danzé	ferns ferns Non-marattialean	(Upper) (Lower) Ernestine Member	Modeste Béthune, pit N° 2,	NA	Marie NA	Danzé (1956, p. 461); Loboziak (1971, p.
	Renaultia villosa Crépin Sphenopteris dissymetrica Danzé	Non-marattialean Non-marattialean ferns	Ernestine Member Ernestine Member (Middle)	Liévin, pit N° 1, Lens, pit N° 1, coal seam	Ernestine Member (Upper) Liévin, pit N° 3, Céline Dalinval (1960, pp. 185-186) NA NA Danzé (1956, p. 408); Dalinval (1960, p. 92-93)
		ferns ferns	(Middle) (Upper)	Omérine Céline undetermined			194)
	Reticulopteris sp. Laveine Sphenopteris dixonii Kidston Sphenopteris pulcherrima Crépin	Medullosales Non-marattialean Non-marattialean	Dusouich Member Dusouich Member Six-Sillons Member	Liévin, pit N° 3, coal Liévin, pit N° 3, Undetermined	NA NA Six-Sillons Member	NA NA Undetermined	Laveine (1967, p. 224) Danzé (1956, p. 476) Danzé (1956, p. 469)
		ferns ferns	(Lower) (Lower) (Lower)	seam undetermined Arago	(Upper)
	Rhodea subpetiolata (Potonié) Sphenopteris douvillei Zeiller Sphenopteris rotundiloba Danzé	Non-marattialean Non-marattialean Non-marattialean	Modeste Member Six-Sillons Member Six-Sillons Member	Aniche, Pit Béthune, pit N° 4, Drocourt, pit N° 4,	NA Dusouich Member Ernestine Member (Upper) Béthune, pit N° 4, coal NA Lens, pit N° 9,	Bertrand (1914, p. 222) Danzé (1956, pp. 488-489); Loboziak Danzé (1956, p. 430); Loboziak (1971, p.
	Zeiller	ferns ferns ferns	(Lower) (Lower) (Upper)	Archevéque, coal seam coal seam	(Middle)	Dusouich seam undetermined	(1971, p. 92-93) 92-93)
				Gabrielle undetermined undetermined
	Samaropsis fluitans (Dawson) Weiss Cordaitales Sphenopteris dufayi Danzé Non-marattialean Sphenopteris rutaeifolia Gutbier (= Non-marattialean	Pouilleuse Member Olympe Member Six-Sillons Member	Undetermined Undetermined Bruay, pit N° 5, coal	Six-Sillons Member Olympe Member (Upper) Undetermined Undetermined Dusouich Member Liévin, pit N° 1,	Zeiller (1888, p. 692); Corsin and Corsin Danzé (1956, p. 418) Danzé (1956, pp. 401, 405); Loboziak
	Sphenopteris stipulata Gutbier -vide	ferns ferns	(Upper) (Lower) (Upper)	seam N° 18	(Middle) (Middle)	Dusouich	(1970, pp. 231-232) (1971, p. 92-93)
	Sigillaria acuta Zeiller Sphenopteris formosa Gutbier Brousmiche, 1983)	Lycopsids Non-marattialean	Chandeleur Member Modeste Member	Undetermined Aniche, pit Gayant,	Meunière Member NA	Undetermined NA	Zeiller (1888, p. 691); Corsin and Corsin Danzé (1956, p. 529); Bouroz (1969, p. 52)
	Sphenopteris cf. selbyensis Kidston	ferns Non-marattialean	(Upper) (Middle) Chandeleur Member	Cécile Anzin, pit Ledoux, 9	(Middle) Pouilleuse Member		(1970, pp. 231-232)
	Sigillaria bretoni (Bertrand) Corsin Sphenopteris cf. fragilis Schlotheim Non-marattialean Lycopsids ferns	Marie Member (Lower) Undetermined Chandeleur Member Aniche, pit (Lower) Paumes	Marie Member (Upper) NA (Upper)	Undetermined NA	Corsin (1962, p. 2); Corsin and Corsin Bertrand (1914, p. 219); Danzé (1956, p.
		ferns	(Lower)	Vuillemin, Vieille			(1970, p. 243) 448)
				Marie

Acknowledgements

We thank Jessie Cuvelier for her technical assistance during both the documentation process and the study of palaeobotanical collections of the University of Lille; We also thank Zbynek Simunek and an anonymous reviewer for providing helpful comments and suggestions. AMS further thanks the support provided by the collaboration grant UVEG 1883981. Research funded by EARTHGREEN project (ANR-20-CE01-0002-01).

Undetermined

Edouard Member (Lower) Liévin, pit N° 1, Edouard Dalinval (1960, pp. 193-194); Laveine (1987, p. 89) Pecopteris bourozii Dalinval Marattialean ferns Meunière Member (Lower)

Lens, pit N° 18, coal seam N° 12

Ernestine Member (Lower) Douai, pit N° 8, Hélène Dalinval (1960, p. 133) Danzé (1956, p. 261)


Abhijeet Lale

Colman Buckley

Vincent Kermène

Agnès Desfarges-Berthelemot

Frédéric Dumas-Bouchiat

Emmanuel Mignard

Fabrice Rossignol

Holographic photopolymerization combined to microfluidics for the fabrication of lab-in-lab microdevices and complex 3D micro-objects

Keywords: Holographic photopolymerization, microfluidic devices, 3D-fabrication, acrylate resins, hydrogels

We show here brand-new possibilities of lab-in-lab fabrication while combining holographic photopolymerization and microfluidics. One shot real-time 3D-printing can produce 3D architectured microchannels, or free-standing complex micro-objects eventually in flow. The methodology is very versatile and can be applied to e.g., acrylate resins or hydrogels.

Introduction

A lack of easy scalable manufacturing techniques has been a roadblock in uptake of microfluidic technologies from academic research to commercially viable products. The capabilities of existing methods for fabricating devices, such as etching (for glass and silicon), embossing, and injection molding (for thermoplastics), are constrained by the diversity of features they can produce. ( [START_REF] Bhattacharjee | The upcoming 3d-printing revolution in microfluidics[END_REF][2] [START_REF] Yao | Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay[END_REF]). The paradigm of bottom-up building, also known as additive manufacturing (AM), is revolutionizing three-dimensional (3D) fabrication ( [START_REF] Nguyen | Dylla-Spears, 3d-printed transparent glass[END_REF], [START_REF] Ngo | Additive manufacturing (3d printing): A review of materials, methods, applications and challenges[END_REF]). Most commonly used AM techniques are either based on point-by-point or layer-by-layer deposition which allows broad geometric versatility. This offers an ability to produce complex structures that are not possible to manufacture using subtractive methods ( [START_REF] Bhargav | Applications of additive manufacturing in dentistry: A review[END_REF][7] [START_REF] Kirk | Phase-only complex-valued spatial filter[END_REF]).

Current AM processes create 3D geometries by way of iterative 1D or 2D unit operations ([9] [START_REF] Hull | Apparatus for production of three-dimensional objects by stereolithography[END_REF]). Transitioning from 2.5D structures (which have differing widths but identical depth) to 3D structures results in extended processing durations and a decrease in achievement rates. Other earlier techniques like soft lithography, although today well developed, require dust-free environment. Hence, they have a high cost and are time-consuming. Other manufacturing techniques like molding, fused filament fabrication (FFF), multijet modelling (MJM) and conventional laser stereolithography have a resolution starting typically from 20 µm (there are however micro-stereolithographic systems with better resolutions [START_REF] Choi | Fabrication of 3D biocompatible/biodegradable micro-scaffolds using dynamic mask projection microstereolithography[END_REF]). Other techniques like nanoimprint lithography, E-beam lithography have resolutions typically from 5 nm to 600 nm. In addition, in the case of stereolithography and FDM, the process is quite slow since it follows a layer-by-layer approach and a pixel-by-pixel approach, respectively. These methods restrict output, deteriorate surface smoothness, limit geometric potential, amplify demands for subsequent processing, which in turn escalates expenses and time commitments. Furthermore, they might induce variations in properties such as mechanical strength. Introducing a manufacturing method that can create all points within any given 3D shape concurrently would present an alternative approach to tackle these challenges and supplement established AM techniques. Several new studies based on Computed Axial Lithography (CAL) and Xolography ( [START_REF] Tumbleston | Additive manufacturing. continuous liquid interface production of 3d objects[END_REF][13][14] [START_REF] Shusteff | One-step volumetric additive manufacturing of complex polymer structures[END_REF]) offer ways to avoid problems created by traditional layer-by-layer approach by providing a volumetric printing solution. A 3D object is fabricated by irradiating a liquid photopolymer volume from the side with computed light patterns representing projections of the object to fabricate. However, none of these methods provide possibility of volumetric printing inside a microfluidic channel with a micron size resolution.

Materials and methods

Here we describe the use of a new photopolymer based additive manufacturing technique that allows formation of 3D volumetric structures as single units, i.e., a single exposure polymerization leading to complete build-up of 3D structures in one shot to develop microfluidic devices inside square capillaries with micron size resolutions. The method can also be adapted as a continuous printing process for micro-parts manufacturing. The printing or manufacturing process is executed by overlaying patterned optical fields generated from multiple light beams projected into a photosensitive resin. Alternatively, a single patterned optical beam can be utilized, as explained in the work by Kelly et al. [START_REF] Kelly | Volumetric additive manufacturing via tomographic reconstruction[END_REF].

Holographic printing set-up

The structuration of the light beams is performed by a holographic projection system [START_REF] Kirk | Phase-only complex-valued spatial filter[END_REF] schematically detailed in Figure 1, using a phase only Spatial Light Modulator (SLM, Meadowlark P1920-0532-HDMI), seeded by a continuous wave green laser (532 nm) or blue laser diode (405 nm) depending on the photoinitiator. This optical system generates two shaped beams overlapping inside the square section capillary from orthogonal direction, to polymerize a 3D form. The SLM displays a computed hologram, two patterns related to the shaped beams, modulated by a phase grating (grating period 10 pixels of 9.2 µm size). In the Fourier plane of the SLM, a filter blocks the unwanted diffraction orders. An afocal system images through this filter the two diffracted beams into the capillary. Both beams overlap in a 1 mm 3 volume, matching the inner part of the square capillary of 1 mm 2 cross section (10 cm long). The 2 mm diameter of the beam block pinhole limits the resolution of the shaped beam in the capillary to 30 µm (Rayleigh criterion). Therefore, the shape and size of the beams of plane wavefront do not change along the capillary cross section. However, the resolution of the 3D polymerized shape can be smaller depending on the characteristics of the photopolymer and light exposure.

This optical system can be used with a single beam to polymerize a 2.5D structure all along the cross section of the capillary, either in the horizontal or the vertical axis. Then, the structure is anchored to the opposite walls of the capillary. Involving both shaped beams, the holographic projection system is able to generate true 3D form, anchored or not to the capillary walls, where the beams overlap.

Materials

The 3D printing system was experimented with different crosslinkable materials such as hydrogels, Polymer Derived Ceramics (not reported here) and acrylate resins. This is with these latter resins that the polymerized shapes shown in Figures 2, 3 and4 were obtained. Exposures in this system were carried out in a resin volume of 0.1 ml contained in a 10 cm long silica glass square capillary with a 1 mm x 1 mm inner cross-section and 0.2 mm wall thickness bought from CM scientific, UK (VitroCom's Vitrotubes). The resin consisted of low molecular weight poly(ethylene glycol) diacrylate (PEGDA; Mn=250 g/mol, Sigma-Aldrich, Germany), with 0.05 to 0.2% (w/w) of Bis(2,6-difluoro-3-(1-hydropyrrol-1-YL)phenyl) titanocene (Apollo Scientific, UK) as photoinitiator sensitive at the 532 nm wavelength of the laser source and 10 to 15% (w/w) of hydroquinone (Sigma Aldrich, Germany) as radical inhibitor. The process involved the manipulation of the laser power at the source and the exposure time. The two parameters are bundled into a single variable, the power-time product for the sake of simplicity. The process involves three key elements. First, structuration of the laser light beams such that all required locations in the resin, as per the shape, receive adequate power dose needed to stimulate simultaneously the polymerization throughout the all volume. Second, limitations on the axial resolution of the beams must be compensated by optimizing the lateral intensity profile of each beam. This optimization is also beneficial for depth-dependence of energy absorption in the resin. Lastly, we use polymerization inhibiting species to allow for the non-linearity necessary for "threshold" behavior in the polymerization process. Indeed, the role of molecular oxygen (O2) dissolved in the resin is known to influence the polymerization inhibiting behavior. The successful feedstock formulations presented in this paper are the result of upstream optimizations which are themselves not reported. The criteria defining a good quality feedstock were the possibility to get polymerized features approaching 10 m in size and, at the same time, able to sustain the post-process of washing without any failure when attached to the wall of the microfluidic channel.

Follow-up of the photopolymerization of acrylates by Raman

Raman spectroscopy was carried out using a 532nm laser wavelength. A drop of feedstock material was placed on a microscope glass slide and the glass slide itself was placed in the holder position of an InVia Reflex spectrometer (Renishaw, UK). The laser in the Raman spectroscopy machine was used for dual purpose: (i) activating polymerization and (ii) measuring the changes in peak intensities during the measurement.

The band at about 1625-1680 cm -1 corresponded to the strong signal of the carbon-carbon double bond of acrylates, while the one, weaker, characteristic of the CH2 group was at about 1405-1455 cm -1 . The decreasing signal of the C=C is commonly used to monitor radical polymerizations, and we already did it with success as shown in one of our previous papers [START_REF] Lorber | Droplet-Based Millifluidics as a New Miniaturized Tool to Investigate Polymerization Reactions[END_REF]. Here the exact conversion rate was not measured. The change in the CH2 signal intensity relies to the fact that what changes with polymerization is that in the CH2 vicinity there is an alkane bond and not an alkene anymore.

Demonstration of feasibility

Shaping ability

While combining a high-resolution flow control system (e.g., Elveflow ® , Elvesys, France) which is not shown in Figure 1, the structures can be realized in a continuous or sequentially interrupted flow to allow for synchronization with external stimuli (e.g., laser illumination or other processing steps). Beyond the synthesis of 3D objects in the static mode, the dynamic mode configuration (continuous flow + spatial modification of the polymerizable zone) makes it possible to consider the development of complex curved objects. Figures 2a to 2b, Figure 3a to 3b and Figure 4 show various 2.5D, true 3D and a standalone 3D complex shape, respectively, that were prepared using this technique inside a 1 mm x 1 mm square silica capillary. In the specific case of 3D structures, the beam was split in two perpendicular beams. Importantly, there are no limitations on the dimensions of span, bridge, and cantilever components, and curves can be generated without encountering layering imperfections. These are notable hurdles in conventional additive manufacturing methods that build layer by layer. Additionally, there is no need for a supporting substrate during the construction process, although one can be employed if preferred. Most structures generated using this route are robust and within tolerance limits but detailed mechanical study on the structures is out of the scope of this paper. A preliminary study on a use of the architectured microfluidic channel of Figure 2b as robust mixers for fluids was done as a proof of concept (not reported here).

Our studies indicate that structures can be generated inside the capillaries using exposure times ranging from 0.3 to 10 secs and total incident power ranging from 10 mW to 40 mW. The smallest feature generated using this process is a cylindrical pillar with a diameter close to 15 µm. The smallest possible feature achievable using this technique is limited by the diffraction limit of the optical system, which is 30 µm for the current system. Although, we state the smallest possible feature printable to be close to 15 µm, there is likely a possibility to print features even smaller than 15 µm. This is achievable as the exposure time for certain shapes is very short (<0.5s) and the polymerization response of the feedstock is quite fast. If a high dose of power is provided for a very short duration, i.e., less than that required for complete polymerization, it results in formation of features which are smaller than the actual size of the projected hologram. This hack to print features which are smaller than the resolution limit of the process works only with symmetric simple shapes.

Figure 5 shows a percentage change in intensity of (C=C)acrylate and CH2 bonds in PEG-DA measured using Raman spectroscopy. As already said, the Raman laser at 532 nm was used both to cause photopolymerization and to collect the Raman data (100 mW). It is clear from this graph that polymerization stabilizes after some time and does not reach 100% conversion. It also shows that the photopolymerization is non-linear, due to oxygen inhibition both from dissolved oxygen and presence of hydroquinone. The 3D shapes made with the 2 illumination beams exploit the non-linearity of the process highlighted in Figure 5 to reach the polymerization threshold only in the target volumes.

Lab-in-lab device

There is a growing interest in microfluidic devices for the biological domain to model microphysiological interactions in so-called organ-on-chips (OOC) [START_REF] Leung | A guide to the organ-on-a-chip[END_REF]. To be closer to a physiological behavior, the conventional microfluidic chips have to be functionalized by adding structured biomaterials such as hydrogels [START_REF] Liu | Advances in hydrogels in organoids and organs-on-a-chip[END_REF]. These biomaterials are much more representative of the properties of the extracellular matrix than standard OOC materials like polydimethylsiloxane (PDMS), thermoplastics or 3D printing resins. For example, hydrogels can be used as a membrane for 2D cell culture or to build more complex systems for 3D cell culture [START_REF] Caliari | A practical guide to hydrogels for cell culture[END_REF] [START_REF] Vera | Engineering tissue barrier models on hydrogel microfluidic platforms[END_REF]. With our experimental setup (Figure 6b), we polymerized an hydrogel membrane into a double-flow microfluidic chip (Figure 6a) made with the Form 3B+ printer and the BioMed Clear resin (FormLabs, MA, USA). This membrane can mimic the epithelial-endothelial barrier between the two microfluidic channels of the chip. The hydrogel was a 10% concentration gelatin methacrylate (Advanced BioMatrix PhotoGel ® , CA, USA) with a 2% (w/w) LAP photoinitiator, sensitive to our blue laser (405 nm). The laser beam exposure time was 7s at 1.4 mW power level. The adjustable dimensions of the membrane are 1 cm long for a 350 µm thickness in the example shown in Figure 6c). Due to the swelling properties of the hydrogel, this thickness can evolve depending on its environment (air, cell culture media). The hydrogel was injected in the chip through its channels. The unpolymerized parts was cleaned with dry air flow through these channels.

Concluding remarks and future scope

Combining holographic photopolymerization and microfluidics offers brand new opportunities of micro-fabrication. Real time 3D-printing can produce in one shot 3D architectured microchannels or free-standing complex micro-objects eventually in flow. The methodology can be seen as a lab-in-lab fabrication platform. It can be applied to e.g., various types of photopolymerizable organics such as acrylates or biocompatible hydrogels.

As perspectives, future works will be focused on the exploration of the versatility of the technology. Interesting preliminary results were already obtained using other photopolymerizable feedstocks such as polymer-derived ceramics that can be further pyrolyzed to produce allceramic 3D micro-objects and lab-in-lab devices. We also started investigating the possibility to add functional and/or structural fillers to the feedstocks, such as nanoparticles. Figure 5: Change in % of intensity of bands at 1625-1680 cm -1 and 1405-1455 cm -1 corresponding to (C=C) acrylate and CH2 bonds, respectively, in PEG-DA subjected to photo-polymerization, as measured using Raman spectroscopy at 532 nm (100 mW laser, PEG-DA with 0.2% photo-initiator and 10% inhibitor). The exact conversion rate was not estimated.

Figure 1 :

 1 Figure 1: a) Diagram of the experimental setup. The afocal system (L1, L2) expands the beam of the laser diode. The hologram displayed on the SLM (spatial light modulator) splits the large laser beam in two shaped beams. A beam block in the SLM Fourier plane selects only the first order of both diffracted beams. The second afocal system (L3, L4) images the SLM into the capillary. VB, vertical shaped beam. HB, horizontal shaped beam. M, mirror. b) Photography of the experimental setup equipped here with the blue laser (405 nm). c) Zoom on the glass capillary under laser beam illumination.

Figure 2 :

 2 Figure 2: a) pillars of varying diameters and b) deflectors to be used as fluid mixers, both anchored inside a microfluidic channel of a 1 mm x 1 mm cross section (irradiation: green laser power of 1W and exposure time of 0.4 second).

Figure 3 :

 3 Figure 3: supported true single shot 3D shapes: a) mesh inclined at 45° to the channel axis and b) T shape, both anchored inside a microfluidic channel of a 1 mm x 1 mm cross section (irradiation: green laser power of 1W and exposure time of 0.4 second).

Figure 4 :

 4 Figure 4: example of an unsupported true 3D Tetris shape fabricated using a sequentially interrupted flow (irradiation: green laser power of 1 W and exposure time of 0.4 second).

Figure 6 :

 6 Figure 6: a) 3D printed microfluidic chip with 2 channels, b) microfluidic chip under laser illumination to photopolymerize an hydrogel membrane, c) side-view of the microfluidic chip with the hydrogel membrane separating the 2 channels.

Acknowledgements

This research was performed with the financial support of the French Nouvelle Aquitaine Region (Holofluid-3D and 3DEFI projects). We want to acknowledge Pierre Lidon, Gérald Glisson and Pierre Guillot of the LOF laboratory (CNRS UMR 5258, Solvay, 178 Avenue du Dr Albert Schweitzer, 33600 Pessac, France) for their support in microfluidic technologies. We also want to acknowledge Nicolas Védrenne and Florent Di Meo (INSERM U1248 Pharmacology & Transplantation, ΩHealth Institute -Univ. Limoges, 2 rue du Prof. Descottes, 87000 Limoges, France) for their support for the design of the double-flow microfluidic chip.

Declaration of Competing Interest

The authors declare no competing interests.