Dominique Marguerie
email: dominique.marguerie@univ-rennes1.fr

Teresa R Krause

Cathy Whitlock

What was burning in the past? Charcoal identifications supplement an early-Holocene firehistory reconstruction in Yellowstone National Park, USA

Keywords: charcoal, wood anatomy, fire, early-Holocene, vegetation history, Yellowstone National Park, USA -

Analysis of charcoal particles preserved in lake sediments has been frequently used to reconstruct fire, vegetation and climate history. Larger macroscopic particles (>125 µm) are attributed to local fires, whereas microscopic charcoal particles (<125 µm), observed on pollen slides, are a signal of regional fire. Taxonomic identification of the charcoal particles adds to the fire-history reconstruction by providing information about fuel composition and past fire conditions. Identification of charcoal can unravel one of the longstanding questions regarding past fire regimes, namely what was burning. This paper describes an analysis of charcoal particles preserved in the sediments of a small closed-basin lake in Yellowstone National Park. Blacktail Pond (44.954°N, 110.604°W; 2012 m elev) is located in steppe vegetation surrounded by conifer forest. Previous studies of the site have presented a multiproxy environmental history of the last 14,650 years. The focus of this investigation was on the fire history of the early-Holocene (11,500-9900 cal yr BP). Thirty-nine sediment samples were taken at 0.5 cm intervals to examine macroscopic charcoal particles, with attention on two large charcoal peaks at 10,970 and 10,200 cal yr BP that registered large or near-site fires. Twenty-eight samples contained 243 large-enough particles (180-250 µm) to be examined with scanning electron microscopy. We were able to identify charcoal of conifers (Pinus, Picea, and Abies or Juniperus), Artemisia and unidentified deciduous shrubs, and unidentified monocotyledons. The first fire burned a mixture of fuels, whereas the second episode burned Artemisia, shrubs and herbs but no conifers. The results are consistent with previous pollen interpretations at Blacktail Pond that indicate an open mixed-conifer forest in the early Holocene prior to 10,750 cal yr BP, replaced by Pinus contorta forest on the slopes and steppe in the valley. The relative dominance of Pinus contorta-type charcoal points to the importance of fire as a catalyst for the spread of this species.

includes the charcoal preserved in lakes or wetlands, because these sedimentary particles are usually small (<0.25 mm). Charcoal particles in lake and wetland deposits are often identified as conifer, hardwood, shrub, or herb and, in some cases, assigned to genera (Quercus sp., Pinus sylvestris-type, Salix sp., Betula sp., Ericaceae) [START_REF] Whitlock | Postglacial vegetation, climate and fire history along the east side of the Andes (lat 41-42.5oS), Argentina[END_REF]. In a study of Holocene fire history, [START_REF] Olsson | A continuous record of fire covering the last 10,500 calendar years from southern Sweden -The role of climate and human activities[END_REF] identified charcoal particles from a 10,500 yr-long record from a peat and lake sediment deposit at Storasjö and Stavsåkra in southern Sweden. They suggested that both microscopic and macroscopic charcoal records represented local fire history and showed three major phases of high fire activity controlled by climate during the early and middle Holocene and by humans during the late Holocene. These charcoal identifications offered a partial list of fuels because a high quantity of the particles were unidentifiable. The objective of this paper is to better understand past vegetation and fire conditions through the use of charcoal analysis on sediment cores taken from a small lake in northern Yellowstone National Park, USA. Blacktail Pond (44.954°N, 110.604°W; 2012 m elevation) is surrounded by steppe with conifer forest growing above the site on adjacent rocky buttes and at higher elevations. A ca. 14,650-yr-long record of vegetation and fire history was reconstructed at this site and described in [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF] and [START_REF] Huerta | Holocene vegetation-fire-climate linkages in northern Yellowstone National Park, USA[END_REF]. Moreover, [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF] present geochemical and isotopic data for the late-glacial to early Holocene period, as part of a multi-proxy investigation of environmental history. The period of interest for the present study is the early Holocene from 11,500 to 9900 cal yr BP at Blacktail Pond when summers were becoming progressively warmer and drier as a result of increasing summer insolation [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF]. Early postglacial warming shifted the vegetation from late-glacial parkland to early-Holocene mixed conifer forest and then steppe and closed pine forest [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF]) (Fig. 2). Fire activity, as evidenced by the charcoal record, was also increasing during this transition in response to warmer conditions and greater fuel biomass (Fig. 3). Two significant fire episodes occurred at ca. 10,970 and 10,200 cal yr BP, based on large charcoal peaks [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF]. In this paper, we describe charcoal particle identifications that reveal fuel and fire conditions of the early Holocene and supplement previous understanding of the vegetation and fire history at Blacktail Pond by answering the question: what was burning?

Study area 2.1. Current vegetation and fire regimes

The vegetation of northern Yellowstone is strongly influenced by elevation, aspect and geology [START_REF] Despain | Yellowstone Vegetation: Consequences of Environment and History in a Natural Setting[END_REF]. Today, grassland and steppe communities dominated by Artemisia tridentata (big sagebrush), Festuca idahoensis, Ericameria nauseosa (rabbitbrush), Sarcobatus vermiculatus (greasewood, on saline soils), and various herbs are present generally below 1700 m elevation. Montane and subalpine forest occurs between 1700 and 2900 m elevation and is replaced by alpine tundra at elevations above 2900 m. Within the forest zone, Pinus flexilis (limber pine) and Juniperus scopulorum (Rocky Mountain juniper) occur at low elevations (1700-1900 m elevation), Pseudotsuga menziesii (1900-2000 m elevation) and Pinus contorta (2000-2400 m elevation) are present at middle to high elevations. Picea engelmannii (Engelmann spruce), Abies lasiocarpa (subalpine fir), and Pinus albicaulis (whitebark pine) dominate high elevation forests and parkland (2400-2900 m elevation). Juniperus communis is present in the understory at all elevations. Blacktail Pond lies within calcareous glacial outwash and is surrounded by Artemisia tridentata steppe, which grows at this elevation on fertile soils. Pseudotsuga forest is present on adjacent rocky slopes of basalt and andesite, and Pinus contorta forest grows on local rhyolite exposures and throughout the central part of Yellowstone National Park. Small populations of Abies and Picea are found in valleys with cold-air drainage, and stands of Populus tremuloides (quaking aspen) grow on lower slopes in moist areas. Salix spp, Betula occidentalis (water birch), Scirpus americanus, Carex and Typha latifolia are present along the lake margin. Fires, both lightning and human caused, are common in western U.S. forests, and Yellowstone National Park became internationally famous for large fires in 1988 that burned 3213 km 2 , or about 40% of the park [START_REF] Schullery | The Fires and Fire Policy[END_REF]. Regional studies, based on lakesediment studies, indicate that fire activity increased during the late-glacial and early Holocene periods and then decreased after ca. 9000 and again at 5000 cal yr BP [START_REF] Iglesias | Past vegetation dynamics in the Yellowstone region highlight the vulnerability of mountain systems to climate change[END_REF]. The conifers of the region have different adaptations to fire that allow for their survival. For example, the thick bark of Pseudotsuga enables these trees to survive lowintensity surface fires. Similarly, Pinus flexilis and P. albicaulis tolerate surface fires as the trees mature and their bark thickens. P. contorta is adapted to high-severity fires, because, even though the trees are killed, its serotinous cones open immediately and release thousands of seeds. Abies and Picea have thin bark with branches to the ground and thus are highly vulnerable to fires. These conifers grow in wet regions where fires tend to be infrequent crown fires, and regeneration occurs by seed [START_REF] Knight | Mountains and Plains; the Ecology of Wyoming Landscapes[END_REF].

Lithology and chronology

This study used core BTP08B of [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF], which was collected at the western margin of the lake from the fen. The sediment from 5.25 m to 8.10 m depth was the focus of this study. The chronology for Blacktail Pond record was developed from seven AMS radiocarbon dates on pollen residues, charcoal, and plant macrofossils, and two known tephra ages [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF]. The environmental reconstruction was based on pollen, geochemical properties (elemental geochemistry, oxygen isotopes from authigenic carbonates), magnetic susceptibility, and macroscopic charcoal. The pollen and charcoal data and radiocarbon ages are available at Neotomadb.org. The original charcoal analysis focused on particles >125 µm in diameter to capture highseverity fires occurring within a few kilometers of the lake [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF][START_REF] Huerta | Holocene vegetation-fire-climate linkages in northern Yellowstone National Park, USA[END_REF]. Charcoal accumulation rates (CHAR; particles cm -2 yr -2 ) were calculated using CharAnalysis [START_REF] Higuera | Frequent fires in ancient shrub tundra: implications of paleorecords for arctic environmental change[END_REF]. Charcoal concentrations and deposition times were interpolated into contiguous 25-yr bins (the median resolution of the record), and CHAR was determined by dividing the time-interpolated concentrations. Long-term trends in CHAR (background CHAR or BCHAR) were separated from positive deviations (charcoal peaks), with a 500-yr loess smoother. BCHAR is considered an indicator of woody fuel biomass [START_REF] Marlon | Fire-fuel-climate linkages in the northwestern USA during the Holocene[END_REF]. Charcoal peaks were assigned to the positive residuals remaining after BCHAR is removed from the time series and identified above a 95 th percentile of the noise distribution [START_REF] Higuera | Peak detection in sedimentcharcoal records: impacts of alternative data analysis methods on fire-history interpretations[END_REF]. These peaks are interpreted as fire episodes (one or more fires during the time span of the peak).

Previous information on fire and vegetation dynamics

Pollen data from [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF] describe the late-glacial to early-Holocene vegetation history at Blacktail Pond (Fig. 2). The period from ca. 14,650 to 11,500 cal yr BP was a period of cooler-than-present summer conditions, but temperatures were steadily increasing with rising summer insolation. The postglacial landscape was initially sparsely vegetated with little soil development. After ca. 12,900 cal yr BP, conditions were warm enough for Picea engelmannii to form a parkland at the site, and after ca. 12,500 cal yr BP, Picea, Abies, and Pinus (both P. contorta and P. albicaulis/flexilis pollen types) were present in open mixed-conifer forest communities. Beginning at ca. 11,500 cal yr BP, the carbonate d 18 O record from Blacktail Pond indicates a sharp increase in summer temperatures, and the pollen record suggests a more closed forest at ca. 11,350 cal yr BP. By 9900 cal yr BP, Pinus contorta replaced Picea engelmannii as the dominant conifer on hill slopes near Blacktail Pond. Given soil constraints, Artemisia steppe probably still covered the valley floor. The charcoal-based fire history followed the vegetation history closely, showing few fires before 12,000 cal yr BP when vegetation cover was sparse. This was followed by a steady CHAR rise, suggesting a maximum in fire activity between ca. 12,000 and 10,500 cal yr BP, and a slight decline thereafter. Noteworthy are the two large fire episodes at ca. 10,970 cal yr BP and 10,200 cal yr BP (Fig. 3). It is likely rising fire activity due to increasing summer insolation facilitated the transition from mixed-conifer forest to Pinus contorta, given the fact that Picea and Abies have little adaptation to fire. The two prominent fire events may have been important triggers in this transition. The first peak occurred early in the period of mixed-conifer forest, while the second peak occurred just before P. contorta became the dominant species near Blacktail Pond at 9900 cal yr BP, where it likely occupied upslope rocky areas and steppe was locally present [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF].

Methods

For this study, a new macroscopic charcoal analysis was undertaken on the sediments above and below the two charcoal peaks. Twenty-six samples of 5 cm 3 were taken every 0.5 to 1 cm in 0.5-cm-thick sections from 671.5 (ca. 11,510 cal yr BP) to 653.5 cm (ca. 10,620 cal yr BP) depth around the first peak and 13 samples were also collected every 0.5 to 1 cm in 0.5-cm-thick sections from 643 (ca. 10,230 cal yr BP) to 631 cm (ca. 9910 cal yr BP) depth around the second peak (Table 3). The sediment samples were disaggregated in 30 ml of 5% sodium metaphosphate for 20 minutes before 30 ml of 6% bleach (sodium hypochlorite, NaClO) was added. The samples were soaked in this solution for another 24 hours to further deflocculate clays and oxidize organic matter. This procedure made the samples easier to analyze. The samples were then gently washed through two sieves with mesh sizes of 180 and 250 µm [START_REF] Whitlock | Charcoal as a fire proxy[END_REF]. The residues after sieving were subsequently placed in a gridded petri dish and examined under a stereomicroscope. Of the 39 samples examined under a stereomicroscope, 28 contained relatively large charcoal particles (>180 µm in diameter). A total of 243 particles were mounted on stubs for SEM observation.

Identification

The classical anatomical criteria used to identify modern wood may also be applied to charcoal [START_REF] Marguerie | Charcoal analysis and dendrology: data from archaeological sites in western France[END_REF]. The identification is based on diagnostic characters of the wood microstructure and comparison of fossil material with modern reference collections [START_REF] Trouy | Anatomie du bois, formation, fonctions et identification[END_REF]. Identification of charcoal requires examination of the threedimensional arrangement of the cell elements that comprise hardwood or softwood. The three anatomical planes of the ligneous structure are transverse (Tr), radial (R) and tangential (Tg). The transverse plane is most easily discovered because of the visibility of the growth rings. It also is the most important section for observing microscopic features and ring distinctness. Within a longitudinal section (i.e., the vertical orientation of the cells in tree trunk) are the radial and tangential planes. The radial plane is a vertical surface running from the pith outwards towards the bark; the tangential plane is perpendicular to the rays, and tangential to the growth rings. Given the small size of the charcoal particles (about 180-250 µm in diameter) preserved in the Blacktail Pond core, usual identification criteria were not systematically possible. The first step was to determine if a specimen was ligneous (woody) or not. This identification was done from exposed planar sections produced by natural breakage. Generally, the charcoal particles offered only one or two anatomical sections visible along the radial or tangential planes, and it was impossible to split the particle along a new surface. We had to work with the existing fractures to observe all the aspects of the wood microstructure. Identification keys were consulted for the details of charcoal particle characteristics (e.g., pits, perforation plates). The more fragmented the particle was, the more difficult it was to identify or even find diagnostic features for identification. In contrast, large "mesoscopic" charcoal particles (180-500 µm) often contain two anatomical sections allowing distinction of monocotyledons, dicotyledons and gymnosperms [START_REF] Marquer | Charcoal scarcity in Epigravettian settlements with mammoth bone dwellings: the taphonomic evidence from Mezhyrich (Ukraine)[END_REF]. These longitudinal sections (either radial or tangential) are particularly informative for identifying of some conifer genera.

In addition, processes related to transport and deposition of charcoal in the lake lead to fractures, deformities, and impurities that often obscure anatomical features. At Blacktail Pond, a few mesoscopic charcoal particles were recovered, including isolated wood fibers and vascular elements that enabled conifer identification. Attention to the anatomical criteria preserved on the longitudinal planes was critical, even when only a few bundles of fibers were preserved. In conifers and broad-leaved trees and shrubs, axial tracheids and vessels form the main part of the wood tissue and these groups cannot be separated. However, longitudinal sections reveal different combinations of pits that are diagnostic. All wood anatomical features preserved in the charcoal particles at Blacktail Pond were recorded according to the IAWA list of microscopic features for hardwood and softwood identification [START_REF] Wheeler | IAWA list of microscopic features for hardwood identification[END_REF][START_REF] Richter | IAWAlist of microscopic features for softwood identification[END_REF]. Taxonomic identification relied on identification keys and illustrated atlases of microscopic thin sections (e.g. [START_REF] Schweingruber | Microscopic Wood Anatomy[END_REF]. To observe cross-field pitting in the radial section required high magnification, and the charcoal particles were examined through a SEM using magnifications of up to 5000x to view these very fine anatomical characters. The SEM sessions took place at the Montana State University (USA) and the University of Rennes 1 (France). Wood anatomical features were compared with genera in the charcoal reference collection (Archeosciences Laboratory, CReAAH, CNRS, University of Rennes 1) and several wood anatomical atlases. The low number of identified charcoal particles at Blacktail Pond (243 particles) did not allow a quantitative reconstruction, but rather documented the presence of some taxa and the type of vegetation burned. Charcoal accumulation rates (CHAR) were calculated for particles >125 in diameter and ranged from 0.2 to 2.6 particles cm -2 yr -1 (Krause and Whitlock, 2013). There were few particles in the >180 µm size fraction.

Collection of fresh wood samples around the site

The characteristics of the fossil wood anatomy were checked against modern, charred, and wood specimens. In addition, a reference collection was also made of wood from living trees and shrubs in the vicinity of Blacktail Pond. Wood samples from several plants of each species were charred in an open furnace after drying at 50°C for 30 hours. After carbonization (at 350°C with the time depending on the size of the wood), three anatomical sections were obtained by scalpel or simply by breaking the charcoal by hand. The fragments of charred tissue were mounted on metal stubs for SEM examination. 4. Results and Discussion

Wood anatomy of living trees and shrubs around Blacktail Pond

We collected modern wood specimens from three pines and four broad-leaved shrubs from the northern Yellowstone region: Pinus albicaulis, P. contorta, P. flexilis, Artemisia tridentata, and Sarcobatus vermiculatus. For these taxa, wood identifications were based on burned living woody plants. Other conifers were not sampled: Juniperus scopulorum, Picea engelmannii, and Abies lasiocarpa. Information about the wood anatomy of the living conifers at the species or genus level, especially the unsampled taxa, was obtained from several atlases [START_REF] Panshin | Textbook of Wood Technology[END_REF][START_REF] Barefoot | Identification of modern and tertiary woods[END_REF][START_REF] Hoadley | Identifying Wood. Accurate results with simple tools[END_REF]. In addition, we made use of the atlases of [START_REF] Schweingruber | Microscopic Wood Anatomy[END_REF] and [START_REF] Vernet | Guide d'identification des charbons de bois préhistoriques et récents : Sud-Ouest de l[END_REF], although neither deals with western U.S. trees. The European collection of wood at the laboratory of Rennes, France was also examined for this project. Two papers on identification of North American wood species [START_REF] Kukachka | Identification of Coniferous Woods[END_REF][START_REF] Minnis | Identification of Wood from Archaeological Sites in the American Southwest. I. Keys for Gymnosperms[END_REF] and a special issue of IAWA journal [START_REF] Richter | IAWAlist of microscopic features for softwood identification[END_REF] were also consulted. For each charcoal type, descriptions were made in accordance with the on-line InsideWood database (IAWA Committee, 1989;[START_REF] Insidewood | Inside Wood -Database[END_REF]. 4.1.1. Wood anatomy of the conifers The anatomical distinction between conifer (softwood) and angiosperm (hardwood) is straightforward. Conifer wood is less complex because tracheids function for both conduction and support, and their shape is rather different from that of hardwood vessels. Identification of different conifer taxa, however, is generally more difficult than for hardwoods [START_REF] Kukachka | Identification of Coniferous Woods[END_REF]. Wood anatomy criteria allow conifer identification to genus, but species-level identification is difficult or even impossible. Among the conifer genera likely to be identified in the Blacktail Pond core, the primary diagnostic features was the presence or not of resin ducts, the height of rays, and the nature of cross-field pitting (i.e., pits intersecting walls of an axial tracheid and a ray parenchyma cell) (Table 1). Five types of pits occur in the earlywood or springwood and vary in terms of frequency, arrangement, form and size (Ritcher et al., 2004;[START_REF] Schweingruber | Microscopic Wood Anatomy[END_REF][START_REF] Schweingruber | Atlas of Stem Anatomy in Herbs, Shrubs and Trees[END_REF]. The types range from a single large pit that occupies most of the area of the cross-field, as in the case of some pines, to very small pits that occupy only a small portion of the cross-field areas, as in Picea.

In the case of small charcoal particles where it is impossible to observe the three anatomical sections, the radial plane is often the most obvious one. It is also the crucial section for discerning decisive features for the identification of conifer wood. For example, the characteristics used to identify small fragments of Abies are the absence of resin canals, transversal tracheids, nodular ray parenchyma, and the presence of two to four taxodioid pits per cross-field. Crystals appearing in the form of regular prisms, cubes, or octahedra are commonly found in Abies species [START_REF] Schweingruber | Microscopic Wood Anatomy[END_REF]. In Juniperus wood, resin canals and transversal tracheids are also absent, and key features are ray parenchyma that have abundantly nodular ends walls and cupressoid cross-field pitting. In Picea, resin canals are surrounded by rather thick-walled epithelial cells, the ray tracheids are present with a smooth and wavy wall, ray parenchyma cell walls are thick, and cross-field pitting is piceoid-type [START_REF] Marguerie | Distinction anatomique du bois du Mélèze (Larix laricina), de l'épinette blanche (Picea glauca) et de l'épinette noire (Picea mariana) en vue de l'analyse des macrorestes[END_REF][START_REF] Schweingruber | Microscopic Wood Anatomy[END_REF]. Crystals in ray cells occur occasionally in Picea wood. Finally, small fragments of Pinus wood in radial section show abundant and large resin canals with thin-walled epithelium cells, ray tracheids that are dentate to reticulate, and fenestriform or pinoid pits in the cross-fields (Table 1). Species identification of Pinus is difficult or impossible. However, the genus can be subdivided into two groups on the basis of anatomical traits like ray cells, ray tracheids and the type of cross-field pitting, all of which can be discerned under high magnification (Table 2). Pinus contorta wood has rays of medium height in tangential section (1 to 12 cells, mainly 5) (Fig. 4b), ray tracheids that are distinctly dentate to reticulate walls, and one to four, but generally two, pinoid ray pits per cross-field. Each pit has an irregular shape and size (Fig. 4c and4d). Pinus albicaulis wood has short rays with only one to ten cells (although sometimes more), with an average height of three to four cells (Fig. 4f). It also generally has one large, simple, fenestriform (window-like) ray pit per cross-field, rarely two (Fig. 4g and4h). Pinus flexilis wood has short rays in tangential section with one to eight cells (three to four on average) (Fig. 4j). Cross-field pitting is also fenestriform, with on average one or two pits per earlywood cross-field (Fig. 4k and4l). Using these criteria, we were able to identify Pinus, Picea, and Juniperus or Abies genera in the charcoal particles at Blacktail Pond. It was also possible to separate Pinus contorta from P. albicaulis and/or P. flexilis on the basis of the cross-field pitting and the walls of ray tracheids. 4.1.2. Wood anatomy of the broad-leaved shrubs In contrast to gymnosperms, the xylem of angiosperm woods contains vessels and is thus easy to distinguish from conifers by its more complex wood anatomy. Four common shrubs found in the steppe vegetation of northern Yellowstone National Park were examined. The wood of Artemisia tridentata, an Asteraceae shrub, is semi-ring porous (Fig. 5a and5b) and contains vessels with spiral thickenings and simple perforations. Rays are large (6-8 cells) and heterogenous (Fig. 5c and5d). In the case of small charcoal particles in the sedimentary record, the key feature was vessel walls: numerous, big, lentoid, with lined-up pits (Fig. 5e). Ericameria nauseosa, another Asteraceae, has ring porous wood with large (5 to 11 cells) (Fig. 5f, 5g and 5h), heterogenous (square cells) rays (Fig. 5j), and simple perforations in the vessels (Fig. 5i). Vessel walls are adorned by rounded pits (Fig. 5i). Purshia tridentata, a Rosaceae shrub, has a semi-ring porous wood (Fig. 5k and5l) with thin (1-3 cells) homogenous rays (Fig. 5m and5n). Perforations inside the vessels are simple and inter-vessel walls bear medium sized and few pits (Fig. 5m and5n). Sarcobatus vermiculatus (greasewood), an Amaranthaceae shrub, has semi-ring porous wood (Fig. 5o), thin rays (1 cell) (Fig. 5p), simple perforations in vessels (Fig. 5r and5s), and rounded pits frequently organized into lenticular openings (Fig. 5q, 5r and5s). So, in the case of the wood of broad-leaved shrubs near Blacktail Pond, the density, arrangement, shape, and size of vessel pits were diagnostic features for identification.

Charcoal particle analysis in the Blacktail Pond sediment core

Charcoal >180 µm in diameter was present in low amounts and variable condition and comprised 28 of the 243 samples. Despite the small size of most particles, wood anatomy was often well preserved. Most of the charcoal particles were pieces of burned wood (67.1% of wood + herb), and some could be identified to genus (11.4%). Samples from the two large CHAR fire peaks had the most identifiable particles and also were the most taxonomically diverse. These two fire episodes suggest large fires or fires near the lake over a short period of time. Samples from the period before the first peak that dated from ca. 11,350 to 11,170 cal yr BP also contained abundant and diverse wood (Table 3). 4.2.1. Remains of monocotyledons Charred remains of herbs, including parenchymatous organs that resembled stems, were commonly observed in three-dimensional views under high-magnification SEM (Fig. 6a to 6f) [START_REF] Hather | An archaeobotanical guide to root and tuber Identification[END_REF]. Some elements were identified as stems when viewed in trilobal crosssection (Fig. 6b) and laterally (Fig. 6a and6d). The vascular tissue or xylem of herbs was preserved, almost intact and without serious distortion in some specimens. Phloem was also recognized by its position relative to the xylem. In Figure 6b, the phloem is internal and forms a cylinder surrounded by a crown of xylem. The xylem elements have a polygonal transversal section (Fig. 6c). Sometimes, deformation of the xylem revealed its staged elements laterally (Fig. 6e). Figure 6d shows stomata in the lower epidermis of an aerial stem. 4.2.2. Remains of broad-leaved taxa Among the burned wood of broad-leaved shrubs in the Blacktail Pond record, two specimens (at ca. 10,970 and 10,185 cal yr BP) had well preserved vessels and wall ornamentation similar to the pitting structure observed in Artemisia tridentata wood (Fig. 5e). Numerous lined-up, lentoid pits were easily distinguished from other plant tissue (Fig. 6g, 6h, 6i). Several vessel wall pits were very simple and could not be identified (Fig. 6j, 6k, 6l).

Remains of conifers

The presence of vertical or longitudinal tracheids with uniseriate (very common) or biseriate opposite big bordered pits was characteristic of conifer wood (Fig. 7a to 7e). Small charcoal particles with tangential sections showed resin canals with epithelial cells and high rays (Fig. 7f,7g). Some tracheids had helical cracks in their walls, which is a common feature of compression wood in conifers. The radial section was best for distinguishing among conifers. This plane is achieved through natural breakage along the rays and was found in several charcoal fragments from Blacktail Pond. Pinus species were identified by the cross-field ray pits. Two types of pits were observed in the Pinus wood. The first type was fenestriform pitting, which is window-like, with a large, simple ray pit occupying almost the entire cross-field. Rarely do wood fragments have two fenestriform pits per cross-field (Fig. 7h,7i,7j). However, in the Yellowstone region Pinus albicaulis and/or P. flexilis share this feature (Fig. 4g,4h,4k,4l). The second type, pinoid pitting, is made by ray pits that are simple and more or less large. Usually two to four pinoid ray pits can be observed per cross-field. This type of pitting is characteristic of Pinus contorta (Fig. 7k,7l,7m). Wood fragments with ray tracheids and without fenestriform or pinoid pits in the cross-fields were assigned to Picea (Fig. 7d,7n). A third type of cross-field ray pits was taxodioid: large pit aperture with narrow border. When charcoal particles had such pits and no tracheids, we assigned them to either Abies or Juniperus (Fig. 7o to 7r). In conifer tracheids, two types of cell contents were present, one of which forms pyramidal crystals or druses (Fig. 7s,7t). This common feature was not diagnostic. 4.2.4. Unidentifiable charcoal 16.5% of charcoal particles in the large size range (180 -250 µm) lacked any discernible cellular structure. Remains of leaves were often found as part of charred plant tissues (11.5%), and hairs (trichomes) and stomata were also observed (Fig. 6m, 6n, 6o). Similarly, small fragments of vesicular charcoal were noted and could have come from any parenchymous tissue.

Charcoal analysis results

The charcoal particles that could be identified include Pinus; Picea; Abies or Juniperus; a deciduous shrub, most likely Artemisia; and unidentifiable monocotyledons. Pinus albicaulis and/or P. flexilis (these species cannot be distinguished by their pollen) was present in the vegetation at ca. 11,350 cal yr BP, during a time of mixed-conifer forest (Table 3). P. albicaulis and/or P. flexilis charcoal was also noted at ca. 9980 cal yr BP, when these taxa were less abundant locally and may have come from more distant fires. The presence of Pinus contorta charcoal at ca. 11,180 cal yr BP aligns well its rising abundance in the pollen record. P. contorta and/or Picea were also identified in three charcoal samples dating to ca. 10,780 cal yr BP. Artemisia charcoal was identified at ca. 10,990, 10,180, and tentatively at 9920 cal yr BP, but shrub charcoal was present throughout the record, as was charcoal from herbaceous taxa. The presence of both hardwood and softwood charcoal suggests that most fires burned both forest and steppe. However, remains of hardwood and monocotyledons comprised most of the charcoal from two samples dating to ca. 10,650 cal yr BP, suggesting that steppe was the main fuel at that time. It is interesting that the first large fire episode at 10,970 cal yr BP consisted of burned conifers (Picea, Abies and/or Juniperus), shrubs and herbs, but no Pinus charcoal was identified. This assemblage is consistent with an open mixed-conifer forest, in which pine was not dominant. The second fire episode at 10,200 cal yr BP contained shrub and herb charcoal and no conifer charcoal, implying a local fire that primarily burned steppe vegetation around the lake (Table 3). Two centuries before the first fire peak (from ca. 11,150 to 10,970 cal yr BP) and one century after the second fire peak (from ca. 10,200 to 10,085 cal yr BP), the cores contained no charred macroscopic plant remains.

Conclusion and perspectives

The Blacktail Pond study contributes new information to our understanding of the fire history of northern Yellowstone, and the development of vegetation in the northern Rocky Mountains during the late-glacial and early-Holocene periods. The pollen and charcoal records show that increased fire activity played a significant role in the transition from late-glacial Picea parkland to early-Holocene vegetation of Pinus contorta forest and steppe. The charcoal identifications enrich this reconstruction by showing that large fire episodes may have facilitated the transition. In the case of the fire at ca. 10,970 BP, conifers and shrubs were both fuel types near the lake. The second fire episode at ca. 10,200 cal yr BP, the presence of hardwood charcoal and no softwoods implies the development of steppe vegetation near the site with conifers restricted to more distant settings on rocky buttes and at higher elevations. The second fire must have been fairly local but probably intense to produce so much charcoal. Another interesting insight is the presence of Pinus contorta charcoal as early as ca. 11,180 cal yr BP, even before it became the dominant vegetation type. Fire and P. contorta are closely linked at present, given its ability to rapidly regenerate after fire. The record at Blacktail Pond suggests that fire facilitated its increasing dominance in the early Holocene.

This study shows that it is possible to identify a part of the very small charcoal remains preserved in the lake sediments with SEM identification. The results provide unique insights as to the fuel composition. The next phase of research will focus on examining charcoal from other sites in the Yellowstone region to see if comparable shifts in fuel were occurring at times of changing climate, vegetation, and fire activity. 5.1. Perspectives on the identification of very small fragments of burnt wood Identification of charcoal fragments is a valuable tool in reconstructing past environments and determining disturbances. This paper illustrates the potential for the identification of charcoal particles to be part of paleoenvironmental reconstructions. We believe that this approach deserves consideration in other settings. The analysis of charcoal in lake or wetland sediment cores is challenged by the smallness and relative scarcity of suitable material as well as preservation issues related to charcoal transport and deposition into such sites. These data complement other quantitative proxies in building an environmental reconstruction (e.g., pollen, macroscopic charcoal, lithologic and geochemical data). Other techniques can improve the results in this study. Reflected Light Microscopy (RLM) is used to identify sizable and numerous charcoal fragments by studying fresh break surfaces [START_REF] Marguerie | Charcoal analysis and dendrology: data from archaeological sites in western France[END_REF]. In case of very small fragments, Scanning Electron Microscopy (SEM) is best suited for detailed observation of small irregular surfaces with high relief. The increasing number of tabletop SEMs in laboratories now enables this type of analysis at high magnifications and great depth of field. Because it is difficult to make transversal or longitudinal sections by breaking, splitting or slicing to observe wood anatomical features on small charcoal particles, High-Throughput Xray Computed Tomography (HT-µCT) also opens up new opportunities to reveal fine anatomical characters. It has been shown to be effective in imaging anatomical features that cannot be observed on exposed surfaces [START_REF] Haneca | X-Ray sub-micron tomography as a tool for the study of archaeological wood preserved through the corrosion of metal objects[END_REF]. The ability to yield images of internal wood structure without having to break or cut the sample is a powerful tool to identify diagnostic features (3D observation) [START_REF] Haneca | X-Ray sub-micron tomography as a tool for the study of archaeological wood preserved through the corrosion of metal objects[END_REF]. The technique allows the production of unlimited virtual sections and clean digital cuts in any direction on very small samples (Van den [START_REF] Van Den Bulcke | X-ray tomography as a tool for detailed anatomical analysis[END_REF][START_REF] Hubau | Complementary imaging techniques for charcoal examination and identification[END_REF]. The resolution obtained is about 2 µm, which is sufficient to reveal many features of interest for wood anatomy [START_REF] Mayo | Micron-scale 3D imaging of wood and plant microstructure using high-resolution X-ray phase-contrast microtomography[END_REF]. However, the resolution of HT-µCT is not as high as with SEM, and fine details in the vessels and fibres, and pitting may not be visible [START_REF] Hubau | Complementary imaging techniques for charcoal examination and identification[END_REF]. Another related technique, nanotomography or X-ray nano Computed Tomography (nanoCT), offers higher resolution and is also excellent for seeing features that are not clearly visible under SEM because they are concealed by coatings or mineral deposits (Van den [START_REF] Van Den Bulcke | X-ray tomography as a tool for detailed anatomical analysis[END_REF][START_REF] Hubau | Complementary imaging techniques for charcoal examination and identification[END_REF]. -----------------------------------Acknowledgements SEM sessions took place in the Imaging and Chemical Analysis Laboratory (ICAL) at Montana State University, Bozeman, thanks to Nancy Equall and the Centre de Microscopie Electronique à Balayage et de microAnalyse (CMEBA) of the University of Rennes 1, thanks of Francis Gouttefangeas and Loïc Joanny. Yann Rantier carried out systematic quality adjustments of SEM images at the ECOBIO research unit of the CNRS and University of Rennes 1. We also acknowledge Peter Kováčik from the PaleoResearch Institute at Golden, Colorado (USA) and Alan Crivellaro from the Department Land, Environment, Agriculture and Forestry (TESAF), University of Padova (Italy) for their advice and help on problematic specimens. The MSU research was supported by NSF grant (0966472) to CW. We thank two anonymous reviewers for their helpful comments on the manuscript. -------------------Figure [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF] Figure 3. Fire proxies at Blacktail Pond (ex. [START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF] Figure 4. Scanning electron microphotographs of burnt wood of three pine species in the northern Rocky Mountains: a. Pinus contorta, transverse section; b. P. contorta, tangential section; c, d. P. contorta, radial section; e. P. albicaulis, transverse section; f. P. albicaulis, tangential section; g, h. P. albicaulis, radial section; i. P. flexilis, transverse section; j. P. flexilis, tangential section; k, l. P. flexilis, radial section Figure 5. Scanning electron microphotographs of burnt wood of four shrubs in the northern Rocky Mountains: a, b. Artemisia tridentata, transverse section; c. A. tridentata, tangential section; d, e, A. tridentata, radial section; f, g. Ericameria nauseosa, transverse section; h. E. nauseosa, tangential section; i, j. E. nauseosa, radial section; k, l. Purshia tridentata, transverse section; m. P. tridentata, tangential section; n. P. tridentata, radial section; o. Sarcobatus vermiculatus, transverse section; p, q, r. S. vermiculatus, tangential section; s. S. vermiculatus, radial section

Figure 1 .

 1 Figure 1. Location of Blacktail Pond.

Figure 2 .

 2 Figure 2. Pollen diagram from Blacktail Pond (ex.[START_REF] Krause | Climate and vegetation change during the late-glacial/early-Holocene transition inferred from multiple proxy records from Blacktail Pond[END_REF]

Figure 6 .

 6 Figure 6. Charred remains of monocotyledons, broadleaf woods and leafs from Blacktail Pond by a scanning electron microscope: a. Stem of monocotyledon; b, c. Trilobal crosssection stem of monocotyledon with visible phloem and xylem; d, e. Details of xylem elements of stem; f. Xylem and phloem in longitudinal section; g, h, i. Particular pits of the vessel walls Artemisia sp.; j, k, i. Simple pits of the vessel walls in unidentifiable broadleaf; m, n, o. Fragments of leaves with hairs (trichomes) and stomata

Figure

  Figure

Table 1 .

 1 Trilobal crosssection stem of monocotyledon with visible phloem and xylem; d, e. Details of xylem elements of stem; f. Xylem and phloem in longitudinal section; g, h, i. Particular pits of the vessel walls Artemisia sp.; j, k, i. Simple pits of the vessel walls in unidentifiable broadleaf; m, n, o. Fragments of leaves with hairs (trichomes) and stomata Figure 7. Charred remains of conifers from Blacktail Pond a, b, c, d, e. Woody fragments of conifers; d, n. wood fragment of conifer with ray tracheids; f. Tangential section showing low rays of conifer; g. Possible resin canal and rays in tangential section; h, i, j. Pinus with fenestriform cross-field pitting; k, l, m. Pinus with pinoid cross-field pitting; o, p, q, r. Conifer with taxodioid cross-field pits; s. Crystals in tracheids; t. Other type of crystals in tracheids Main wood anatomical features of the common genera of conifers of the northern Rocky Mountains around Blacktail Pond

Table 2 .

 2 Main key characteristics of microscopic anatomical features in the wood of three northern Rocky Mountains pines around Blacktail Pond

Table 3 .

 3 Results from charcoal analyses of the sediment of Blacktail Pond

	Purshia tridentata
	Sarcobatus vermiculatus

Conflict of Interest

All the authors signing this paper "Charcoal identifications supplement an early-Holocene fire-history reconstruction in Yellowstone National Park, USA" declare that they approve the submission of their work to Quaternary International.

Dr. Dominique Marguerie, dominique.marguerie@univ-rennes1;fr Dr. Teresa Krause, krausete@augsburg.edu Dr. Cathy Whitlock, whitlock@montana.edu


Nikolaos A Burger

Antonios Mavromanolakis

Gerhard Meier

Patrick Brocorens

Roberto Lazzaroni

Laurent Bouteiller

Benoit Loppinet

Dimitris Vlassopoulos

Stabilization of Supramolecular Polymer Phase at High Pressures

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

structure of the filaments is rationalized by using the Clapeyron equation which yields an associated volume change of about 8 Ǻ 3 /molecule. This observation is rationalized by means of Molecular Dynamics simulations of the two phases, which show a decrease in the molecular volume at the filament-tube transition, originating from increased intermolecular contacts in the tube with respect to the filament. These new findings offer insights into the role of pressure in stabilizing self-assemblies.

I. INTRODUCTION

Pressure is often considered to be a forgotten thermodynamic variable, in part because pressuredependent experimentation is far more challenging than its temperature-dependent counterpart.

Yet, over the years several investigations of static and dynamic properties of soft materials at high pressures have been reported in the literature. For example, moderate pressures of the order of 100 bar were found to influence the second virial coefficient and radius of gyration of flexible polymers in different solvents. [START_REF] Gaeckle | Effect of Pressure on the Second Virial Coefficient and Chain Dimensions in Polymer Solutions[END_REF] Increasing pressure (up to about 4 kbar) was reported to increase the intrinsic viscosity of polymer solutions, with the Huggins coefficient exhibiting a nonmonotonic dependence with a broad minimum. [START_REF] Schmidt | Pressure Dependence of Intrinsic Viscosities and Huggins Constants for Polystyrene in tert-Butyl Acetate[END_REF] Clearly, pressure affects the miscibility in polymer mixtures, promoting miscibility in LCST polymer solutions [START_REF] Wolf | Improvement of polymer solubility: Influence of shear and of pressure[END_REF] and in UCST polymer blends [START_REF] Beiner | Pressure-Induced Compatibility in a Model Polymer Blend[END_REF] due to volume changes on mixing. More complicated is the effect of pressure on triblock copolymer micelles, where it is found to promote crystal to liquid transition, but often the micellar liquid is topologically interacting. [START_REF] Kloxin | High-pressure phase diagram of an aqueous PEO-PPO-PEO triblock copolymer system via probe diffusion measurements[END_REF] Furthermore, pressure is found to affect the glass transition and associated alpha relaxation (typically increasing the glass temperature) in supercooled liquids 6 and a wide range of shape-persistent polymeric materials with intrinsic orientational order, for which the lack of thermal energy rather than free volume is proposed to be responsible for vitrification [7]. In general, soft materials experience high pressures in a variety of applications or during their transformation, which renders relevant studies necessary.

For example, the application of pressure was found to increase the yield stress and strain of polymeric solids undergoing tensile deformation. [START_REF] Christiansen | The mechanical behavior of polymers under high pressure[END_REF] Additional examples involve processing and capillary flow of polymer melts, [START_REF] Guan | Structure and properties of self-reinforced polyethylene prepared by oscillating packing injection molding under low pressure[END_REF][START_REF] Berthe | High pressure rheology for high pressure lubrication: A review[END_REF][START_REF] Couch | High pressure capillary rheometry of polymeric fluids[END_REF][START_REF] Hatzikiriakos | Start-up Pressure Transients in a Capillary Rheometer[END_REF][START_REF] Mackley | Experimental Observations on the Pressure-Dependent Polymer Melt Rheology of a Linear Low-Density Polyethylene using a Multipass Rheometer[END_REF][START_REF] Koran | A high pressure sliding plate rheometer for polymer melts[END_REF] drilling operations with fracturing fluids, [START_REF] Barati | A review of fracturing fluid systems used for hydraulic fracturing of oil and gas wells[END_REF] the use of high pressure to obtain protein crystals from spider silk [START_REF] Peng | Pressure-Induced Crystal Memory Effect of Spider Silk Proteins[END_REF] and the high-pressure preparation of dairy products (emulsions). [START_REF] Rastogi | Opportunities and challenges in high pressure processing of foods[END_REF] Despite the above advances, it appears that the role of pressure on the properties of selfassemblies, and in particular their phase behavior, has not received much attention. Recently, pressure was reported to stabilize dynamic supramolecular assemblies of the host-guest type (enhancing their binding equilibria) and maintaining a constant viscosity (in contrast to crosslinked fluids, which may suffer pressure-induced viscosity thinning). [START_REF] Appel | Decoupled Associative and Dissociative Processes in Strong yet Highly Dynamic Host-Guest Complexes[END_REF] That work shows that pressure is an important variable that may affect supramolecular associations and exploiting its consequences is an outstanding challenge. In this letter, we present a methodology to address this challenge by using as archetype a reversible supramolecular polymer built from 2,4-bis(2ethylhexylureido)toluene (EHUT), based on a bis-urea moiety. Its synthesis, thermodynamic and rheological properties have been discussed quite extensively in the literature. [START_REF] Ducouret | Rheological characterisation of bis-urea based viscoelastic solutions in an apolar solvent[END_REF][START_REF] Lortie | Structural and rheological study of a bis-urea based reversible polymer in an apolar solvent[END_REF][START_REF] Simic | Highly cooperative formation of bis-urea based supramolecular polymers[END_REF][START_REF] Bellot | Thermodynamic description of bis-urea self-assembly: competition between two supramolecular polymers[END_REF][START_REF] Shikata | Structure and dynamics of a bisurea-based supramolecular polymer in n-dodecane[END_REF][START_REF] Sabadini | Bis-urea-based supramolecular polymer: The first self-assembled drag reducer for hydrocarbon solvents[END_REF][START_REF] Francisco | Tuning the Viscoelastic Properties of Bis (urea)-Based Supramolecular Polymer Solutions by Adding Cosolutes[END_REF][START_REF] Van Der Gucht | Brownian particles in supramolecular polymer solutions[END_REF][START_REF] Louhichi | Humidity affects the viscoelastic properties of supramolecular living polymers[END_REF][START_REF] Jacob | Large amplitude oscillatory shear of supramolecular materials[END_REF][START_REF] Pinault | Solvents with similar bulk properties induce distinct supramolecular architectures[END_REF] In non-polar solvents such as toluene, cyclohexane or dodecane, at high enough concentrations EHUT selfassembles into long supramolecular polymers, which have tube structure at lower temperatures and filament structure at higher temperatures. Their viscoelastic behavior is similar to wormlike surfactant micelles whose rheology depends on the bonding lifetime and overall length (which controls terminal relaxation). [START_REF] Lortie | Structural and rheological study of a bis-urea based reversible polymer in an apolar solvent[END_REF][START_REF] Shikata | Structure and dynamics of a bisurea-based supramolecular polymer in n-dodecane[END_REF][START_REF] Sabadini | Bis-urea-based supramolecular polymer: The first self-assembled drag reducer for hydrocarbon solvents[END_REF][START_REF] Francisco | Tuning the Viscoelastic Properties of Bis (urea)-Based Supramolecular Polymer Solutions by Adding Cosolutes[END_REF][START_REF] Van Der Gucht | Brownian particles in supramolecular polymer solutions[END_REF][START_REF] Louhichi | Humidity affects the viscoelastic properties of supramolecular living polymers[END_REF][START_REF] Jacob | Large amplitude oscillatory shear of supramolecular materials[END_REF][START_REF] Pinault | Solvents with similar bulk properties induce distinct supramolecular architectures[END_REF][START_REF] Hendricks | Non-monotonic stress relaxation after cessation of steady shear flow in supramolecular assemblies[END_REF] A similar bis-urea was recently found to exhibit thickening of high-pressure (about 345 bar) supercritical propane with significant implications in fracturing fluid processing for enhanced oil recovery operations. [START_REF] Hansch | Thickening compressed liquid and supercritical propane with bisurea DMHUT N[END_REF] Here we investigate the effects of pressure on the thermoreversible supramolecular assemblies of EHUT in cyclohexane, which combine different length and time scales depending on temperature and concentration.

II. MATERIALS

The synthesis of EHUT was achieved by reacting racemic 2-ethylhexylamine with 2,4-toluene diisocyanate [20]. An apolar solvent, cyclohexane (99.7 % pure) was used as received, obtained from Sigma Aldrich. Its dielectric constant is 2.02. [START_REF] Reichardt | Solvents and Solvent Effects in Organic Chemistry[END_REF] The solutions were prepared under conditions of atmospheric humidity and room temperature, by adding the EHUT powder to the solvent and stirring for at least 48 hours. [START_REF] Louhichi | Humidity affects the viscoelastic properties of supramolecular living polymers[END_REF]

III. METHODS

III.A. High-Pressure DLS

A homemade high-pressure cell was coupled to a single scattering DLS setup (from ALV, Germany) for the high-pressure DLS (HP-DLS) experiments (Fig. 1). Here we provide a brief description of the main components: the pressurized chamber comprises 6 optical windows corresponding to scattering angles of 45, 90 and 130⁰, and the light beam was focused with an 50x objective lens before reaching the sample, in the middle of the cylindrical cell. Nitrogen was compressed by means of a membrane compressor and entered the cell from the top. Details concerning the cell, windows and sealing are described elsewhere. [START_REF] Meier | A high pressure cell for dynamic light scattering up to 2kbarswith conservation of plane of polarization[END_REF][START_REF] Meier | SANS and dynamic light scattering to investigate the viscosity of toluene under high pressure up to 1800 bar[END_REF] The experiments were performed at pressures ranging from 1 to 1200 bar and temperatures from 20 to 70 0 C (the latter were controlled by means of a recirculating water/alcohol bath and measured with a thermocouple attached at the surface of the sample cell). Since the pressure-transmitting medium is compressed nitrogen, special care was taken for the measurement time and the selected sample volume in order to avoid diffusion of nitrogen molecules into the polymer sample up to the measurement point (where the laser beam enters the sample). More specifically, the diffusion coefficient of nitrogen molecules in liquids such as organic solvents, at ambient conditions (P = 1 bar, T = 20 0 C) is in the order of 10 -9 m 2 /s, [START_REF] Ferrell | Diffusion Coefficients of Nitrogen and Oxygen in Water[END_REF] therefore for distances of about 1 cm (between the free surface of the solution and the measurement point), the corresponding time 𝑡 = $% ! & '( , is in the order of days, ensuring that the measurements are unaffected by possible dilution of the sample. Further evidence is provided in Fig. S1 of the supporting information (SI) which shows identical where DLS data over long times for a similar system (EHUT in dodecane). A cw laser at 514.5 nm was used and the scattering angle was fixed at θ = 90 0 . A mono-mode optical fiber was employed to feed the scattered light into an avalanche photodiode (Perkin-Elmer) operating in photon counting mode (Fig. 1). The time autocorrelation function of the scattering intensity was obtained in real time by means of an ALV 5000E digital correlator, and the electric field autocorrelation function (or intermediate scattering function, ISF) 𝐶(𝑡) was recorded and analyzed (see also the supporting information, SI and Fig. S2). The pressure was kept constant over long times and data were highly reproducible.

To better appreciate the difference in dynamics between tube (viscoelastic network) and filament (predominantly viscous) phases, high-pressure microrheology experiments [START_REF] Dennis | operation, and validation of a microrheology instrument for high-pressure linear viscoelasticity measurements[END_REF]37 were conducted as well. To this end, polymethylmethacrylate (PMMA) particles were added to an EHUT solution in cyclohexane at a volume fraction of about 10 -4 vol%, to act as effective tracers for following their motion. They were chemically grafted with polyhydroxy stearic acid (PHSA) chains (of about 10nm in length) to ensure stability of the dispersion, their hydrodynamic radius was R=130 nm (measured in the dilute regime by dynamic light scattering, DLS) and their polydispersity was 10%. [START_REF] Royall | In search of hard spheres[END_REF] The dual goal of adding these particles was to stay in the single scattering limit (i.e., photons reaching the instrument detector being scattered by a probed particle and not also re-scattered by neighboring particles) [START_REF] Schärtl | Light Scattering from Polymer Solutions and Nanoparticle Dispersions[END_REF] and reach a scattering intensity at least 50 times higher compared to that of EHUT solutions in the absence of particles. Details of the microrheological measurements are presented in the SI (see also Fig. S3). Note that our approach is similar to that of Ref.36 but differs in the pressurizing system (we used gas instead of oil) and the detection method (we used DLS in the single scattering instead of diffusive wave limit).

III.B. Molecular Dynamics simulations

Molecular Dynamics (MD) simulations were performed with the Materials Studio (MS) software on a tube and a helical filament of EHUT molecules. [START_REF] Shikata | Structure and dynamics of a bisurea-based supramolecular polymer in n-dodecane[END_REF][START_REF] Biovia | Biovia Materials Studio[END_REF][START_REF] Brocorens | Conformational Plasticity of Hydrogen Bonded Bis-urea Supramolecular Polymers[END_REF] Details are given in the SI. The volumes of conformations generated during the simulations were determined by rolling on the molecules a dummy atom that acts as a spherical probe (Fig. S7). The contact is defined between the Van der Waals radius of the probe and the Van der Waals radii of the atoms (as implemented in the Dreiding force field [START_REF] Mayo | A Generic Force Field for Molecular Simulations[END_REF] used to do the MD simulations). The radius of the probe can be chosen arbitrarily: (i) if it is set to zero, the probe simply explores the surface defined by the Van der Waals radii of the atoms in the molecule and the corresponding volume is called the Van der Waals volume (see gray area in Fig. S7); (ii) if the radius of the probe is non-zero, some parts of the molecular surface become inaccessible to the probe, its trajectory then defines a Connolly surface (see the black curve in Fig. S7) and the corresponding volume is called the Connolly volume. Its value depends on the radius of the probe. In this work, the radius of the probe was given different values: 0 Å, 0.8 Å, 1.6 Å (typical of a hydrogen atom), and 2.4 Å. In addition to the tube and helical single filament, a molecule was extracted from the simulated assemblies, and its conformation was used to evaluate its van der Waals and Connolly volumes in the absence of packing with neighbors, i.e., as an isolated molecule. For the three systems, the volumes were calculated on six different conformations and averaged.

IV. RESULTS AND DISCUSSION

The phase diagram of EHUT solutions in cyclohexane is schematically depicted in Fig. 2.

Depending on the concentration and temperature, EHUT solutions may be monomeric (unassembled) or form self-assembled filaments or tubes, under atmospheric pressure, qualitatively similar to the respective behavior in toluene. [START_REF] Bellot | Thermodynamic description of bis-urea self-assembly: competition between two supramolecular polymers[END_REF][START_REF] Shikata | Structure and dynamics of a bisurea-based supramolecular polymer in n-dodecane[END_REF][START_REF] Alvarenga | Unexpected Solvent Influence on the Rheology of Supramolecular Polymers[END_REF] Both filament and tube structures are long chains of molecules held together by hydrogen bonds, but the cross-section of the former contains a single EHUT monomer, whereas that of the latter contains three monomers, which leads to a slowing-down of the chain scission process and eventually to viscoelastic behavior.

Of interest in this work is the specific concentration c=4 g/L, where a tube-to-filament transition is observed at 49°C. In fact, SANS and IR spectroscopy experiments indicate a structural thickness transition at T≈52 0 C, P=1 bar and c=10.4 g/L. [START_REF] Bouteiller | Thickness transition of a rigid supramolecular polymer[END_REF] Characterization details of the tubefilament transition at low pressures are given in Table S1 of the SI. The effects of pressure will be discussed below. Note that there is no macroscopic signature of phase separation in this solvent, as the samples remain visually transparent. We have also measured the pressuredependent solvent viscosity (see Fig. S4 of the SI) and accounted for it, as well as for the respective temperature dependence in the presentation of the experimental data in order to emphasize the net effect of pressure on the supramolecular polymer organization. Indeed, in Fig. 3 below the dynamic light scattering data or dynamic viscoelastic data have been appropriately normalized by the solvent viscosity (in their time or frequency axes, respectively) at the respective pressure and temperature (see also in Fig. S5 of the SI the effect of pressure on the relative viscosity of a 4g/L EHUT/cyclohexane solution). Moreover, since the thrust of the present investigation is the effects of pressure on the EHUT-based reversible supramolecular polymers, we work in conditions of ambient humidity without considering its effects on the viscoelastic properties of the solutions. The cartoons illustrate the molecular structure of the EHUT monomer, and the self-assembled filaments and tubes (the dotted lines illustrate the links (hydrogen bonds) between units which are shown by circles). The red filled circle indicates the observed structural transition from filament to tube for c=4 g/L and T=49 0 C, which is in excellent agreement with the literature [START_REF] Bouteiller | Thickness transition of a rigid supramolecular polymer[END_REF] (see also Table 1).

First, we investigated the dynamics at atmospheric pressure. As can be seen in Fig. 3a, there is a transition from unimodal ISF at high temperatures (T ³ 49 0 C) to bimodal ISF at low temperatures (T<49 0 C). A quantitative analysis of the relaxation modes (amplitutes and times) is presented the SI (Figs. S2,S6 and Table S2). We attribute the presence of a second, slow relaxation process to the appearance of self-assembled EHUT tubes, which are known to exhibit strong viscoelasticity accompanied by a plateau modulus [START_REF] Van Der Gucht | Brownian particles in supramolecular polymer solutions[END_REF][START_REF] Louhichi | Humidity affects the viscoelastic properties of supramolecular living polymers[END_REF][START_REF] Jacob | Large amplitude oscillatory shear of supramolecular materials[END_REF] ; note that entangled polymers have been reported to reveal a second slow mode in their ISF, assigned to the relaxation of the entanglement network. [START_REF] Jian | Coupling of Concentration Fluctuations to Viscoelasticity in Highly Concentrated Polymer Solutions[END_REF][46][START_REF] Wang | Reexamination of the Origin of Slow Relaxation in Semidilute Polymer Solutions-Reptation Related or Not?[END_REF] Note further that the second mode is readily observed by eye. Hence, single-mode ISF refers to filaments and bimodal ISF to tubes. Moreover, this distinction is also corroborated by the total scattering intensity as a function of temperature, which is depicted in the inset of Fig. 3a upon heating and cooling at 1 bar; the tube-to-filament transition is marked by a change in intensity. Note that in some cases (higher pressure or lower temperature) a weaker third slower mode is detected (Table S2), which reinforces the assignment to tube phase. Consequently, we use the existence of slow mode(s) in the ISF as a signature of the formation of tubes, in order to construct a phase diagram, which is depicted in Fig. 4 below. To this end we examine the shape of the ISF of a given EHUT/cyclohexane solution (c=4 g/L) at different temperatures and pressures (see also Fig. S6 and Table S2 of the SI). For example, the typical ISFs at three temperatures and P=325 bar (Fig. 3b) indicate that at 49 0 C we have a tube phase while at 58 0 C (where the slow mode progressively disappears) we have a filament phase, hence there is a shift of the tube-filament transition from about 49 0 C (at atmospheric pressure, Fig. 3a) to about 58 0 C (at 325 bar), where the slow mode progressively disappears. At 600 bar, the supramolecular solution forms only tubes throughout the examined temperature range (Fig. 3c). Furthermore, an examination of the microrheological linear viscoelastic spectrum indicates sharp differences between low (1 bar) and high (600 bar) pressures, i.e., filaments and tubes (Fig. 3d and Fig. S5 of the SI). At this particular semidilute concentration of 4 g/L, a loose arrangement of selfassembled filaments is formed at 49 0 C and 1 bar (see also Fig. 2 and Ref.27), which exhibits the rheological signature of a predominantly viscous liquid (with low viscosity of ~ 3 mPas, see Fig. S5 of the SI) The same response is expected at higher temperatures, well into the phase dominated by filaments. However, on increasing the pressure to 600 bar at the same temperature, the viscoelastic spectrum changes drastically, signifying the response of an entangled network of supramolecular tubes [START_REF] Louhichi | Humidity affects the viscoelastic properties of supramolecular living polymers[END_REF] (and a slow mode in the ISF). The eventual terminal regime of this network gives rise to a viscosity which exceeds that of the filaments by a factor of about 330 (Fig. S5). Finally, we note that for both tubes and filaments, the EHUT solutions were completely transparent without visual hint of phase separation. The interesting finding of Fig. 4 is that high pressures appear to stabilize further the tubes, which are the only self-assembled structure at this concentration. It is also important to emphasize that the observed tube-filament transitions are reversible and all measurements are performed in equilibrium conditions (very fast kinetics due to the dynamic character of this supramolecular system).

Finally, we note that the high-frequency viscoelastic response of tubes and filaments in Fig. 3d compares favorably with that of wormlike surfactant micelles and other semiflexible polymeric materials. The latter were reported to exhibit an intermediate and high-frequency power-law dependence of G'' with exponents of 5/9 (Rouse-Zimm) and 3/4 (internal bending modes), respectively, with the transition occurring at the scale of the persistence length, as well as good agreement between conventional rheometry and passive microrheology data. [START_REF] Willenbacher | Broad Bandwidth Optical and Mechanical Rheometry ofWormlike Micelle Solutions[END_REF][START_REF] Willenbacher | Dynamics and structure of complex fluids from high frequency mechanical and optical rheometry[END_REF] The analysis of the high-frequency response is presented in the SI (Fig. S7). In general, the effect of pressure can be accounted for by approximating it as a first order transition and invoking the Clapeyron equation

-. -/ = ∆1 / * ∆3
where 𝑇 * is the tube-filament transition temperature at reference (atmospheric pressure), ∆𝐻 is the latent heat per molecule associated with the tube-to-filament transition and ∆𝑉 the associated difference in molecular volumes. The experimental results of Fig. 4 suggest that upon increasing the pressure to 300 bars, i.e., ∆𝑃 ≈ 300 bar, the transition temperature approaches 60°C, i.e., ∆𝑇 ≈ 10𝐾. This suggests that ∆1 ∆3 ≈ 9.7x10 [START_REF] Christiansen | The mechanical behavior of polymers under high pressure[END_REF] Pa. Note that ∆𝐻 is about 1.7𝑘 ? 𝑇 at low pressures (see Table S1 and Ref. 47), and we assume to a first approximation that it has the same dependence on pressure with ∆𝑉. This leads to ∆𝑉 ≈ 8 Ǻ 3 /molecule.

In order to rationalize that volume change, MD simulations were carried out on model systems for the filament and the tube (see details in SI), along with calculations on an isolated EHUT molecule as reference. The simulations indicate that the van der Waals volume decreases from the isolated molecule to the filament, as the interactions between the molecules, especially the formation of H bonds, leads to an overlap of the van der Waals volumes (see also Fig. S7). The volume is further reduced when the filament evolves to a tube, on the order of 10 Ǻ 3 /molecule, which conforms to the experimental results (Table 1). The volume decrease from filament to tube reflects the increase of contacts between neighbors. In both structures, the hydrogen bonds between urea moieties drive the assembly, but in the filament, the cores interact by p-p stacking and segregate from the alkyl chains that protrude in the environment, while in the tube, the cores and alkyl chains intercalate to form a continuous, dense medium minimizing its exposure to the environment (Fig. 5). . In this approach, the possible reorganization of the solvent has not been considered explicitly.

Nevertheless, we investigated the accessibility of cavities in the EHUT assemblies to solvent molecules, we used probes with different radii and found that an isolated molecule has the smallest Connolly volume, while it has the largest van der Waals volume, when compared to the two assemblies. The larger Connolly volume in the assemblies is explained by the formation of new cavities inaccessible to solvent upon molecular packing. When comparing the Connolly volumes of the filament and tube, the volume changes vary with the probe size, as expected.

More importantly, the tubes always appear more compact (i.e., have a higher density) than the filaments (see Table 1 and Fig. S8), again pointing to the higher density of intermolecular contacts in the tubes.

An important question is whether the stabilization of the tube structure at high pressures reflects a kind of host-guest effect, due to more favorable interactions between the tube and the included solvent, or if it is a more general phenomenon. Concerning the former situation, we note that solvent interactions inside and outside the tubes have been found to be different. [START_REF] Willenbacher | Dynamics and structure of complex fluids from high frequency mechanical and optical rheometry[END_REF] In the latter case, it should also occur in other hydrogen-bonded assemblies by enhancing bonding at high pressures. Another challenge is to determine the consequences of the above pressure-dependent structural changes on the viscoelastic properties of the supramolecular assemblies. These questions will be addressed in the future.

In summary, high-pressure DLS in the single scattering limit and passive microrheology were employed to construct the PT phase diagram of a supramolecular polymer. The 2,4-bis(2ethylhexylureido)toluene (EHUT) self-assembles into stable viscoelastic network tube or viscous filament phases in apolar solvent cyclohexane, depending on temperature and concentration. By choosing a specific concentration, we spanned the temperature-pressure parameter space and identified tubes and filaments. We found that pressure substantially affects the phase behavior by promoting the tube phase. Indeed, at high levels of pressure (in the range 600 bar) the tube phase is stabilized throughout the entire range of temperatures. The tube-tofilament transition is rationalized as a quasi-first order transition and the associated volume change, in the order of 10 Ǻ 3 /molecule, can be ascribed to a higher density of intermolecular contacts in the tube, as determined is consistent with independent findings from molecular modeling simulations.

Figure 1 :

 1 Figure 1: Schematic illustration of the experimental high-pressure DLS setup. Shown, out of scale, are: the gas (nitrogen) tank, compressor, chamber cell with different glass windows for scattering at different fixed angles, gas inlet to DLS cell (P), inlet of recirculating fluid to cell chamber for maintaining constant temperature (T with arrows), temperature meter (T), laser (with green incident beam), scattered beam entering the photomultiplier tube (PMT), whose signal is collected and analyzed by a correlator on a PC.

27

 27

Figure 2 :

 2 Figure 2: Schematic illustration of the phase diagram for EHUT solutions in cyclohexane at atmospheric pressure. Three regimes are identified: monomeric (unassembled), self-assembled thin filaments and self-assembled tubes.The cartoons illustrate the molecular structure of the EHUT monomer, and the self-assembled filaments and tubes (the dotted lines illustrate the links (hydrogen bonds) between units which are shown by circles). The red filled circle indicates the observed structural transition from filament to tube for c=4 g/L and T=49 0 C, which is in excellent agreement with the literature 44 (see also Table1).

Figure 3 :

 3 Figure 3: Experimental data of EHUT/cyclohexane solution at 4 g/L, with the time divided by the temperature-or pressure-dependent solvent viscosity (see also SI). (a) Intermediate scattering functions (ISF); the open (filled) symbols correspond to filament (tube) structures. Heating from

Figure 4 .

 4 Figure 4. Phase diagram of a 4 g/L EHUT / cyclohexane solution in the (T, P) space. Star symbols correspond to passive microrheological data (see Fig.S2 in the SI). Circles correspond to DLS (without added particles). The black line is drawn to guide the eye. Blue (black) color indicates tubes (filaments).

Figure 5 .

 5 Figure 5. Pieces of filament (left), tube (center), and tube longitudinally cut in two to expose its inner part (right). The assemblies are of the same length (72 Å); the atoms are rendered as spheres having the Van der Waals radius of the elements (CPK representation). The urea moieties are in red, the toluene cores in blue, and the alkyl chains in green.

Table 1 :

 1 Average van der Waals volume per molecule with standard deviation, for the tube, the filament and the isolated molecule.

		van der Waals volume per	Connolly volume per molecule (Ǻ 3 )
		molecule (Ǻ 3 )
			0.8 Ǻ probe	1.6 Ǻ probe	2.4 Ǻ probe
	Tube	638.3 ± 0.7	689.8 ± 1.6	729.9 ± 2.6	766.3 ± 2.9
	Filament	649.3 ± 0.8	701.6 ± 1.4	763.9 ± 2.7	805.6 ± 3.5
	Isolated molecule	670.4 ± 2.8	688.1 ± 3.6	714.8 ± 4.7	741.0 ± 6.0

ACKNOWLEDGMENTS

Partial support has been received by the European Commission (Horizon2020-INFRAIA-2016-1, EUSMI grant no. 731019) and the Greek Secretariat for Research and Technology (INNOVATION program-AENAO). We thank J. K. G. Dhont (Forschungszentrum Jülich) for donation of part of the instrumentation and A. Vagias (Technical University of Munich) for early experimental tests. The modelling activities are supported by FNRS EOS 2Dto3D project (grant 30489208) and Consortium des Équipements de Calcul Intensif -CÉCI (Grant 2.5020.11) and by the Walloon Region (ZENOBE Tier-1 supercomputer, grant 1117545).