Soizig Le Stradic

Pauline Hernandez

G Wilson Fernandes

Elise Buisson

Regeneration after fire in campo rupestre: short-and long-term vegetation dynamics

Keywords: Biomass, campo arenoso, campo pedregoso, rupestrian grasslands, vegetation recovery, wildfire

The Cerrado (Brazilian savanna) is the second largest biome in Brazil, recovering 22% of the country, and campo rupestre is one of the most biodiverse ecosystem. Campo rupestre are extremely old mountaintop tropical ecosystems, composed of a mosaic of herbaceous, shrubland and savanna vegetation, generally located above 900 m above sea level characterized by shallow, acidic and nutrient-poor soils. In the context of increased land-use changes, effective conservation and management projects appear necessary to guarantee the conservation of these ecosystems. Although fire is a natural disturbance in campo rupestre, the effects of fire on vegetation dynamics remain poorly understood. Our objective was to assess the effects of fire on vegetation recovery and plant composition at both the short-and longterm on the main herbaceous vegetation types: the sandy and stony grasslands. We monitored plant community composition before and after a wildfire in order to assess the short-term vegetation recovery. Diachronic analyses of grasslands burnt at various dates were used to understand the effects of fire on longer-term vegetation dynamics. Our results highlighted a rapid recovery of campo rupestre vegetation after wildfires, suggesting a high adaptation to fire of plant communities. We did not find a significant variation in species richness of sandy grasslands according to time after fire, whereas higher species richness was observed in the recently burnt stony grasslands. No changes in plant composition of campo rupestre in response to fire was found, probably due to the high heterogeneity of this ecosystem. After fire, biomass gradually increased over time in both vegetation types. High biomass accumulation could lead to stronger fires. Further studies are necessary to understand the relationship between biomass accumulation and fire intensity in campo rupestre in order to set up adapted fire management strategies to conserve campo rupestre biodiversity.

Introduction

The Brazilian Cerrado is the world's most species-rich savanna, which originally covered over 2 million km 2 [START_REF] Oliveira | The Cerrados of Brazil. Ecology and natural history of a neotropical savanna[END_REF]. It is composed of a mosaic of ecosystems influenced by the interaction between climate, soil and fire [START_REF] Oliveira | The Cerrados of Brazil. Ecology and natural history of a neotropical savanna[END_REF], ranging from the most open ecosystems, such as campo limpo grasslands, to ecosystems that show higher tree density, i.e. cerradão woodlands [START_REF] Coutinho | O conceito de cerrado[END_REF]. The Cerrado has already lost ca. 50% of its area to soybean and maize plantation and to cattle raising [START_REF] Carvalho | The Cerrado into-pieces: habitat fragmentation as a function of landscape use in the savannas of central Brazil[END_REF][START_REF] Fernandes | Ecology and conservation of mountaintop grasslands in Brazil[END_REF], but only 2.85% of its total surface is located in fully protected conservation units [START_REF] Bustamante | Política de clima negligencia o Cerradomais uma vez[END_REF]. The protection of the remaining land therefore appears as a high conservation priority [START_REF] Bitencourt | The worrying future of the endemic flora of a tropical mountain range under climate change[END_REF][START_REF] Mittermeier | Global biodiversity conservation: the critical role of hotspots[END_REF].

In savanna ecosystems, fire is a common endogenous disturbance (sensu [START_REF] Mcintyre | A framework for conceptualizing human effects on landscapes and its relevance to management and research models[END_REF], i.e. disturbances to which the system has been exposed repeatedly through evolutionary time and that can maintain the biodiversity under an appropriate regime [START_REF] Bond | What controls South African vegetationclimate or fire? South African[END_REF][START_REF] Hoffmann | Ecological thresholds at the savanna-forest boundary: how plant traits, resources and fire govern the distribution of tropical biomes[END_REF][START_REF] Mistry | World savannas: ecology and human use[END_REF][START_REF] Ratnam | When is a "forest" a savanna , and why does it matter ?[END_REF][START_REF] Sankaran | Determinants of woody cover in African savannas[END_REF]. In such fire-prone environments, plants species may present adaptive strategies that can improve their resilience capacity (i.e. implying a notion of both resistance and recovery sensu [START_REF] Hodgson | What do you mean, "resilient"?[END_REF] in the face of recurrent fires. These adaptive strategies include belowground meristematic and storage organs, thick corky bark, sclerophyllous leaves, fire resistant seeds, ability to resprout, and specialized flowering and fruiting phenologies [START_REF] Bond | Fire as a global "herbivore": the ecology and evolution of flammable ecosystems[END_REF][START_REF] Miranda | The fire factor[END_REF][START_REF] Oliveira | Ecophysiology of Campos Rupestres Plants[END_REF].

Since the Cerrado flora, like many other savannas, has been assembled along with recurrent fires [START_REF] Oliveira | The Cerrados of Brazil. Ecology and natural history of a neotropical savanna[END_REF][START_REF] Simon | Recent assembly of the Cerrado, a neotropical plant diversity hotspot, by in situ evolution of adaptations to fire[END_REF], it seems essential to include fire in conservation management plans [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF][START_REF] Overbeck | Fine-scale post-fire dynamics in southern Brazilian subtropical grassland[END_REF][START_REF] Ramos-Neto | Lightning fires in a Brazilian savanna national park: rethinking management strategies[END_REF][START_REF] Ribeiro | Uma abordagem histórica do fogo no parque nacional da Serra do Cipó, Minas Gerais -Brasil[END_REF]. This requires the identification of adequate fire regimes (e.g. season of fire, frequency) as variation in fire regimes can induce changes in plant community composition or structure and/or in ecosystem functioning (Gorgone- [START_REF] Barbosa | A relict species restricted to a quartzitic mountain in tropical America: an example of microrefugium?[END_REF][START_REF] Lehmann | Spatio-temporal trends in tree cover of a tropical mesic savanna are driven by landscape disturbance[END_REF]. By removing biomass, fire opens gaps necessary for germination and reduces competition for light and the dominance of few species, allowing the establishment and persistence of numerous other species [START_REF] Fidelis | Population biology and regeneration of forbs and shrubs after fire in Brazilian Campos grasslands[END_REF][START_REF] Grubb | The maintenance of species-richness in plant communities: the importance of the regeneration niche[END_REF][START_REF] Knox | Fire season and intensity affect shrub recruitment in temperate sclerophyllous woodlands[END_REF][START_REF] Overbeck | Fine-scale post-fire dynamics in southern Brazilian subtropical grassland[END_REF]. Fire ecology (i.e. fire effects and fire regimes) of some Cerrado ecosystems has been widely studied [START_REF] Hoffmann | The effects of fire and cover on seedling establishment in a Neotropical Savanna[END_REF][START_REF] Hoffmann | Ecological thresholds at the savanna-forest boundary: how plant traits, resources and fire govern the distribution of tropical biomes[END_REF][START_REF] Miranda | The fire factor[END_REF][START_REF] Mistry | Indigenous fire management in the cerrado of Brazil: the case of the Krahô of Tocantíns[END_REF][START_REF] Oliveras | Effects of fire regimes on herbaceous biomass and nutrient dynamics in the Brazilian savanna[END_REF][START_REF] Ramos-Neto | Lightning fires in a Brazilian savanna national park: rethinking management strategies[END_REF]. However, such precise information is not available for campo rupestre grasslands. Understanding how fire impacts vegetation composition and structure can help to improve environment policies related to fire and thus future actions within reserves and national parks [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF][START_REF] Ribeiro | Uma abordagem histórica do fogo no parque nacional da Serra do Cipó, Minas Gerais -Brasil[END_REF].

Campo rupestre are part of the Cerrado biome and can be defined as a mosaic of herbaceous and savanna vegetations with shrubland on rocky outcrops (Fig. S1). This ecosystem establishes at altitudes above 900 m a.s.l., mainly along the Espinhaço range in eastern Brazil, [START_REF] Silveira | Ecology and evolution of plant diversity in the endangered campo rupestre: a neglected conservation priority[END_REF] and originally covered 66,447 km 2 [START_REF] Bitencourt | The worrying future of the endemic flora of a tropical mountain range under climate change[END_REF][START_REF] Silveira | Ecology and evolution of plant diversity in the endangered campo rupestre: a neglected conservation priority[END_REF]. Campo rupestre are species-rich ecosystems, established on shallow and oligotrophic, quartzite-derived soils with successive periods of water logging during the rainy season and water deficit during the dry season (Le Stradic et al., 2015a;[START_REF] Oliveira | Mineral nutrition of campos rupestres plant species on contrasting nutrientimpoverished soil types[END_REF].

Previous studies have already evaluated fire effects at the population-level for some species [START_REF] Alves | Vegetation strategy of Vellozia crinita (Velloziaceae)[END_REF][START_REF] Conceição | Massive post-fire flowering events in a tropical mountain region of Brazil: high episodic supply of floral resources[END_REF][START_REF] Conceição | Post-fire flowering and fruiting in Vellozia sincorana, a caulescent rosette plant endemic to Northeast Brazil[END_REF][START_REF] Ferreira | Alocação preferencial de recursos e morfologia de órgãos subterrâneos em plantas resistentes ao fogo em vegetação campestre[END_REF][START_REF] Miola | The effect of fire on phenology of Syagrus glaucescens Glaz. ex Becc. (Arecaceae)[END_REF][START_REF] Neves | Revisão sobre os efeitos do fogo em Eriocaulaceae como subsídio para a sua conservação[END_REF][START_REF] Simon | Mimosa splendida Barneby (Mimosoideae, Leguminosae) rediscovered in Central Brazil: preliminary studies for conservation of a rare species[END_REF]. Overall, the effects of fire on campo rupestre grassland plant communities are still poorly understood [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF][START_REF] Ribeiro | Uma abordagem histórica do fogo no parque nacional da Serra do Cipó, Minas Gerais -Brasil[END_REF]. To our knowledge only two studies described the effects of fire on campo rupestre plant communities. One of them, was based on a floristic survey complemented by plant collection and field observation [START_REF] Kolbek | Impacts of cattle, fire and wind in rocky savannas, southeastern Brazil[END_REF] whereas the other one discussed the strategies of regeneration and investigated the composition and structure vegetation in a recently burnt area but did not present pre-fire data [START_REF] Neves | Campo rupestre recém-queimado na Chapada Diamantina, Bahia, Brasil : plantas de rebrota e sementes, com espécies endêmicas na rocha[END_REF]. These studies concluded that vegetation cover and plant family composition in burnt campo rupestre recover rapidly after fire, mainly because many plant species have various adaptations to resprout from underground storage organs after fire.

However, it appears that fire, in combination with grazing, can lead to invasion by an African grass (Melinis minutiflora) (Gorgone- Barbosa et al., 2016[START_REF] Barbosa | A relict species restricted to a quartzitic mountain in tropical America: an example of microrefugium?[END_REF].

In this study, we aim to provide a precise assessment of the effects of fire on vegetation recovery and composition at both the short-and long-terms on the main herbaceous vegetation types: the sandy and stony grasslands. On the short-term, plant community composition was monitored before and two and five months after a wildfire to assess vegetation recovery. We used diachronic analyses of grasslands burnt at various dates to study the effects of fire on vegetation dynamics on the long-term (up to 25 years after the last fire).

Material and methods

Study sites

The study was conducted in a campo rupestre located along and within the National Park of Serra do Cipó, about 100 km northeast of Belo Horizonte, southeastern Brazil (19° 15′-25′S and 43° 30′-40′W). The climate of the area is tropical altitudinal climate with an average annual rainfall of ca. 1622 mm mainly concentrated during the rainy season extending from November to April; the dry season extends from May to October [START_REF] Madeira | Reproductive phenology of sympatric taxa of Chamaecrista ( Leguminosae ) in Serra do Cipo[END_REF]. The annual average temperature is 21.2°C [START_REF] Madeira | Reproductive phenology of sympatric taxa of Chamaecrista ( Leguminosae ) in Serra do Cipo[END_REF]. All study sites are at altitudes between 1100 and 1420 m and 12 km separate the most distant sites. We studied the two dominant herbaceous vegetations of campo rupestre: the sandy grasslands (Sa) and the stony grasslands (St) (Fig. S1). These grasslands are subjected to frequent fires, leading to a mosaic of grassland patches burnt at different dates. According to [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF] the peak frequency of fire return time in the Serra do Cipó National Park is seven years, indicating that the area is moderately susceptible to fire.

Plant community composition and aboveground biomass sampling

In order to obtain accurate data on short-term fire effects on plant communities, we monitored permanent plots in one stony and one sandy grassland site (Table 1), before and after a wildfire which occurred in August 2011 (sites are highlighted by * in Table 1). In each grassland site, vegetation surveys were carried out in ten 1-m² quadrats prior to fire, in February 2009, and twice after the fire, in October 2011 (i.e. two months after fire) and January 2012 (i.e. five months after fire).

To study long-term vegetation dynamics after fire, we sampled 12 stony and 12 sandy grassland sites between January and March 2012. Three replicates sites of four different times since last fire constituted the 12 sites of each vegetation type: (a) burnt less than a year ago, in 2011, (b) burnt 5 years ago, in 2007, (c) burnt 11 years ago, in 2001 and (d) unburnt in the past 25 years, last burn dated at least in 1987 (Table 1). The sites were selected using a map of fire occurrence made from satellite images collected over the past 25 years and made available by the National Park of Serra do Cipó. On each site, ten 1-m² randomly placed quadrats were used to assess the plant community.

For both short-term and long term study, within each quadrat all angiosperm species were identified and their percent cover visually assessed based on the vertical projection of all aerial plant parts [START_REF] Mueller-Dombois | Aims and methods of vegetation ecology[END_REF].

Above-ground plant biomass was assessed in three 0.4 × 0.4 m randomly placed quadrats in each site. All biomass above 5mm of the ground was clipped and bagged separately for each quadrat. Living and dry biomass were separated in the laboratory. Biomass was weighed after oven-drying at 60 °C for 48 h.

Statistical analyses

Statistical analyses were conducted separately for the sandy grasslands (Sa) and the stony grasslands (St) considering the large difference in species composition between the two types of campo rupestre (Le Stradic et al., 2015a). All statistical analyses were performed in software R Version 3.2.1 (R Core Team, 2015), using stats, vegan [START_REF] Oksanen | Vegan: community ecology package[END_REF] and lme4 [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF] packages.

Short-term effect of a fire passage

We ran generalized linear mixed models (glmer in the R package lme4) with a Poisson error distribution, a log-link function and random effect for quadrats [START_REF] Crawley | The R Book[END_REF], to assess variation of species richness before and after fire (prior to fire in February 2009, two months after fire in October 2011, and five months after fire in January 2012). For each grassland type, analyses of variance (ANOVA) for repeated measures and pairwise comparisons with p values adjustments according to Holm correction were used on the data of percentage cover over time, including quadrats as random effect. Percentage cover data were arcsine transformed to meet the assumptions of statistical analyses.

Dissimilarities between vegetation composition before and after fire were assessed using ANOVA-style analysis Adonis. Adonis (i.e. Multivariate ANOVA based on dissimilarities) is an analysis which implements a multivariate analysis of variances using distances matrices, to study the differences in group means and performs permutation tests to inspect the significances of the differences observed [START_REF] Oksanen | Vegan: community ecology package[END_REF]. Adonis function informs whether the groups consistently differ in their community composition. Adonis analyses were performed using Bray-Curtis dissimilarity to calculate distance matrices based on vegetation data (quadrat × species abundance matrices); it ranges from 0 and 1 and tends towards 1 as the dissimilarity between plant communities increases [START_REF] Bray | An ordination of the upland forest communities of southern Wisconsin[END_REF].

Correspondence Analyses (CA) were performed to detect and visualize changes in plant community composition, structure, and recovery for each vegetation type. The two CAs (i.e. one for each vegetation type, with the matrix of species cover in sandy grasslands = 30 observations × 81 species and the matrix of stony grasslands = 30 observations × 69 species) were performed using data before fire (February 2009) and after fire (October 2011 and January 2012).

Long-term fire effects

Effects of time since last fire on mean species richness (number of species/m²) and mean vegetation cover were assessed using Generalized Linear Models (glm) with a Poisson distribution for richness and a Gaussian distribution for vegetation cover. Time since last fire ((a) burnt less than a year ago ( 2011), (b) burnt 5 years ago ( 2007), (c) burnt 11 years ago ( 2001) and (d) unburnt in the past 25 years) was defined as the categorical explanatory variable.

Analyses of variance (ANOVA) and Tukey post-hoc tests were performed to compare biomass (i.e. total and dry biomass) according to time since last fire. Parametric conditions were tested for each grassland type using Shapiro-Wilk test of normality and Bartlett's test for homogeneity of variance. Where necessary, data were log-transformed to meet the assumptions of statistical analyses.

As previously explained, dissimilarities between vegetation composition according to time since last fire were analyzed using ANOVA-style analysis Adonis. We performed Constrained Correspondence Analyses (CCA, i.e. Correspondence Analyses with including a conditioning matrix) to detect and visualize changes in plant community composition and structure with time since last fire, including a conditioning matrix of altitude, the effect of which was removed [START_REF] Oksanen | Multivariate Analysis of Ecological Communities in R : vegan tutorial[END_REF]. The two CCAs (i.e. one for each grassland type, with the matrix of species cover in sandy grasslands = 120 observations × 229 species and the matrix of stony grasslands = 120 observations × 233 species) were thus performed on the vegetation data collected in 2012 to assess differences between sites according to time since last fire (i.e. burnt less than a year ago (2011), burnt 5 years ago, burnt 10 years ago and unburnt in the past 25 years).

Results

Short-term effect of fire

Species richness and vegetation cover recovered quickly after fire. For both vegetation types, species richness showed a clear significant decrease just after fire from more than 30 species/m 2 on average in February 2009 and less than 20 species/m 2 in October 2001 (Fig. S1). However, five months after fire, in January 2012, species richness significantly increased to values close to pre-fire ones (glmer zSa = 59.69 pSa <0.001; glmer zSt = 62.91, pSt <0.001, Fig. 1a, Fig. S1).

Vegetation cover was low two months after fire but increased significantly (FSa = 68.59, pSa <0.001; FSt = 19.88, pSt <0.001, Fig. 1b, Fig. S1) to reached, five months after fire, values similar to pre-disturbance level on the stony grassland and values close to the pre-disturbance level on the sandy grassland (Fig. 1b, Fig. S1).

Five months after fire, vegetation composition and structure of both communities, did not differ from the pre-disturbance state, and returned to be very similar to the pre-disturbance state (Table 2, Fig. 2 & 3, Table S1). No significant difference in terms of composition was found between plots sampled prior to fire, and two and five months after fire (Table 2). The CA revealed that the species composition of each quadrat of both grassland types sampled before fire and five months after fire were very similar (Fig. 2 & 3).

Long-term vegetation dynamics after fire

Species richness remained stable in sandy grasslands over 25 years (glm procedure zSa = 96.27, p=0.9) but decreased with time since last fire on the stony grasslands (glm procedure zSt = 133.04, p < 0.001), with a higher species richness on recently burnt grasslands (i.e. less than 5 years ago, in 2011 and 2007) and lower species richness on grasslands burnt more than 11 years ago (Fig. 4a). Total vegetation cover strongly increased with time since last fire on sandy grasslands (glm procedure FSa = 97.27, p <0.001, Fig. 4b) while it slightly decreased on stony grasslands (glm procedure FSt = 34.76, p <0.001, Fig. 4b).

Species composition and community structure differed according to the date of the last fire (Table 3). For both vegetation types, species composition in the most recently burnt sites (< 1 year) and sites burnt less than 5 years before were more similar (Table 3). In the two vegetation types, main differences in terms of vegetation composition were found between grasslands burnt 11 years prior to the study ( 2001) and grasslands unburnt in the past 25 years (Table 3).

CCA performed on sandy grassland data highlighted that recently burnt areas (2011 and 2007 i.e. burnt less than 5 years ago) were similar in terms of species composition (Fig. 5), and two of the three sites that were unburnt in the past 25 years were grouped together (Fig. 5, see also Table S2). On stony grasslands, only two of the three sites unburnt in the past 25 years showed a divergent composition compared to the other sites (Fig. 6). All other sites, although different in species composition, did not show divergent plant composition according to time since last fire (Fig. 6, see also Table S2).

There was almost twice more biomass in sandy grasslands than on stony grasslands in sites unburnt at last for 25 years (Fig. 7). Total biomass and dry biomass increased significantly with time since the last fire in both vegetation types (Fig. 7). While total and dry biomass increased gradually over time on stony grasslands (F = 6.30, p <0.001 for total biomass, and F = 5.69, p <0.001 for dry biomass; Fig. 7), on sandy grasslands, both the total and dry biomass remained stable for the first 11 years but doubled between 1987 and 2001, due to an important increase of dry biomass (F = 12.74, p <0.001 for total biomass and F = 13.37, p <0.001 for dry biomass; Fig. 7).

Discussion

The sandy and stony grasslands are two different habitat types within campo rupestre ecosystem (Le Stradic et al., 2015a), and both of them recover quickly, c.a. of five months, after wildfire in terms of species richness and community composition, which corroborates previous studies [START_REF] Kolbek | Impacts of cattle, fire and wind in rocky savannas, southeastern Brazil[END_REF][START_REF] Neves | Campo rupestre recém-queimado na Chapada Diamantina, Bahia, Brasil : plantas de rebrota e sementes, com espécies endêmicas na rocha[END_REF]. The high resumption capacity of both plant communities implies that most of the species are able to rapidly regenerate after fire. Vegetation cover on stony grasslands is naturally lower than that on sandy grasslands, because quartzitic rocks overlay a large share of the ground (Le Stradic et al., 2015a), that is why, in stony grasslands, vegetation reaches pre-fire cover values in only five months. On the other hand, vegetation cover is denser on sandy grasslands and, although our results show a delay for vegetation recovery in sandy grasslands, vegetation cover reaches values similar to pre-disturbance quite quickly as well.

Our study shows that fire is not related to an increase in species richness in campo rupestre, and community composition remains very similar before and after fire, contrary to subtropical grasslands in southern Brazil where fire can lead to a short-term increase in species richness, mainly through a reduction in plant competition and an increase in recruitment right after the fire [START_REF] Overbeck | Fine-scale post-fire dynamics in southern Brazilian subtropical grassland[END_REF]. While some species seem to be slightly favored in the early postfire environment, such as Poaceae or Cyperaceae which produce flowers and fruits in the weeks following a fire (personal observation), no post-fire colonization by opportunistic species has been observed. In many fire-prone environments, the recruitment processes of many species are restricted to the first post-fire year, as these processes are stimulated by factors related to fire [START_REF] Bond | Variation in seedling recruitment of Cape Proteaceae after fire[END_REF][START_REF] Bond | Fire and plants[END_REF][START_REF] Hobbs | Fire-related dynamics of a Banksia woodland in south-western western Australia[END_REF][START_REF] Knox | Fire season and intensity affect shrub recruitment in temperate sclerophyllous woodlands[END_REF]. In Australia or South Africa, fire regimes increase seedling emergence of shrubs [START_REF] Bond | Variation in seedling recruitment of Cape Proteaceae after fire[END_REF][START_REF] Bond | Fire survival of Cape Proteaceae -influence of fire season and seed predators[END_REF][START_REF] Hobbs | Fire-related dynamics of a Banksia woodland in south-western western Australia[END_REF][START_REF] Knox | Fire season and intensity affect shrub recruitment in temperate sclerophyllous woodlands[END_REF]. Although no study has dealt so far with recruitment processes after fire in campo rupestre, potential increase in recruitment of some campo rupestre species may not be due to fire-triggered germination [START_REF] Fidelis | To resist or to germinate? The effect of fire on legume seeds in Brazilian subtropical grasslands[END_REF]Le Stradic et al., 2015b) but instead because some species present firestimulated flowering and fruiting [START_REF] Conceição | Post-fire flowering and fruiting in Vellozia sincorana, a caulescent rosette plant endemic to Northeast Brazil[END_REF][START_REF] Fidelis | Does fire induce flowering in Brazilian subtropical grasslands?[END_REF][START_REF] Lamont | Fire-stimulated flowering among resprouters and geophytes in Australia and South Africa[END_REF]Le Stradic et al., 2015b).

In the present case, the regeneration of the original community appears mostly driven by the persistence of resprouters. In fire-prone environments, plant community assemblages contains species that are able 1) to resprout from any plant structure such as rhizomes, root buds, stem buds, etc. and persist via vegetative regeneration, i.e. resprouters, or 2) to thrive in and to recolonize a site through sexual reproduction and to persist at species level by seeds, i.e. seeders [START_REF] Bell | Ecological response syndromes in the flora of southwestern Western Australia: Fire resprouters versus reseeders[END_REF][START_REF] Keeley | Role of fire in regeneration from seed[END_REF]Pausas et al., 2004). Whereas it has been usually neglected in favor of the 'regeneration niche', our results support the idea of the 'persistence niche' i.e. regeneration and persistence in situ is mainly due to species resprouting ability [START_REF] Bond | Ecology of sprouting in woody plants : the persistence niche[END_REF]. Resprouting ability is a key trait for persistence at the population level, enabling species to survive diverse disturbance regimes. It may result in a strong impact on community re-assembly, especially in fire-prone environments [START_REF] Bond | Ecology of sprouting in woody plants : the persistence niche[END_REF][START_REF] Clarke | Resprouting as a key functional trait: how buds, protection and resources drive persistence after fire[END_REF]. Most of the species in sandy and stony grasslands are perennial species that resprout quickly after fire (Le Stradic et al., 2015a), which could explain the limited changes in terms of species richness observed in our plant communities; once established, perennial species do not necessary rely on seed germination to be maintained in the system, and therefore might be less impacted by biomass accumulation and shade.

No significant change in species richness over time was found in sandy grasslands, while only a slight decrease in species richness was observed in stony grasslands unburnt for more than 25 years. A decrease in species richness with time since last fire was expected [START_REF] Bond | Fire as a global "herbivore": the ecology and evolution of flammable ecosystems[END_REF][START_REF] Uys | The effect of different fire regimes on plant diversity in southern African grasslands[END_REF], especially because some plant species with high light requirements should be negatively affected by shade produced by increasing biomass.

Competition generated by some dominant species and the accumulated biomass over time may limit the germination of annual species [START_REF] Overbeck | Fine-scale post-fire dynamics in southern Brazilian subtropical grassland[END_REF], as well as the persistence and growth of some herbaceous campo rupestre species that are not tolerant to shade. Stony grassland species might be more sensitive to competition for light because stony grasslands are naturally more open vegetation, inducing a negative correlation between species richness and biomass production [START_REF] Overbeck | Fine-scale post-fire dynamics in southern Brazilian subtropical grassland[END_REF].

While vegetation composition varied between the different study sites, including between recently burnt areas and unburnt areas, no clear pattern was observed in terms of fire impacts.

It can result from the fact that vegetation composition analysis highlighted first the natural heterogeneity of campo rupestre plant communities (Le Stradic et al., 2015a) rather than the effect of fire on community composition. Campo rupestre are patchy plant communities under the control of local topography, nature of substrate, soil depth and fires [START_REF] Fernandes | Ecology and conservation of mountaintop grasslands in Brazil[END_REF]Le Stradic et al., 2015a;[START_REF] Neves | Campo rupestre recém-queimado na Chapada Diamantina, Bahia, Brasil : plantas de rebrota e sementes, com espécies endêmicas na rocha[END_REF][START_REF] Silveira | Ecology and evolution of plant diversity in the endangered campo rupestre: a neglected conservation priority[END_REF]. Campo rupestre are located on mountaintops and represent isolated islands where species endemism and narrow species endemism are common [START_REF] Alves | Plant species endemism in savanna vegetation on table mountains (Campo Rupestre) in Brazil[END_REF][START_REF] Barbosa | A relict species restricted to a quartzitic mountain in tropical America: an example of microrefugium?[END_REF], leading to very high heterogeneity in plant community composition even at small spatial scale. Studies of longterm fire impacts on vegetation dynamics is therefore challenging. Natural heterogeneity of community composition and fire patchiness hampers greatly to detangle the effects of fire from the geographic localization of the plots. Long-term experimental studies using controlled burnings would be more appropriated to study the impact of fire on plant species composition in campo rupestre.

Similarly to our results, studies have shown that fire occurs due to fine fuel accumulation and many ignition sources [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF]. As stony grasslands naturally present a higher level of bare ground compared to sandy grasslands (Le Stradic et al., 2015a), biomass accumulation in this type of vegetation is lower. Values of total biomass in campo rupestre varied between 200 g.m -2 and 400 g.m -2 , which are similar to those found in campo sujo (i.e. open vegetation physiognomy) [START_REF] Conceição | Biomassa combustível em campo sujo no entorno do Parque Nacional da Chapada Diamantina, Bahia, Brasil[END_REF]. However, without any fire for a long time period (i.e. >25 years), total biomass in sandy grasslands doubles, reaching values above 800 g.m -2 , which are similar to those in wet grasslands in Brazilian savanna without fire occurrence for four years [START_REF] Fidelis | Above-and below-ground biomass and carbon dynamics in Brazilian Cerrado wet grasslands[END_REF]. This implies that fire suppression over a long time period should lead to high biomass accumulation. Throughout the world, prescribed burning policy in fire-prone environments have resulted in a large accumulation of biomass, an increase of fuel load and in severe wildfires [START_REF] Van Wilgen | Fire management and research in the Kruger National Park, with suggestions on the detection of thresholds of potential concern[END_REF]. In campo rupestre, the absence of sustainable fire management will probably lead to biological impoverishment [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF].

Whereas fire should be managed as a necessary conservation tool, prescribed burning is a matter of debate in Brazil [START_REF] Durigan | The need for a consistent fire policy for Cerrado conservation[END_REF][START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF]. Fire can be used to protect sensitive vegetation, some campo rupestre species [START_REF] Conceição | Massive post-fire flowering events in a tropical mountain region of Brazil: high episodic supply of floral resources[END_REF][START_REF] Conceição | Post-fire flowering and fruiting in Vellozia sincorana, a caulescent rosette plant endemic to Northeast Brazil[END_REF] and to maintain community dynamics [START_REF] Oliveras | Effects of fire regimes on herbaceous biomass and nutrient dynamics in the Brazilian savanna[END_REF]. However, fire can also lead to severe damages for some populations in case of inappropriate fire regimes [START_REF] Silveira | Impact of wildfires on the megafaune of Emas National Park, central Brazil[END_REF]. [START_REF] Oliveras | Effects of fire regimes on herbaceous biomass and nutrient dynamics in the Brazilian savanna[END_REF] suggested that an interval of at least 3 to 4 years between each burn can be a sustainable fire regime, allowing the reestablishment plant communities to a pre-burn condition of some Cerrado ecosystems. In the case of campo rupestre, further studies are needed to assess what fire regimes should be applied to maintain biodiversity and ecosystem services and what fire management strategy should be taken to avoid deleterious fires and conserve campo rupestre (but see [START_REF] Figueira | Fire in rupestrian grasslands: plant response and management[END_REF]. In order to guarantee conservation of their biodiversity and ecosystems services that have been severely threatened by fire suppression policy, the definition and implementation of a clear fire management policy is urgently necessary, especially in conservation units of cerrado and associated vegetation as the campo rupestre [START_REF] Durigan | The need for a consistent fire policy for Cerrado conservation[END_REF].

Table 1. Information about study sites including type of vegetation (sandy or stony grasslands), sites name as presented in Fig. 5 & 6, altitude (m), year of the last fire and time since last fire (yr): (<1) burnt less than a year ago, in 2011, (5) burnt 5 years ago in 2007, (11) burnt 11 years ago in 2001 and (>25) unburnt in the past 25 years, with a last burn dated at least in 1987. * indicates the two sites which were followed more precisely before (February, 2009)

/ Sa-K -3 -2 -1 0 1 2 -1 0 1 2 3 CCA1 CA1

Fig. 1 .Fig. 2 .

 12 Fig. 1. Short-term impact of a wildfire on a) species richness (in 1m 2 ) and b) vegetation cover (cover percentage in 1m 2 ) in two types of campo rupestre vegetation at Serra do Cipó (Brazil): the sandy grasslands, represented with light grey bars, and the stony grasslands with dark grey bars. Quadrats were sampled before fire (Feb 2009), two months after fire (Oct 2011) and five months after fire (Jan 2012). Means ± standard errors are shown. Bars with no letter in common are significantly different according to the glmer procedures and pairwise comparisons (p < 0.05)

Fig. 3 .

 3 Fig. 3. Correspondence Analysis (CA) based on vegetation survey in ten 1m 2 permanent plots on stony grassland [30 observations × 69 species] before fire (Feb 2009), two months after fire (Oct 2011) and five months after fire (Jan 2012). For clarity, only the most correlated species are shown. Barycenters of each sampling date are represented.

  Fig. 4. Long-term impact of wildfires on a) species richness (in 1 m 2 ) and b) vegetation cover (cover percentage in 1 m 2 ). Two types of campo rupestre vegetation at Serra do Cipó (Brazil) are represented: the sandy grasslands, with light grey bars, and the stony grasslands with dark grey bars, according to time since last fire are: unburnt in the past 25 years (1987), burnt 11 years ago (2001), burnt 5 years ago (2007) and burnt less than one year (2011). Means ± standard errors are shown. Bars with no letter in common are significantly different according to the glm procedures (p < 0.05).

Fig. 5 .

 5 Fig. 5. Constrained Correspondence Analysis (CCA) based on vegetation survey performed on 120 1-m² vegetation plots on sandy grasslands [120 observations × 229 species] in sites unburnt in the past 25 years (1987), burnt 11 years ago (2001), burnt 5 years ago (2007) and burnt less than one year (2011). For clarity, barycenters of each sampling site are represented on above graph and only the most correlated species are shown on below graph. Abbreviations (i.e. St-A, B, C, D, E, F, G, H, I, J, K) refer to site name (see table 1).

Fig. 6 .

 6 Fig. 6. Constrained Correspondence Analysis (CCA) based on vegetation survey performed on 120 1-m² vegetation plots on stony grasslands [120 observations × 233 species] in sites unburnt in the past 25 years (1987), burnt 11 years ago (2001), burnt 5 years ago (2007) and burnt less than one year (2011). For clarity, barycenters of each sampling site are represented on above graph and only the most correlated species are shown on below graph. Abbreviations (i.e. St-A, B, C, D, E, F, G, H, I, J, K) refer to site name (see table 1).

  ).

Fig. 7 .

 7 Fig. 7. Total plant biomass (light grey) and dry plant biomass (dark grey) in sandy and stony grasslands at Serra do Cipó (Brazil) in sites unburnt in the past 25 years (1987), burnt 11 years ago (2001), burnt 5 years ago (2007) and burnt less than one year (2011). Means ± standard errors are shown. Results of Tukey post-hoc tests performed on total biomass are indicated with letters and were equal than Tukey post-hoc tests performed on dry biomass.

Table 2 :

 2 and after the fire (October 2011 and January 2012). Mean Bray-Curtis dissimilarity based on floristic composition between plots before fire (February 2009) and after fire (October 2011 and January 2012), for the two kinds of vegetations, the sandy and the stony grasslands. Results of Adonis analysis (i.e. difference in group means) based on Bray-Curtis dissimilarity (vegetation data) in February 2009, October 2011 and January 2012).

		Sandy grasslands			Stony grasslands
	Site	Altitude	Year of	Time	Site	Altitude	Year
	name	(m)	the last	since	name	(m)	of the
			fire	last fire			last
				(yr)			fire

Table 3 :

 3 Mean Bray-Curtis dissimilarity based on floristic composition between plots burnt in 2011: burnt less than a year ago, 2007: burnt 5 years ago, 2001: burnt between 11 years ago and 1987: unburnt in the past 25 years, for the two kind of vegetations, the sandy and the stony grasslands. Results of Adonis analysis (i.e. difference in group means) were based on Bray-

	Curtis dissimilarity (vegetation data).
			1987	2001	2007 Adonis -F	p-value
	Sandy	2001	0.83
	grasslands	2007	0.76	0.79	10.32	0.0049 **
		2011	0.71	0.79 0.68
			1987	2001	2007 Adonis -F	p-value
	Stony	2001	0.87
	grasslands	2007	0.85	0.83	10.99	0.0049 **
		2011	0.83	0.84 0.76

Acknowledgments

We thank the National Park of Serra do Cipó for having made their fire map available to us and two anonymous reviewers and Fernando O. A. Silveira for improving greatly the manuscript. This work was carried out with grants from the Conselho Nacional de Pesquisa (CNPq) [CNPq 561883/2010-6], Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), RTP CNRS-CEMAGREF "Ingénierie Ecologique" [Ecological Engineering] SAVER -SAVanna Ecological Restoration, Natural Reserva Vellozia and the Ministère Français des affaires étrangères et européennes (EGIDE)[EGIDE 2009/657176K].

Appendix A. Supplementary material Supplementary Table S1: List of plant species occurring on sandy grassland (Sa-C) and stony grassland (St-C) surveyed to study short-term fire effects on plant communities, including family, species name, plant form and the presence of each species for the three sample date: before fire (Feb 2009), two months after fire (Oct 2011) and five months after fire (Jan 2012). Supplementary Table S2: List of plant species occurring on sandy grasslands and stony grassland surveyed to study long-term fire effects on plant communities, including family, species name, plant form and the presence of each species in sandy or stony grasslands.

Supplementary Figure S1: a


Jianshu Yang

Jie Deng

Cedic Troadec

Thierry Ondarçuhu

Christian Joachim

Solid-state SiO 2 nano-gears AFM tip manipulation on HOPG

On a native graphite surface, 15 nm thick solid state nanogears are nanofabricated with an outer diameter down to 30 nm and 6 teeth. In a tapping mode, the nanogears are manipulated one at a time by the tip of an atomic force microscope using the sample stage displacements for the manipulation to construct mechanism like a long nanogears train. In absence of a central shaft per nanogear, gearing between nanogears is limited to a few 1/12 turns for 6 teeth. For step height below 3.0 nm, nanogears are manipulated up and down native graphite surface step edges.

When the step is higher than 3 nm, a rack and pinion mechanism was constructed along a graphite step edge with a 90 nm nanogear pinion.

Introduction

Solid state gears are widely used in various mechanical machineries [START_REF] Uicker | Theory of Machines and Mechanisms[END_REF]. They are currently micro-fabricated down to the micron scale [START_REF] Garcia | [END_REF]3,4]. Following the work by Y.J. Yun and coworkers on solid state gear miniaturization 5, 6], we have miniaturized solid state gears by reporting the nanofabrication of amorphous SiO2 nanogears down to 60 nm in diameter with a 30 nm thickness [7]. Those diameter and thickness are not compatible with the prospect of interconnecting mechanically a solid state nanogear with a single molecule-gear or a single molecule-motor [8,10].

In this paper, we present the nanofabrication of solid state SiO2 nanogears down to 30 nm in diameter with a 15 nm thickness on flat highly ordered pyrolytic graphite (HOPG) terraces .

Aside from this diameter reduction, one important aspect of this new process is the reduction of the nanogear thickness down to 15 nm with also the objective to explore the limit of solid state classical mechanics [9]. In a first section, our new process is presented approaching the nanogear thickness limit by a systematic exploration of the resist deposition conditions. In section 2, atomic force microscope (AFM) tip manipulations of the nanogears are presented in air with the recording of the characteristics AFM signals during a manipulation sequence (tapping mode oscillations amplitude, cantilever deflection). In section 3, some nanogears mechanics are presented. On the HOPG surface, we demonstrate that a native 10 graphene layers step edge height is required to play the role of a nanoscale rack for a 15 nm thick nanogear.

Further developments of this work are presented in conclusion.

The nanogear fabrication process

In our initial hydrogen silsesquioxane (HSQ) nanogears fabrication process on a silicon surface [7], an intermediate Au layer was introduced between the HSQ layer and the silicon surface.

This layer was important to chemically release the nanogears from the surface. Without this sacrificial layer, it was extremely difficult to manipulate a single nanogear using a scanning tunneling microscope (STM) or an AFM tip, even after hardening the HSQ gears by annealing the sample [7]. For the new process presented in this section, an HOPG surface was selected with the objective to avoid those friction and anchoring effects. In effect and on HOPG, there is no need for an intermediate layer between HSQ and the HOPG surface, making the nanofabrication process simple and clean in view of future ultra-high vacuum (UHV) applications. In the following, the nanogears are fabricated out of a negative tone e-beam resist.

The HSQ resist is directly spin coated on the HOPG surface. Our new and very simple process flow is presented in Fig. 1a. A freshly peeled-off HOPG surface was spin-coated with an HSQ resist (XR1541 from Dow Corning, 4000 rotation per minute) and baked at 90 C for 5 min.

Then, the HSQ / HOPG sample was e-beam exposed at a line dose of 0.48 nC/cm with a commercial ELS-7000 (Elionix) e-beam nanolithography system using an e-beam acceleration voltage of 100 kV and a current intensity of 50 pA. The exposed samples were developed in MF CD-26 (Shipley) for 60 seconds, then washed out with 1:9 CD-26 to de-ionized (DI) water for 10 seconds and finally rinsed in DI water during 30 seconds.

The fabricated HSQ nanogears were directly observed on the HOPG surface and first imaged using a high resolution scanning electron microscope (SEM). Fig. 1b shows a regular array of HSQ nanogears with an outer diameter ranging from 150 nm to 30 nm. The Fig. 2a SEM zoomed image is presenting two lines of five nanogears. On the ELS-7000, the diameter of the nanogear and the number of teeth was varied. For a 15 nm minimum thickness (see the discussion below), the smallest 30 nm in diameter nanogears obtained have 6 teeth and each tooth width is about 8 nm, the minimum observed with this process. Tapping mode AFM images of the same HOPG surface location confirm the 15 nm  1 nm thickness of the HSQ nanogears (see the profile line Fig. 2b). The smallest nanogears are often found not aligned on HOPG as compared with the e-beam mask indicating a weak surface interaction between the nanogears and the HOPG surface. In an AFM tapping mode and in SEM images, native steps of different heights (mostly 1 nm / 3 ML to 3.3 nm / 10 ML) can normally be found on the freshly cleaved HOPG surface depending on how the HOPG was peeled off. As presented in Fig. 2, our process preserves those steps edge because the end cleaning of the process was very well optimized and that AFM scanning in a tapping mode was used, avoiding any eventual manipulation and accumulation of surface residues. This was also confirmed by SEM images after the process.

It was also important to explore the HSQ resist thickness limits of our process in view of the possible use of solid state nanogears to transfer motion from / to a molecule-gear whose chemical structure height will not be larger than a few nm [10]. For this purpose, a defined nanogear e-beam pattern with various nanogear diameters from 30 nm to 150 nm was used with different HSQ layer thicknesses. This thickness was varied from 15 to 90 nm by changing only the HSQ concentration. With a 1% HSQ solution, a uniform 15 nm thick HSQ film can still been obtained on HOPG. Concentrations lower than 1% results in non-uniform HSQ films with many pin-holes indicating a de-wetting of HSQ on the HOPG surface. Following this procedure, the HSQ nanogears were SEM imaged and their sizes systematically measured. Fig. 3a is presenting the variation of the smallest size nanogears obtained (outer diameter) as a functio n of the HSQ resist layer thickness. For a given HSQ layer thickness, the smallest nanogears were selected when at least a 1:1 ratio is reached between the teeth length and the teeth width.

Below this 1:1 ratio, deformed or incomplete nanogears were not considered for Fig. 3. With a 15 nm thick HSQ layer, the smallest solid nanogears have an outer diameter of 30 nm with 6

teeth (each about 8 nm in width) as illustrated in Fig. 3b. For example, with an HSQ layer thicker than 50 nm, the smallest nanogear was about 60 nm in outer diameter.

Single solid state nanogear AFM tip manipulation

The small interaction between a nanogear and the HOPG surface opens the possibility to manipulate a nanogear one at a time and with an AFM tip in air. To reduce the piezo delay effects and the compensation which occurs when using single tube scanners to scan the surface over large distances, the nanogear manipulations were performed by scanning the sample stage instead of scanning the AFM tip. For this purpose, the HOPG sample was mounted on a XYZ piezo-controlled nano-positioning table (positioning error Δ < 2 nm) inserted under a Dimension 3000 Bruker AFM head [11]. In this configuration, the AFM tip is used to push on the selected nanogear by moving the sample stage. Fig. 4 presents a 90 nm nanogear manipulated on an HOPG terrace. Here, a fresh nanogear (previously un-manipulated) was firstly selected using a tapping mode AFM image recorded in standard conditions, the other imaged nanogears being used as reference during the manipulation sequence. For a manipulation in a pushing mode, the tip apex was positioned about 60 nm away from the selected nanogear (marked by a cross on Fig. 4a). The manipulation starts by disengaging the AFM feedback loop and by a 10 nm tip apex approach towards the surface. Then, the piezo table is moved in the X direction for about 80 nm. In Fig. 4a, the initial set point voltage of the oscillation amplitude is 1.22 V (corresponding to a 27.3 nm oscillation amplitude). After pushing on the nanogear, the AFM feedback loop is turned on in normal tapping mode imaging conditions and the table brought back to its initial position. In Fig. 4b, the recorded AFM image demonstrates that the selected nanogear had been moved by 40 nm to the left with a very small apparent clockwise rotation. After this first manipulation, Fig. 4c reports a second identica l manipulation sequence also in a pushing mode over 50 nm with now no apparent rotation during the manipulation.

For nanogears between 57 nm and 153 nm in diameter (SEM measured) and following the above manipulation protocol, hundreds of single nanogear manipulations were performed demonstrating a 70% success of manipulating a nanogear by moving the sample stage. Below a diameter of 57 nm, AFM tip manipulations were not reproducible. This may be due to the tip apex used, not sharp enough in this case. Another observation is that after a first manipula tio n of a 90 nm fresh nanogear, the following manipulations require to start with only a 6 nm initia l tip apex vertical displacement. This implies that after the nanofabrication process, the fresh 90 nm nanogears are still anchored on the HOPG surface.

The manipulation sequence from images Fig. 4a to 4b was analyzed by recording in time and during the manipulation sequence, the variations of the AFM cantilever oscillations amplitude and deflection (Fig. 4d and4e). Instead of converting the timeline in sample stage displaceme nts, the experimental timing was kept for the abscise in Fig. 4d and4e showing also the manipulation signal during a short period of time before and after the starting of the manipulation sequence itself.

The solid line signal Fig. 4d is giving the time dependent variation of the cantilever oscilla tio n amplitude during the first Fig. 4a to Fig. 4b manipulation of a fresh nanogear. From times T1 to T2, the tip scanning was stopped and the AFM feedback loop disengaged. As expected, the chosen 1.22 V (23.7 nm) oscillation amplitude voltage remains constant during this time interval. At time T2, this amplitude decreases abruptly to 0.6 V (13.4 nm) because the tip apex was approached to the surface by 10 nm. After this approach, the piezo table was moved 80 nm to the right for the tip apex to approach the nanogear by its edge and between two teeth. The amplitude remains at 0.6 V (13.4 nm) until time T3 where the nanogears and the tip apex start to interact. From T3 to T4, this interaction gradually increases up to time T4 where the AFM tip apex is touching the nanogear edge and starts to push resulting in a zero value of the oscillation amplitude signal. Then, the piezo table was moved to the right up to time T5. Here, the oscillation amplitude signal remains at zero volts for a few seconds more indicating that the nanogear and the tip apex are still in interaction long after stopping the piezo table. From Fig.

4b to 4c, the same time dependent signal was recorded during a second manipulation of the same nanogear. The starting oscillation amplitude is slightly higher in voltage (1.62 V). Notice that during this manipulation sequence, the time dependent signal is similar to the one recorded during the first manipulation.

To get more insights on the manipulation mechanisms, the cantilever deflection signal was also . From T1 to T2, the cantilever deflection is almost constant since it is free to oscillate. The same deflection is also observed from T3 to T4 since the 10 nm vertical tip approach to the surface was just chosen to be in the range of the nanogear thickness. At time T4, the tip apex is touching the nanogear edge. Here, the deflection signal is characteristics of a quasi-stick-slip motion behavior during the follow up manipulation [12,13].

With an intensity of 2 mV, the first large maximum (larger than the other up and down quasiregular deflection features) indicates that the static friction is higher than the kinetic one. The cantilever alternatively bend up and down during this manipulation sequence. At time T5, the piezo table was stopped leading to a cancellation of the large oscillations. However, the average deflection does not return to its T1 value indicating that the cantilever is still bent down and the tip apex still interacting with the nanogear.

For the second manipulation from Fig. 4b to 4c, the time dependent dash curve deflection signal in Fig. 4e is similar to the first one with a notable small intensity of the first large initia l deflection maximum at the beginning of the manipulation. This is indicative of a smaller static friction for this second manipulation as compared to the first one which was performed on a fresh nanogear. Notice as already discussed that it is difficult to manipulate a fresh nanogear without a little rotation along the manipulation pathway. After the first manipulation and by selecting a good tip apex location around the nanogear, a translation without rotation is possible.

This confirms that the freshly nanofabricated nanogears are anchored on the HOPG surface.

This may come from the nanofabrication process residues like non-polymerized HSQ remaining or because the HSQ polymerization step leads to an anchoring of the nanogears on HOPG surface native defects [14,15,16].

The two other possible sliding and pulling modes of manipulation have also been explored. A sliding mode is possible by positioning the tip apex on top of a nanogear and exerting a gentle pressure before moving the piezo table. Generally, this leads to very irregular stick-slip motions of the nanogear and consequently to a non-precise positioning. After approaching the tip apex to a nanogear as performed in Fig. 4 but now by the left, it was not possible to pull the nanogear by moving the piezo table.

Series of single nanogear manipulations were also performed for different tip apex heights while the tip apex was step by step approached towards the HOPG surface. The recording of this kind of manipulation signals is well known for single atom or molecule STM manipulatio ns [17,18]. As presented in Fig. 5b and5c, the oscillation amplitude of the cantilever and its deflection were simultaneously recorded as a function of the piezo table displacement. On those figures, z = 0 corresponds to the reference tip apex height before disabling the AFM feedback loop (corresponding here to an 18.8 nm oscillation amplitude i.e. a 0.84 V set point voltage).

The relative position of the nanogear before and after the manipulation is given in Fig. 5a. The 300 nm lateral displacement of the piezo table was the same for all the z value selected. Notice that the piezo table was moved only in X direction to keep the deflection of the laser beam only in one direction to get the maximum sensitivity for the output signal.

When the tip apex is far away from the surface (z = -8 nm to z = -4 nm), the Fig. 5b oscilla tio n amplitude along a line scan is corresponding to the awaited variation in a constant height mode.

It decreases when the tip approaches and passes over the nanogear [19]. The corresponding Fig. 5c deflection signal does not show up any changes during a given scan, indicating that the tip apex was not in direct contact with the nanogear and that the cantilever was not bent. As the tip is brought closer to the sample surface (z = -2 nm), the oscillation amplitude decreases slightly more as the tip passes over the nanogear. The deflection signal presents a small dip corresponding to an interaction increase with the top of the nanogear. This deflection can be interpreted as a small bending down of the cantilever due to the attractive interaction between the tip apex and the nanogear. In air, such an attractive interaction generally originates from surface contamination and from the thin water film present on the surface.

While approaching further the tip apex to the HOPG surface and reaching now z = 0, a saturation of the oscillation amplitude appears when the tip apex is in contact with the nanogear.

Consequently, a bump now appears in the cantilever deflection signal during the piezo table scan since the cantilever is now bending up [20]. When the tip apex and the HOPG surface are getting even closer (z = +2 nm to z = +4 nm), the amplitude oscillation saturation extends further during the piezo table scan. The deflection bump increases even more and the tip apex is pushing on the edge of the nanogear. The nanogear manipulation threshold is reached for z = +6 nm. Here, the oscillation amplitude signal fully saturates while scanning with the piezo table.

After this manipulation at z = +6 nm, the AFM image was recorded by returning to a normal tapping imaging mode showing that the nanogear has been successfully moved laterally by less than 300 nm.

Figure 6 is also presenting the manipulation in a pushing mode of a 90 nm diameter nanogear (15 nm in thickness) up and down a native HOPG step of about 2.7 nm height i.e. 8 graphene layers in height (1 layer = 0.335 nm). This manipulation was accompanied by a rotation along the manipulation pathway. Notice as presented in Fig. 6b that the manipulation process using the piezo table is precise enough to position the nanogear well across the step edge. Without any apparent damage of the step edge and of the nanogear, one can manipulate the nanogear up and down this step edge indicating a rather smooth atomic scale border of the HOPG 8 layers step edge used. A 10 ML step edge will not allow such a manipulation and is used in the next section to construct a rack and pinion mechanism.

Simple mechanical experiments with a few solid state nanogears

With our actual process, below 100 nm in diameter and in absence of a central shaft, a nanogear does not have enough friction on the HOPG surface and in air to rotate around its center [7].

Hence and with their teeth intertwined, a combination of two 90 nm in diameter nanogears was used to assemble by AFM manipulation in a pushing mode a circular rack and pinion device.

By using a very short manipulation sequence, one of the 2 nanogear was pushed and then rotates around the second one. Generally, such a manipulation leads to only a few 1/12 of a turn for a 6 teeth nanogear (see the sequence from Fig. 7c to 7d). A further push on a tooth drives the manipulated nanogear away from the supposed fixed one and leads to a set of entangled nanogears which can only be manipulated together with no control of their relative orientatio n.

To demonstrate how below 100 nm in diameter, a nanogear can be step by step rotate on the HOPG surface, we have used an HOPG native step edge to play the role of a rack in a rack and pinion mechanism. Using the Fig. 6 experimental results, we have first determined the tip apex to surface distance and the minimum step height to avoid any jump of the manipulated nanogear over the step edge. The Fig. 7e to Fig. 7i tapping mode AFM images correspond to a manipulation sequence of a 90 nm in diameter nanogear with 6 teeth step by step rotated along a 3.1  0.2 nm (10 ML) HOPG step edge playing here the role of a nano-rack. The nanogear was first brought in contact with the step edge to ensure a good interaction with one of its teeth (Fig. 7e). The AFM tip apex was positioned in contact with the tooth opposite to the one in interaction with the step edge. After approaching the AFM tip apex 12 nm towards the surface, the piezo table was moved as discussed in section 3 in such a way that the tip apex is pushing on a tooth in a direction parallel to the step edge. By pushing step by step on the nanogear teeth one after the other and in a direction parallel to the step edge, a rotation of the nanogear was observed along this step edge similar to a standard solid state gear rotating along its rack in a rack and pinion mechanical device. From Fig. 7e Furthermore, the 42 nm very long motion of the nanogear center from Fig. 7g to Fig. 7h also indicates a slipping motion probably due to a non-adapted pushing strength of the tip apex as a function of the step edge corrugation. In the prospect of a motion transmission to/from a solid state nanogear to a molecule-gears, the 3.1 ± 0.2 nm native corrugation of the HOPG step edge used is intermediate between the 2.0 nm height of a molecule-motor [8] and the actual 15 nm thickness of the solid state nanogears manipulated here.

Conclusion

A nanofabrication process was presented on an HOPG surface to produce a large number of solid state nanogears. The complete optimization of the nanofabrication process leads to the fabrication of 15 nm thick nanogears with a diameter down to 30 nm. Solid state nanogears down to 57 nm in diameter can be reproducibly manipulated in air by the tip apex of an AFM in a pushing mode. Native HOPG surface steps of at least 3.1  0.2 nm in height can be used as a nanorack for those nanogears showing a nice gearing effect along the step edge. This points out that a minimum 3.1 ± 0.2 nm in height graphene nanogears will be able to gear with the 15 nm thick solid state nanogears studied here opening the way for a transmission of rotation between a molecule-gear and a solid state nanogears. Our nano-mechanics is still missing a technology to fabricate the nano-shaft, an important problem under exploration together with the compatibility of our nanofabrication process with nano-mechanical experiments in UHV conditions to be compatible with the environment of single molecule manipulations. From (e) to (i), the nanogear marked by the white arrow was anchored down to a 3.1 nm height native HOPG surface step edge and then step by step rotated clockwise along this step edge. From (e) to (f): one tooth is interacting with the step edge and then the tip apex displaced the nanogear center by 30 nm equivalent to about 1/12 turn. As a consequence, 2 teeth are now interacting with the step edge in (f). A 29 nm displacement was obtained from (f) to (g) with a consequent 27 clockwise rotation, a 42 nm displacement with a 32 clockwise rotation from (g) to (h) and a 20 nm displacement with a 26 clockwise rotation from (h) to (i). The short dash lines direct the nanogear orientation starting from the perpendicular to the step edge orientatio n in (e). The AFM images (e) -(i) recorded at a 1.22 V oscillation amplitude set point.

Figure captions

  recorded with a voltage sensitivity of 1 mV per 0.165 nm of deflection. The time dependent solid line signal Fig. 4e corresponds to the first manipulation sequence from Fig 4a to Fig. 4b and the dashed line to the second one from Fig. 4b to Fig. 4c. The T1, T2, T3, T4 and T5 time markers are positioned following Fig. 4d

  to Fig. 7i, 4 step by step rotations are presented in clockwise direction. For a rigid body, the 𝜃 rotation angle (in degrees) of a disk of radius R on its support leads to a displacement 𝑑 𝑐 = 2𝜋 360 𝑅𝜃 of its center of rotation. During the 1/12 turn of the 90 nm nanogear from Fig. 7e to Fig. 7f, its rotation center moved by 𝑑 𝑐 = 30 nm  3 nm indicating an effective nanogear rotation of 𝜃 = 38°  2° during its displacement. The displacement of the nanogear center from Fig. 7f to 7g is 𝑑 𝑐 = 29 nm  3 nm, from Fig. 7g to 7h 𝑑 𝑐 = 42 nm  3 nm and from Fig. 7h to Fig. 7i, 𝑑 𝑐 = 20 nm  3 nm. The corresponding rotation angles are respective ly 𝜃 = 27  2°, 𝜃 = 32  2° and 𝜃 = 26  2° i.e. not exactly 1/12 of a turn per step. Around the expected 30° (1/12 of a turn according to the AFM images), the dispersion in 𝜃 is caused by the irregularity of the atomic scale structure of the step edge along the manipulation direction and also by the variations of the teeth length relative to the geometrical center of the nanogear .

Figure 1 .

 1 Figure 1. (a) The process flow for the fabrication of SiO2 nanogears down to a 30 nm outer diameter with 6 teeth. A: the initial graphite substrate with its ultra-thin spin-coated HSQ layer. B: the e-beam lithography step where the nanogear size and shape are electronically exposed by scanning. C: after the HSQ processing (annealing, development), the amorphous SiO2 nanogears remain on the graphite surface. (b) An SEM image of a large series of fabricated nanogears. Each set consists of 20 lines of nanogears. Per line, there are one 153 nm in diameter with 8 teeth and 5 different in diameter nanogears down to 30 nm with 6 teeth. Many nanogears below 57 nm in diameter are not respecting the e-beam pattern alignment.

Figure 2 .

 2 Figure 2. The detail SEM and tapping mode AFM images of 2 nanogear lines fabricated on the HOPG surface. (a) An SEM image where the largest nanogears is 153 nm in diameter with 8 teeth, and the other 4 nanogears per line with 6 teeth are 110 nm, 90 nm, 74 nm and 57 nm in diameter respectively. (b) A tapping mode AFM image of the same two lines recorded with an oscillation amplitude set point of 0.47 V and an oscillation frequency of 321.6 kHz. The downright largest nanogear seats on 3 native HOPG surface steps. (c) A scan line (solid line) crossing the center of the nanogear. The two higher steps underneath (marked step 2 and step 3) are still distinguished. The lateral dash line crossing over the 3 steps act as a reference scan profile of the HOPG surface for this peculiar step profile. Here, the 3 native steps (marked step 1, step 2 and step 3) are 1 nm, 3.1 nm, and 5.6 nm in height respectively.

Figure 3 .Figure 4 .

 34 Figure 3. (a) The minimum in diameter fabricated nanogear as a function of the specific HSQ layer thickness and dilution used in the process. From (b) to (f): the corresponding SEM imageswith diameter (b) 30 nm for 15 nm HSQ, (c) 48 nm for 30 nm HSQ, (d) 58 nm for 50 nm HSQ, (e) 60 nm for 60 nm HSQ and (f) 60 nm for a 90 nm thick HSQ layer. To expect to go down in diameter limit, a new graph will have to be plotted for another resist (if it exists) for a new dewetting limit

Figure 5 .

 5 Figure 5. Variations of the cantilever oscillation amplitude and deflection for different tipsurface distances while moving the piezo table instead of scanning the AFM tip. Tapping AFM images (a) before and (b) after the nanogear manipulation. The dash line in (a) is the trace of the tip scanning over the nanogear. The crosses mark the tip positions before (left) and after (right) the line scans. The white dotted star shows the nanogear location before the manipulatio n. (c) The cantilever oscillation amplitude recorded during the piezo table motion at the differe nt tip-surface distance. z = 0 is the reference tip-surface distance at a 0.84 V oscillation amplitude voltage set point (18.8 nm oscillation amplitude). The first saturation at z = -2 nm is characteristic of a tip apex to nanogear contact. (d) The corresponding cantilever deflectio n recorded during the same piezo table motion.

Figure 6 .

 6 Figure 6. A series of tapping mode AFM images to follow the step by step manipulation of a nanogear across a 3.1 nm height HOPG surface step (oscillation amplitude voltage set point: 0.65 V; oscillation frequency: 321.6 kHz). (a) to (c) the nanogear was manipulated down the step. (d) to (f) the nanogear was manipulated up the same step. The crosses indicate the tip apex positions before the manipulation and the arrows show the manipulation directions opposite to the directions of the piezo table displacements.

Figure 7 .

 7 Figure 7. Two series of a single nanogear rotation. From (a) to (d), the top nanogear marked by a white arrow was pushed towards the down nanogear and step by step pushed around it.

From (b)

  to (c), a first 1/12 (30) of a turn was obtained with the down nanogear preserving its position. From (c) to (d), 1/6 of a turn was obtained for the top nanogear. At the same time, the down nanogear moved down by 42 nm and turn in counterclockwise direction by 21. By pushing again on the top nanogear, both nanogears move following the (c) to (d) sequence before forming one single object (AFM images recorded at a 0.64 V oscillation amplitude set point).

Acknowledgement:

We like to thank the MANA-NIMS project and the A*STAR VIP Atom Tech program for the opportunity to explore single solid state nanogear mechanics.


P A Albouy

S Deville

A Fulkar

K Hakouk

M Impéror-Clerc

M Klotz

Q Liu

M Marcellini

J Perez

Freezing-induced self-assembly of amphiphilic molecules

The self-assembly of amphiphilic molecules usually takes place in a liquid phase, near room temperature. Here, using small angle X-ray scattering (SAXS) experiments performed in real time, we show that freezing of aqueous solutions of copolymer amphiphilic molecules can induce self-assembly below 0°C.

Self-assembly is the process in which objects spontaneously arrange themselves into ordered phases through weak interactions and has emerged as a central notion to understand the organization of matter at the nanoscale. In particular, self-assembly of amphiphilic molecules plays a central role in soft matter, biology, and numerous technological applications. Self-assembly of lipids, surfactants, copolymers, liquid-crystalline molecules, or colloids can be defined as the equivalent of the crystallization phenomenon for atoms or molecules, but at a larger length scale and for objects interacting through weak interactions and not strong ones (like covalent or ionic ones). The story of self-assembly started in the field of biological membranes when it was discovered that lipids can spontaneously form ordered phases, ranging from a simple 1D stack of lipid bilayers in an aqueous medium (lamellar phases) to much more complex 3D periodic architectures such as cubic phases. 1 Cubic phases are now used, for instance, for crystallization of membrane proteins. 2 The field of liquid crystals is certainly a major scientific area based on self-assembly, with many successful technological applications. For example liquid crystal displays (LCD) are based on the self-assembly of liquidcrystalline molecules into corresponding liquid-crystal phases. Such phases exhibit strongly anisotropic properties, used in the devices, because of the specific symmetries of their self-assembled architectures. Self-assembly has also become an inspiring path for bottom-up approaches 3 to obtain many different nano-structured materials, hybrid materials, mesoporous materials, nanoparticle assemblies and so on.

Self-assembly in an aqueous medium is specifically interesting for many practical and biological implications. 4 Here, we report the self-assembly of a copolymer that can be achieved by freezing its aqueous solution below its freezing point. This process is fast, easy to control, and is a new way to induce self-assembly of an amphiphilic molecule. We believe this approach is independent of the copolymer used so far, and could thus be applied to any other type of amphiphilic molecules or building blocks known to organize via self-assembly in a solvent, provided that the freezing point of the solvent is within an easily accessible temperature range at atmospheric pressure.

Using in situ synchrotron Small Angle X-ray Scattering (SAXS), we followed the selfassembly during freezing of aqueous solutions of a commercial block-copolymer composed of ethylene oxide (EO) and propylene oxide (PO) blocks, named P123 (EO20PO70EO20), in the concentration range 5-13 wt% (see Methods section). In these solutions, a phase transition occurs at the critical micellar temperature (CMT), from individual P123 molecules called unimers (below the CMT) to aggregates of P123 molecules called micelles (above the CMT). The value of the CMT decreases with increasing copolymer concentration, but it is always higher than 10 °C for the concentration range investigated here (Fig. S1, ESI †). 5 Prior to freezing, the solution was initially cooled down to 5 °C and it was checked by SAXS that no micelles were present Figure 1: Self-assembly during freezing: evolution of temperature, concentration, and SAXS patterns. Sample concentration of 13 wt%. (A) Evolution of the temperature at the bottom of the sample. The peak at 8 min corresponds to the latent heat release when the first ice crystals are formed. The points 1 to 3 correspond respectively to the nucleation of ice (1), the time when the ice crystals reach the observation window (2) and the appearance of a liquid structure factor that indicates the presence of micelles (3). (B) Increase of the copolymer concentration due to ice formation. (C) Time-lapse evolution of SAXS patterns showing the self-assembly. The following sequence is observed: a concentrated liquid of micelles (structure factor signal) (3), followed by a hcp phase of spherical micelles (4), and a 2D hexagonal phase of cylindrical micelles (5). in solution, confirming that all the P123 molecules were solubilized as unimers. The bottom of the sample was then cooled below 0°C so as to induce an upward moving freezing front into the sample. Freezing at the bottom starts after 8 minutes as revealed by the temperature increase due to the latent heat release (point 1 in Fig. 1). The subsequent displacement of the freezing front was monitored by a video camera (Fig. S2,ESI †). The transmission of the incoming X-rays beam (Fig. S3, ESI †) and SAXS patterns (Fig. 1C) were simultaneously time-recorded at 10 mm above the sample bottom. The growth of vertical needle-like ice crystals at this position was detected by the appearance of narrow horizontal strikes on the SAXS patterns close to the beamstop attributed to the reflection of the X-ray beam on the crystallites surface (point 2 in Fig. 1, time = 10 min).

The P123 molecules are expelled from the growing ice crystals, effectively increasing the P123 copolymer concentration in the remaining liquid phase. The first pattern in Fig. 1C, at 11.5 minutes, shows two different signals: the horizontal narrow strikes (in red, close to the beamstop) linked to the presence of ice crystals, and a broad ring (green/yellow) more intense along the vertical direction. This ring is a background signal due to the extruded plastic sample holder. The extrusion induces a preferential orientation of the polymer chains in the sample holder that results in an anisotropic SAXS signal. These two signals are therefore present on all patterns afterwards. No specific signal of micelles is present, so we assume that this liquid phase is a mixture of P123 and the part of the water molecules which are still in a liquid state. The actual concentration of the P123 molecules (Fig. 1B) can be derived from the transmission of the sample, by comparison with the transmission of a pure water sample during freezing (Fig. S3, ESI †). The concentration effect of the growing ice-crystals induces a very fast self-assembly process, as revealed by the evolution of the SAXS patterns (Fig. 1C): at 12 minutes, a narrower ring (in green) is detected, and is attributed to the main peak of the structure factor of P123 micellar aggregates, revealing that such aggregates form rapidly within just 30 s.

Several very narrow Bragg peaks are then detected, revealing the ordering of the micelles into three dimensional periodic lattices. The positions of the peaks can be unambiguously attributed to two successive periodic phases (Tables S4 andS5, ESI †). First, a transient phase is observed that consists in a hexagonally close packed (hcp) phase of spherical micellar aggregates (space-group P63/mmc).6 Then, a 2D hexagonal packing of cylindrical micelles is stabilized until the end of the freezing experiment (spacegroup P6/mmm). After 30 minutes, the sample was entirely frozen. SAXS was then carried out along the vertical direction (Fig. 2), which corresponds to the growth direction of the ice-front. The 2D hexagonal phase is always present, except at the very top of the sample where the hcp phase is stabilized in a narrow region (Fig. 2C). An important observation is that both phases grow with a preferred orientation. All the domains of the hcp phase have their c-axis perpendicular to the growth direction, as can be deduced from the SAXS pattern (Fig. 2B) which exhibits modulation along the scattering rings. The domains of the 2D-hexagonal phase are preferentially oriented with the a* direction and the cylinder axis perpendicular to the growth direction, as shown by the 6-fold symmetry modulations of the intensity along the 10 scattering ring (Fig. 2A). These preferential orientations could be related to the growth habit displayed by the ice front, which consists of ice needles oriented along the temperature gradient direction (Fig. 2). It is known that the denser planes for a given structure have a tendency to lie parallel to the deposition surface, because the interfacial energy is minimized. [START_REF] Albouy | Comprehensive Guide for Mesoporous Materials, Synthesis and Characterization[END_REF] In the present case, such planes are the (001) planes for hcp and (10) planes for 2D-hexagonal. Furthermore, the micellar cylinders are lying parallel to the growth front, and it may indicate that a horizontal interface is also inducing orientation during the freezing process. Interestingly, these two types of periodic phases (hcp and 2D-hexagonal) have already been observed in the phase diagram of P123 in water, but in a range near room temperature and well-above 0 °C. [START_REF] Wanka | [END_REF]9 To our knowledge, a complete phase diagram of P123 in water below room temperature is not available in the literature. The concentration range for which they are observed in such phase diagrams is in good agreement with the observed sequence during freezing, with the hcp phase around 50 wt% and the 2Dhexagonal phase at higher concentrations (Fig. 3). From our experiments, we can conclude that these organized phases can exist at temperatures below the freezing point with no major modifications of their structures. At this point, we cannot conclude whether these phases are frozen, or if liquid water is still present in these concentrated micellar phases. However, the penetration of ice in confined spaces requires much lower temperatures. 10 Liquid water is therefore probably still present in the concentrated micellar phase at the lowest temperatures achieved here (-7°C).

It is generally accepted that self-assembly in aqueous solution of a copolymer, surfactant, lipid or any other object with weak interactions can occur only when micellar aggregates are already present in solution, and consequently cannot take place below the CMT. Our results show that it is not always true, and that micelles can form at temperatures lower than the initial value of the CMT. The formation of micelles is driven by the increase of the copolymer concentration when ice is formed. Two phases coexist in the sample, both containing water molecules: ice crystals and a concentrated aqueous solution of P123. We assume that the water molecules remaining in solution with the P123 molecules are in the liquid-like state, and are linked by H-bonds to the EO groups of the copolymer. 11 The equilibrium between these two phases is fixed by the chemical potential of the water molecules, which is imposed by the ice phase and depends only on temperature. Note that we verified that the freezing-point depression temperature due to the presence of P123 in solution is small, typically of -0.5 °C. The colligative freezing point depression of P123 is therefore smaller than that of other solutes (Methods). 12 The final P123 concentration depends only on the temperature of the sample and not on the initial P123 concentration. This hypothesis is validated by comparing experiments performed with different initial concentrations and similar final temperatures (Table 1 and Table S5, ESI †). The lattice parameters of the hcp and 2D-hexagonal phase have comparable values for all initial concentrations. This is in good agreement with the previous observations that freezing-induced segregation can increase the concentration of solutes, molecules, or particles by several orders of magnitude. 13,14 Table 1 Effect of the initial concentration in P123 [P123]ini. Estimated concentrations (wt%) of the liquid phase from transmission values (Fig. S3, ESI) are given for different states: micelles, hcp phase and 2D-hexagonal phase. Error bar for concentration is ±5 wt%. The average values of the lattice parameters for the hcp and 2D-hexagonal phase are reported (Table S5 Finally, the evolution with time of the system during freezing-induced self-assembly can be summarized using a concentration-temperature diagram (Fig. 3). First, before ice formation, the concentration of the unimers is constant and temperature is decreasing rapidly. As soon as ice forms (freezing point curve), the concentration of the copolymer starts to increase while temperature nearly stabilizes. Micelles form when the concentration crosses the CMT curve (dashed line) and self-assembly of the micelles into ordered mesophases subsequently occurs at still higher concentrations. Indeed the phase diagram of the P123 copolymer in water is not precisely established below room temperature. [START_REF] Wanka | [END_REF] No information can be found below freezing temperatures, and the frontiers for the phases (fcc, hcp, 2D-hexagonal and lamellar) in this diagram are thus only indicative.

The freezing-induced self-assembly demonstrated here may be important in several domains in soft matter and materials science. A first possibility is the field of ordered mesoporous materials. Indeed, self-assembly has been extensively used for the preparation of mesoporous materials by templating methods. The mechanism evidenced here with freezing recalls the mechanism of evaporation induced self-assembly (EISA) used for the preparation of films, 15 powders, 16 or monoliths, 17 where self-assembly is similarly driven by a concentration effect (Fig. 3). Freezing-induced self-assembly offers a new route for the elaboration of mesoporous materials. Silica 18 and alumina monoliths 19 have already been prepared by the association of ice-templating, P123 self-assembly and solgel chemistry to manufacture materials with hierarchical multi-scale porosity. Mesopores were templated by P123 micelles and macropores by ice crystals. The 2D hexagonal structure of the micelles was retained in the final material, providing a 2D hexagonal organization of the mesopores. The observed orientation of the self-assembled micelles with respect to the ice-front should permit a control of the pore orientation for the material, which can be an important issue. This orientation effect due to minimization of surface energy at interfaces is similar to what has been reported for mesoporous materials grown under confinement within anodic alumina membranes. 20 Freezing provides another pathway for the self-assembly of many other elementary building blocks. The principles shown here with an amphiphilic block copolymer can be extended to other systems where weak interactions can trigger self-assembly when the concentration is high enough. This could be particularly important for systems sensitive to temperature. Unlike evaporation induced self-assembly, which can require temperatures up to 80°C, self-assembly occurs here at low temperature (-5°C). Temperature sensitive building blocks such as lipids could thus be organized with this approach while preserving their integrity. This could be particularly relevant for biological materials. Very stimulating implications could be the formation upon freezing of lipids vesicles linked to prebiotic synthesis theories. 4 Self-assembled vesicles are considered essential components of primitive cells, and prebiotic synthesis from frozen solutions is considered as a serious contender to explain the initial formation of vesicles. 21 The segregation by the ice crystals provides a very efficient mechanism to increase the concentration of the building blocks by several orders of magnitude and avoid an endless dilution of the formed vesicles. Block copolymer vesicles, in particular, have been used for light-driven ATP generation 22 and enzymatic cascade reactions. 23 We expect therefore that our results obtained for freeze-formed micelles can be used in such studies. Investigating whether lipids vesicles of larger dimensions can be formed by freezing is a particularly exciting perspective.

Methods

Solutions of the P123 copolymer (EO20PO70EO20, BASF®) in water (5, 8, 10 and 13 wt%) were prepared without any purification. SAXS experiments were performed at the SWING beamline of the French SOLEIL synchrotron facility. Transmission and SAXS pattern were recorded during freezing using an incident X-ray beam of wavelength 0.124 nm. Exposure time for SAXS was 2 s, with acquisitions every 30 s. Samples were introduced inside a vertical commercial plastic straw of diameter 7.7 mm filled up to 10 mm. Freezing of the sample was obtained using a commercial device (CT160, Deben company) equipped with a Peltier element. For the measure of the freezing point depression, 100 ml of P123 solution (13 wt%) was inserted inside a Dewar and cooled down until ice crystals were formed. A freezing point depression of -0.5°C was measured when heating the solution.

Figure 2 :

 2 Figure2: Self-assembly along the direction of the temperature gradient. Sample concentration of 13 wt%. (A) Scan along the direction of the temperature gradient after 30 minutes. The 2D hexagonal phase is present over almost the entire region where ice is present (5 mm in height). At the top, the hcp phase is present only in a limited region of about 1 mm just below the liquid phase region (without ice crystals). (B) SAXS pattern of the hcp phase: several domains of the hcp phase coexist with a preferential orientation of their c axis perpendicular to the growth direction. (C) Sketch of the self-assembly along the growth direction, including the preferential orientation of the domains. The domains of the 2D-hexagonal phase are preferentially oriented with the a* direction perpendicular to the growth direction.

Figure 3 :

 3 Figure 3: Scheme of the freezing-induced self-assembly of P123 in water in a concentration-temperature diagram. Initially, only P123 unimers are present in solution. As soon as ice-crystals are forming (1-2), the concentration of P123 (inside the liquid phase) is increased and self-assembly takes place (3-4-5). The CMT curve, and the freezing point temperature curve (FP) are extrapolated (dashed lines). Frontiers of the different phases below 15°C are only indicative.

  , ESI). Error bar for lattice spacing is 0.05 nm

						Lattice parameters
	[P123]ini Micelles hcp	hcp+2D-hex 2D-hex (nm) a (hcp) a (2D hex)
	13	43	44-46 46-52	53-80	14.86 12.94
	10	40	40-53 54	54-80	14.86 12.94
	8	50	50-63 63-65	66-80	15.23 13.2
	5	24	30-42 44	47-80	14.99 13.13

Acknowledgements

This project was supported by the European Research Council under the European Community's Seventh Framework Pro-gramme (FP7/2007-2013) grant agreement 278004 (FreeCo). We acknowledge Synchrotron Soleil for allocation of beamtime at the SWING beamline.