Eduardo Fern Andez-Pascual
email: efernandezpascual@gmail.com

Angelino Carta

Andrea Mondoni

Lohengrin A Cavieres

Sergey Rosbakh

Susanna Venn

Annisa Satyanti

Lydia Guja

Ver Onica

F Briceño

Filip Vandelook

Efisio Mattana

Arne Saatkamp

Haiyan Bu

Karen Sommerville

Peter Poschlod

Kun Liu

Adrienne Nicotra

Borja Jim Enez-Alfaro

Eduardo Fern Andez Pascual

The seed germination spectrum of alpine plants: a global metaanalysis

Keywords: alpine, alternating temperature, cold stratification, embryo endosperm ratio, light germination, seed dormancy, seed germination, seed mass

Assumptions about the germination ecology of alpine plants are presently based on individual species and local studies. A current challenge is to synthesise, at the global level, the alpine seed ecological spectrum.

We performed a meta-analysis of primary data from laboratory experiments conducted across four continents (excluding the tropics) and 661 species, to estimate the influence of six environmental cues on germination proportion, mean germination time and germination synchrony; accounting for seed morphology (mass, embryo : seed ratio) and phylogeny.

Most alpine plants show physiological seed dormancy, a strong need for cold stratification, warm-cued germination and positive germination responses to light and alternating temperatures. Species restricted to the alpine belt have a higher preference for warm temperatures and a stronger response to cold stratification than species whose distribution extends also below the treeline. Seed mass, embryo size and phylogeny have strong constraining effects on germination responses to the environment.

Globally, overwintering and warm temperatures are key drivers of germination in alpine habitats. The interplay between germination physiology and seed morphological traits further reflects pressures to avoid frost or drought stress. Our results indicate the convergence, at the global level, of the seed germination patterns of alpine species.

Introduction

Alpine environments occur worldwide above the maximum ele vation at which trees can grow naturally (K€ orner & Paulsen, 2004;K€ orner et al., 2011;[START_REF] Testolin | Global distribution and bioclimatic characterization of alpine biomes[END_REF]. These treeless habitats are characterised by low temperatures, unstable sub strates and short growing seasons (K€ orner, 2003). Although most alpine plants can reproduce clonally (K€ orner, 2003), sexual reproduction by diaspores (i.e. seeds, fruits or fruits with extra ovarian structures attached; hereafter 'seeds') is the main strategy to maintain genetic diversity and to colonise suitable new sites in response to environmental changes [START_REF] Walck | Climate change and plant regeneration from seed[END_REF]. How ever, the short reproductive season of the alpine habitat con strains the phenological timing of flowering, pollination, seed development and seed dispersal. For regeneration by seed to be successful, dispersed propagules must germinate in the most appropriate period to ensure the survival of seedlings [START_REF] Chambers | Alpine seedling establishment: the influence of disturbance type[END_REF]Sch€ utz, 2002;[START_REF] Forbis | Seedling demography in an alpine ecosystem[END_REF]. The physiological pro cess of seed germination is therefore an essential life stage that must be timed to occur when the environment is favourable for subsequent seedling survival and growth [START_REF] Poschlod | Seed ecology and assembly rules in plant communities[END_REF]. In alpine habitats, seed germination will also be a crucial test for plant populations coping with global change, determining whether they will persist or go into extinction.

Natural selection is expected to favour seed germination requirements that reduce the probability of facing environmental conditions that are not appropriate for seedling survival and growth [START_REF] Angevine | Seed germination syndromes in higher plants[END_REF]. Thus, germination is chiefly regulated by environmental cues related to water availability and soil temperature [START_REF] Bewley | Seeds: physiology of development, germination and dormancy[END_REF]. The propagules of many plant species have also developed seed dormancy, by means of which germination is prevented during periods that are only ephemerally favourable, like a short warm spell in the middle of winter (Baskin & Baskin, 2014). Different degrees of dormancy within a seed population ensure the distribution of seedling emergence across time, a form of bet hedging offspring survival against unpredictable environments [START_REF] Venable | Bet hedging in a guild of desert annuals[END_REF]. Other ger mination cues, such as the response to diurnal alternating tem peratures [START_REF] Thompson | Seed germination in response to diurnal fluctuations of temperature[END_REF] and the need for light or darkness [START_REF] Carta | Photoinhibition of seed germination: occurrence, ecology and phylogeny[END_REF], allow for a fine scale detection of germination micro niches and safe sites [START_REF] Jumpponen | Characterization of 'safe sites' for pioneers in primary succession on recently deglaciated terrain[END_REF]. Given that current assumptions about the germination ecology of alpine plants are mainly based on local studies, a cur rent challenge is to synthesise, at the global level, the seed ecologi cal spectrum of alpine plants. The seed ecological spectrum [START_REF] Saatkamp | A research agenda for seed-trait functional ecology[END_REF] is defined as a set of seed traits that deter mines the ability of plants to disperse, persist, germinate and establish in different habitats. A major goal for trait based seed ecology is to study the relationships and trade offs between differ ent aspects of the seed ecological spectrum [START_REF] Saatkamp | A research agenda for seed-trait functional ecology[END_REF], such as the physiological drivers of germination versus key morphological traits like seed mass [START_REF] Moles | A brief history of seed size[END_REF] and the embryo to seed ratio [START_REF] Vandelook | Relative embryo length as an adaptation to habitat and life cycle in Apiaceae[END_REF].

Traditionally, alpine species have been described as having seeds with physiological dormancy, which is a type of dormancy that is caused by the seed's internal balance of phytohormones and requires a long exposure to dormancy alleviating treatments to be terminated [START_REF] Schwienbacher | Seed dormancy in alpine species. Flora-Morphology, Distribution[END_REF][START_REF] Sommerville | Can seed characteristics or species distribution be used to predict the stratification requirements of herbs in the Australian Alps?[END_REF]Baskin & Baskin, 2014). This physiological dormancy would prevent precocious germination of recently dispersed seeds during autumn, when appropriate conditions are not likely to persist for more than a few weeks or days [START_REF] Meyer | Habitat-correlated variation in mountain big sagebrush (Artemisia tridentata ssp. vaseyana) seed germination patterns[END_REF]. A requirement for cold stratification to alleviate dormancy allows seeds to sense the snow season, thereby postponing germi nation to a more favourable period for seedling survival and development. Thus, it can be expected that a cold stratification period is a requirement for seed germination in alpine plants. One of the first experiments on this topic was conducted by S€ oyrinki (1938), who showed how the storage of arctic-alpine species at winter temperatures (i.e. cold stratification) increased seed germination in most species. Nonetheless, [START_REF] Amen | The extent and role of seed dormancy in alpine plants[END_REF] later suggested that cold stratification was not a requirement for seed germination of alpine species from different mountain ranges in the USA, a finding that was supported by subsequent studies [START_REF] Sayers | Germination responses in alpine species[END_REF][START_REF] Marchand | Reproductive strategies of pioneering alpine species: seed production, dispersal, and germination[END_REF][START_REF] Kaye | Seed dormancy in high elevation plants: implications for ecology and restoration[END_REF]. More recently, research on Australian alpine plants found that a cold stratification period only increased germination in half of the species tested [START_REF] Sommerville | Can seed characteristics or species distribution be used to predict the stratification requirements of herbs in the Australian Alps?[END_REF][START_REF] Hoyle | Seed germination strategies: an evolutionary trajectory independent of vegetative functional traits[END_REF]. Cold stratification increased seed germination over a range of temperatures in most of the species studied in the alpine zone of Japan [START_REF] Shimono | Comparisons of germination traits of alpine plants between fellfield and snowbed habitats[END_REF]. Along an elevational gradient in the central Chilean Andes, cold stratification was an important requirement for seed germination in species from lower elevations, while species from higher sites did not germi nate after a single period of cold stratification [START_REF] Cavieres | Assessing the importance of coldstratification for seed germination in alpine plant species of the High-Andes of central Chile[END_REF]. Finally, in a comparative study on germination of unstratified seeds from different habitats of the Austrian Alps, species from alpine grasslands had the lowest final germination proportion, suggesting that they had the highest level of dor mancy [START_REF] Margreiter | Pros and cons of using a standard protocol to test germination of alpine species[END_REF].

Seed dormancy is not the only trait that can prevent, in prac tice, autumn germination. Early studies on the germination ecology of alpine plants demonstrated that, in most species, recently dispersed seeds require relatively high temperatures for germination (S€ oyrinki, 1938;[START_REF] Bliss | Seed germination in arctic and alpine species[END_REF][START_REF] Amen | The extent and role of seed dormancy in alpine plants[END_REF][START_REF] Billings | The ecology of arctic and alpine plants[END_REF]). Warm cued germination has been consid ered as an adaptation to prevent seed germination at the time of seed dispersal (autumn) when temperatures are relatively low and there is a high risk of frost [START_REF] Cavieres | Seed germination response to cold stratification period and thermal regime in Phacelia secunda (Hydrophyllaceae)[END_REF]. Indeed, germination of alpine seeds tends to occur after winter, mainly in early summer (K€ orner, 2003;[START_REF] Mondoni | Climate warming could increase recruitment success in glacier foreland plants[END_REF]. In recent times, an increasing number of studies have reported that fresh seeds of some alpine plants can also germinate at cool incubation temperatures or during cold stratification treatments [START_REF] Schwienbacher | Seed dormancy in alpine species. Flora-Morphology, Distribution[END_REF][START_REF] Hoyle | Seed germination strategies: an evolutionary trajectory independent of vegetative functional traits[END_REF]Fern andez Pas cual et al., 2017a;[START_REF] Cavieres | Assessing the importance of coldstratification for seed germination in alpine plant species of the High-Andes of central Chile[END_REF]. This cold cued germination could be an adaptation to germinate under snow or during snowmelt, which could presumably allow seedlings to develop a deeper root system before topsoil desicca tion in summer (Kammer & M€ ohl, 2002), or to attain an opti mal size for overwintering [START_REF] Billings | The ecology of arctic and alpine plants[END_REF]). Yet, the generality of warm cued germination across different alpine regions and habitats has never been studied systematically, and the relative importance of cold stratification versus warm cued germination remains unclear. A central question is whether physiological seed dormancy plays a relevant role in postponing alpine germination during autumn and winter, or whether the warm cued germination is enough to prevent germination under cool autumn temperatures. The response to this question becomes pertinent under a warming climate, which could result in nondormant seeds germinating readily in autumn. Nondor mant seeds could possibly have an advantage as the growing sea sons become longer, if seedlings have enough time to grow and become sufficiently large to survive the winter snow season. Potentially, having a nondormant or less dormant seed could be a better strategy for alpine plants to thrive under future climate [START_REF] Verd U | Early emergence enhances plant fitness: a phylogenetically controlled meta-analysis[END_REF][START_REF] Mondoni | Climate warming could increase recruitment success in glacier foreland plants[END_REF]. However, we might also expect that seedlings from these species will be exposed to frost damage if snow starts to melt earlier and insula tion during winter disappears [START_REF] Venn | Foliar freezing resistance of Australian alpine plants over the growing season[END_REF] and therefore mechanisms that enable diverse germination timing (i.e. low germination synchrony) may be favoured.

Besides patterns of seed dormancy and germination tempera ture, the response of alpine seeds to diurnal alternating temperatures and light can provide further information on their capacity to detect fine scale environmental cues. Fluctuations of diurnal temperature decrease with increasing burial depths and the depth profile of this decrease depends on soil type, vegetation cover and snow [START_REF] Thompson | Seed germination in response to diurnal fluctuations of temperature[END_REF][START_REF] Van Assche | The role of temperature on the dormancy cycle of seeds of Rumex obtusifolius L[END_REF][START_REF] Saatkamp | Soil depth detection by seeds and diurnally fluctuating temperatures: different dynamics in 10 annual plants[END_REF]. Solar irradiance in physiologically significant quantities penetrates only the first mil limetres of the soil [START_REF] Tester | The penetration of light through soil[END_REF]. Therefore, the response to alternating temperatures and light can indicate to seeds whether they are buried deep in the soil, in rock crevices or under snow. The depth of seed burial is crucial for seedling emer gence, as small seeds lack the resources to survive until they reach the soil surface [START_REF] Bond | Seed size and seedling emergence: an allometric relationship and some ecological implications[END_REF]. A study of 445 species from the Qinghai Tibet plateau found that species from high elevation alpine meadows did not show a significant response to diurnal temperature oscillations [START_REF] Liu | Effect of diurnal fluctuating versus constant temperatures on germination of 445 species from the eastern Tibet Plateau[END_REF]. This is intriguing given that alpine habitats typically have a strong day/night cli matic variation during the growing season. Regarding light and darkness, some local studies have found alpine species to have a preference for germinating in light [START_REF] Densmore | Effect of day length on germination of seeds collected in Alaska[END_REF][START_REF] Mondoni | Germination requirements of the alpine endemic Silene elisabethae Jan: effects of cold stratification, light and GA3[END_REF]. This trait is linked to the creation of a persistent soil seed bank [START_REF] Venn | Soil seedbank composition and dynamics across alpine summits in south-eastern Australia[END_REF][START_REF] Jaganathan | Towards an understanding of factors controlling seed bank composition and longevity in the alpine environment[END_REF] which may be advantageous in the temporally and spatially unpredictable alpine environments [START_REF] Arroyo | Persistent soil seed bank and standing vegetation at a high alpine site in the central Chilean Andes[END_REF][START_REF] Cavieres | Persistent soil seed banks in Phacelia secunda (Hydrophyllaceae): experimental detection of variation along an altitudinal gradient in the Andes of central Chile (33 S)[END_REF]. Indeed, persistent alpine soil seed banks are more frequent than previously supposed, as consistently reported in recent years [START_REF] Venn | Soil seedbank composition and dynamics across alpine summits in south-eastern Australia[END_REF][START_REF] Hoyle | Soil warming increases plant species richness but decreases germination from the alpine soil seed bank[END_REF][START_REF] Jaganathan | Towards an understanding of factors controlling seed bank composition and longevity in the alpine environment[END_REF]. Nevertheless, other alpine species seem to prefer germination in darkness [START_REF] Schwienbacher | Seed dormancy in alpine species. Flora-Morphology, Distribution[END_REF], which may be interpreted as a strategy for detecting safe sites in rock crevices [START_REF] Arslan | Germination requirements of Androsace villosa L. (Primulaceae)[END_REF]. Although some germination studies report laboratory experiments conducted with constant and alternating temperatures, or light and dark conditions, they are very much case dependent, and therefore it is difficult to eval uate whether these factors show a consistent pattern across global alpine ecosystems.

In this work, we make a global collection of primary germina tion data from laboratory experiments conducted with seeds of alpine plant species from extratropical regions. The assembled dataset represents eight seasonal alpine regions belonging to four continents. We perform a meta analysis of this dataset to investi gate the germination response of alpine plants to key environ mental factors: dormancy alleviating treatments (cold stratification, GA 3 and scarification), average germination tem perature, diurnal alternating temperatures and light [START_REF] Bewley | Seeds: physiology of development, germination and dormancy[END_REF]Baskin & Baskin, 2014). Given the heterogeneity of alpine species lineages adapted to different regions, we account for the shared evolutionary history using a phylogenetic compara tive approach [START_REF] Garamszegi | Modern phylogenetic comparative methods and their application in evolutionary biology[END_REF]. We further analyse the rela tionships between germination ecology and two morphological traits that are key drivers of the seed ecological spectrum: seed mass and the embryo to seed ratio [START_REF] Saatkamp | A research agenda for seed-trait functional ecology[END_REF]. Our main aim in conducting this work is to test the consistency of general assumptions about the influence of major environmental cues on seed germination of alpine plants. Specifically, we deter mined at the global scale whether alpine seeds: (1) have physiological dormancy; (2) require cold stratification to alleviate dormancy; (3) need relatively warm temperatures to germinate; (4) do not require alternating temperatures for germination; and

(5) respond positively to light. To understand the ecological con straints of our meta analysis on alpine species pools, we com pared the germination responses of strict alpine species (that occur predominantly above the treeline) and generalist species (that occur above the treeline but also at lower elevations). We can expect that the former group will show a more convergent alpine germination syndrome, while the latter will be more plas tic in their response.

Materials and Methods

Data collection

We conducted a meta analysis of primary data [START_REF] Mengersen | Meta-analysis of primary data[END_REF], a type of meta analysis which consists of pooling together original data from different local studies to synthesise their con clusions at the global level. All data processing, analysis and manuscript production was performed in R [START_REF] Core | R: a language and environment for statistical computing[END_REF]. The original data, plus R code for the analysis and cre ation of the manuscript using R/MARKDOWN, can be accessed at the article's GitHub repository (see Data Availability statement). To gather data, we contacted research groups that had performed experiments on seed germination of alpine plants, asking them to provide raw data from laboratory germination experiments. The submitted data had to report the studied species, the germination treatments, the number of seeds sown, and the number of seeds germinated. We received data (Table 1) on species from the Cantabrian Mountains of Spain, the European Alps, the Italian Apennines, the Northern Caucasus, the Qinghai Tibet Plateau of China, the Chilean Andes and the Australian Alps. We also received data on European Alpine species from ENSCOBASE, the database of the European Native Seed Conservation Network ENSCONET (http://enscobase.maich.gr/index.tml).

We standardised the species names according to The Plant List (The Plant List, 2013) using the package TAXONSTAND [START_REF] Cayuela | Taxonstand: taxonomic standardization of plant species names[END_REF] in R. For each species, we recorded its elevation range from local floras [START_REF] Hegi | Illustrierte Flora von Mitteleuropa, mit besonderer Berucksichtigung von Deutschland, Osterreich und der Schweiz[END_REF][START_REF] Pignatti | Flora d'Italia[END_REF][START_REF] Castroviejo | Flora iberica[END_REF][START_REF] Brach | eFloras: new directions for online floras exemplified by the Flora of China project[END_REF][START_REF] Parolly | Schmeil-Fischen[END_REF], catalogues [START_REF] Rodr Iguez | Cat alogo de las plantas vasculares de Chile[END_REF] and herbaria (Australian National Herbarium, https://www.anbg.gov.au/cgi bin/anhsir). We used the elevation ranges: (1) to remove from the dataset outlier species that do not grow above the treeline; and (2) to classify the rest of the species as strict alpine (i.e. species that grow predomi nantly above the treeline) or generalist (i.e. species that can grow above the treeline but also in the lowlands). For each species, we compiled three additional traits: seed dormancy class, seed mass and embryo : seed ratio.

We classified the species in seed dormancy classes following Baskin & Baskin (2014) and Rosbakh et al. (2020a), using five categories: physiological (germination prevented by the seed's internal balance of phytohormones), morphological (germination prevented by an embryo which is underdeveloped at the time of dispersal), morphophysiological (both physiological and morphological dormancy present), physical (germination pre vented by a water impermeable seed coat) or nondormant. We followed the concept by [START_REF] Vleeshouwers | Redefining seed dormancy: an attempt to integrate physiology and ecology[END_REF] to define dor mancy as an inner seed property (sometimes called 'endodor mancy'), which should be differentiated from the absence of appropriate germination cues in the environment ('ecodormancy'); and the classification system of [START_REF] Baskin | A classification system for seed dormancy[END_REF] based on previous work by [START_REF] Nikolaeva | Pravocnik po prorascivanij pokojascichsja semjan (Reference Book on Dormant Seed Germination)[END_REF] who consider morphological and physical dormancy as classes separated from physiological dormancy.

We collected mean seed mass for each species as provided by the Royal Botanic Gardens Kew Seed Information Database (2017) and additional sources [START_REF] Bu | Seed mass and germination in an alpine meadow on the eastern Tsinghai-Tibet plateau[END_REF][START_REF] Bu | Community-wide germination strategies in an alpine meadow on the eastern Qinghai-Tibet plateau: phylogenetic and life-history correlates[END_REF][START_REF] Liu | Effect of diurnal fluctuating versus constant temperatures on germination of 445 species from the eastern Tibet Plateau[END_REF]. We recorded the embryo to seed surface area ratio for each species. Embryo to seed surface area values were calculated by dividing embryo area by the surface area of the seed, more specifically embryo plus endosperm and perisperm. Measure ments were made using the IMAGEJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] on drawings and photographs of seeds cut in half along the longitudinal axis, retrieved mainly from [START_REF] Martin | The comparative internal morphology of seeds[END_REF] and the Royal Botanic Gardens Kew Seed Information Database (2017), supplemented with own photographs (F. Vandelook, unpub lished). For some species that were missing seed mass or embryo values, we calculated genus or family averages. In the case of seed mass, we used species level values for 473 species, and genus aver ages for another 188 species. For embryo mass, we used 21 species level values, 427 genus averages and 213 family averages.

The collected species traits, including information on which taxo nomic rank was used to calculate the values for each species, is available in the data folder of the GitHub repository (see Data Availability statement).

Germination response variables

We used the final germination proportion (i.e. the number of seeds germinated out of seeds sown) at 8 wk as the main response variable for the germination treatments. The choice of 8 wk was a compromise deadline to unify datasets, as there was a small frac tion of trials that had run for longer times. We further calculated the mean germination time and the germination uncertainty index with the package GERMINAR (Lozano Isla et al., 2019). Equations for these two germination indices are available in Lozano [START_REF] Lozano-Isla | GerminaR: an R package for germination analysis with the interactive web application 'GerminaQuant for R[END_REF]. Mean germination time is the time that it takes for half of the seed lot to germinate, indicating the germi nation speed in each experimental condition. The germination uncertainty index estimates how scattered germination is through time, giving an idea of whether germination is synchronous (i.e. most seeds germinate around the mean germination time) or asynchronous (i.e. germination events are separated through time during the germination experiment). Lower values in the germi nation uncertainty index indicate higher germination synchrony. The uncertainty index is analogous to the synchrony index and we used it because, unlike the latter, its values are not constrained between 0 and 1 (Lozano Isla et al., 2019). To calculate these indices, we used only records that met two conditions: (1) more than 50% final germination percentage; and (2) a mean germina tion time under 4 wk. Within these limits, we were able to calcu late mean germination time and uncertainty for 293 species. We established this conservative approach to prevent the results being altered by a modification of the seed's dormancy status during the germination incubation (e.g. a fraction of the seed lot is non dormant at the beginning of the incubation and germinates shortly after the start of the experiment; while another fraction is originally dormant, loses dormancy in response to the incubation conditions, and germinates in a second wave). The primary ger mination dataset is available in the data folder of the GitHub repository (see Data Availability statement).

Statistical analyses

We performed the meta analysis of the germination data by fit ting generalised mixed models with Bayesian estimation (Markov Chain Monte Carlo generalised linear mixed models, MCMCglmms) using the R package MCMCGLMM [START_REF] Hadfield | MCMC methods for multi-response generalized linear mixed models: the MCMCglmm R package[END_REF]. To model final germination proportions, we used bino mial MCMCglmms, while for mean germination time and the germination uncertainty index we used gaussian MCMCglmms. Models had, as fixed effects, the experimental conditions (stratifi cation, GA 3 , scarification, temperature, alternating temperature and light), plus their interaction with seed mass and embryo : seed, and the strict alpine or generalist character of the species. Random effects included a reconstructed phylogenetic tree for the 661 species, and species identity, seed lot, laboratory (i.e. data provider), and alpine region (Table 1). To create the phylogeny, we used the R package V.PHYLOMAKER [START_REF] Jin | PhyloMaker: an R package that can generate very large phylogenies for vascular plants[END_REF] which contains an updated mega tree of the seed plants based on [START_REF] Smith | Constructing a broadly inclusive seed plant phylogeny[END_REF]. We placed taxa absent from the mega tree at the genus level basal node. The phylogenetic tree is available in the data folder of the GitHub repository (see Data Availability statement). In all models, all variables were scaled so their contribution to the effect sizes could be compared. We used weakly informative priors in all models, with parameter ex panded priors for the random effects. Each model was run for 500 000 MCMC steps, with an initial burn in phase of 50 000 and a thinning interval of 50 [START_REF] Villemereuil | General quantitative genetic methods for comparative biology[END_REF], resulting, on average, in 9000 posterior distributions. From the resulting posterior distributions, we calculated mean parameter estimates and 95% highest posterior density and credi ble intervals (CI). We estimated the significance of model param eters by examining CIs, considering parameters with CIs overlapping with zero as nonsignificant. To estimate the phyloge netic signal of seed germination over all variables, we used Pagels's lambda (k) [START_REF] Pagel | Inferring the historical patterns of biological evolution[END_REF], estimated simultaneously with the models by calculating the mean of the posterior distribution and the 95% CI of k as indicated by [START_REF] Villemereuil | General quantitative genetic methods for comparative biology[END_REF]. When k = 0, related taxa are no more similar than expected by chance, while when k = 1, the trait is evolving follow ing a constant variance random walk or Brownian motion model; intermediate values of k indicate a phylogenetic correlation in trait evolution that does not fully follow a Brownian motion model [START_REF] Pagel | Inferring the historical patterns of biological evolution[END_REF]. Detailed results of the MCMCglmms are available in the results folder of the GitHub repository (see Data Availability statement).

Visualisation of the alpine seed germination spectrum

To visualise the seed germination spectrum of alpine plants, we employed a factorial analysis of mixed data (FAMD) as imple mented in the package FACTOMINER [START_REF] Le | FactoMineR: an R package for multivariate analysis[END_REF]. FAMD ordination combines the properties of PCA and MCA to jointly measure the variation of continuous and categorical variables.

The ordination was performed at the species level, that is calculat ing a series of continuous and categorical traits for each species. We transformed the final germination proportions to create a continuous variable for each germination cue (i.e. stratification, GA 3 , scarification, temperature, alternating temperatures and light). To do so, for each cue and species, we calculated a weighted average of the cue levels (in the case of temperature, cue levels were the temperature treatments; for the other cues the levels were 0 = absence and 1 = presence), weighting by the ger mination proportion at each level. This approach underrepresents the importance of the levels that were not tested for a given species, but can serve as a proxy of the response to the germina tion cues when visualised across the whole dataset; it must be stressed that this stage of the analysis serves only for visualisation and not for hypothesis testing. We also included germination timing traits by calculating single values for the mean germina tion time (the minimum, i.e. the time taken at the most favourable treatment) and the germination uncertainty index (the mean). We conducted the FAMD with the 293 species for which we had mean germination time and germination uncertainty index values. We finally included the continuous variables seed mass and embryo : seed, as well as the categorical variables dor mancy class (physiological, morphophysiological, morphological, physical or nondormant) and distribution (strict alpine vs gener alist species).

Results

Description of the dataset

The final dataset used in our meta analysis contained 9799 records (i.e. germination proportions for a given seed lot of a species, recorded in a set of experimental conditions) contributed by 12 research groups, representing 62 seed plant families and 661 species. From these, 291 were categorised as strict alpine species and 370 as generalist. Seed lots were originally sampled in eight alpine regions of the world (Table 1), at middle latitudes of both hemispheres. Strict species are mostly restricted to the alpine belt, generalist species can be found in the alpine belt but also below the treeline. Seed dormancy classes are abbreviated as follows: MD (morphological), MPD (morphophysiological), ND (nondormant), PD (physiological), PY (physical). Whiskers in the boxplots (b, c) extend to the most extreme data point which is no more than 1.5 times the length of the box away from the box.

Ó Ó

Most species in the dataset were reported as having dormant seeds (Fig. 1a), but the frequency of the dormancy classes did not differ between generalist and strict alpine species. Among those species with dormant seeds, physiological dormancy was the most common dormancy class, followed by morphophysiological and then physical. Both strict alpine and generalist species had a simi lar range of values for seed mass (Fig. 1b) and embryo : seed ratio (Fig. 1c). The values of seed mass ranged from 0.02 mg (Calceolaria purpurea) to 58 mg (Vicia orobus), with a median value of 0.56 mg. Embryo : seed ratio encompassed the full range of potential values, from endospermic species with very small embryos (0.001) to nonendospermic seeds that store nutrients in the cotyledons (1). The median value was 0.34.

The total number of seeds used in the experiments was 365 808. Germination temperatures (i.e. the weighted average of the day and night temperatures, weighted by the duration of each phase) ranged from 0 to 36°C, with 7521 records of constant temperatures (i.e. experiments that used the same temperature during all their duration) and 2278 of alternating temperatures (i.e. experiments where different temperatures were applied dur ing the and the night, in diurnal cycles). Seeds were exposed to light during some part of the diurnal cycle in 8933 records and kept in total darkness in 866 records. The experiments were performed with untreated seeds in 7297 records and, of the rest, the majority (2436) went through cold stratification (< 5°C, in darkness). Scarification (i.e. an abrasion of the seed coat to allow water imbibition) was performed in 119 records, and GA 3 (a plant hormone that promotes embryo growth and stimulates ger mination) in 651 records. according to the MCMC meta analysis of primary data. Dots indicate the posterior mean of the effect size, and whiskers its 95% credible interval. The line of zero effect is shown. When the credible intervals overlap with the zero effect line, the effect is not significant. Some effects that overlapped with zero (i.e. had no effect) and whose credible intervals were excessively wide are not shown for clarity's sake. The figure shows first the main effect, then the effects for the strict alpine and generalist groups, and then the interaction with seed mass and embryo : seed. A negative interaction (e.g. seed mass and light, in the case of the germination proportion) indicates that, as the interacting factor increases (e.g. seed mass) the response to the germination cue (e.g. light) decreases, in comparison with the main effect. GA3, gibberellic acid.

Stratification

Ó Ó

Dormancy-alleviating treatments

In general, stratification significantly increased the final germination proportion (Fig. 2a), as shown by a credible interval not overlap ping zero. This positive effect occurred in both generalist and strict alpine species, although the effect size was larger in the strict alpine group. Stratification slightly reduced the mean germination time (Fig. 2b), which was evident in the generalist species, but not in the strict alpine species. Further, stratification reduced the germination uncertainty index as a general response (Fig. 2c), and slightly more so in generalist species. Seed mass did not interact with stratifica tion, but species with larger embryos had a smaller response to strat ification their final germination proportion and reduced less markedly their mean germination time and germination uncer tainty index. GA 3 had similar effects to stratification, with the main difference being that seed mass affected the response to GA 3 , whereas it did not interact with the response to stratification.

Scarification promoted the germination proportion (Fig. 2a) and reduced the mean germination time (Fig. 2b), but it did not affect the germination uncertainty index (Fig. 2c). Strict alpine species were slightly more responsive to scarification than general ist species. Heavier seeds were more responsive to scarification, while seeds with larger embryos responded less.

Germination temperature

Temperature had a positive significant effect on the final germina tion proportion (Fig. 2a), indicating a preference for warmer tem peratures. Warmer temperatures also significantly decreased mean germination time (Fig. 2b) and the germination uncertainty index (Fig. 2c). The effect of warmer temperatures was slightly higher in strict alpine than in generalist species, although the CI of the two groups of species do overlap. Seed mass and embryo : seed had a negative interaction with temperature, reducing the response to temperature. In other words, heavier and less endospermic seeds had a smaller increase in germination proportion and a smaller decrease in mean germination time when germinating in warmer temperatures. The germination uncertainty index did not show an interaction with seed mass or embryo : seed.

Alternating temperatures

Alternating temperatures had a positive effect on the final germi nation proportion (Fig. 2a), but not on the mean germination time (Fig. 2b) or the germination uncertainty index (Fig. 2c). The positive effect on the germination proportion was slightly higher in the generalist species. As in the case of the mean germi nation temperature, higher seed mass and embryo : seed values reduced the positive effect of alternating temperatures on the final germination proportion. Alternating temperatures resulted in faster germination (reduced the mean germination time) for seeds with low values of embryo : seed.

Light

Light increased the final germination proportion (Fig. 2a), with no differences between strict alpine and generalist species. It did not affect the mean germination time (Fig. 2b), but it did result in more variable germination (increased the germination uncer tainty index) of generalist species (Fig. 2c). Heavier and less endospermic seeds responded less to light.

Random effects and phylogenetic signal

Random factors had a significant effect in the models (Fig. 3). The final germination proportion and the mean germination time were affected more strongly, on average, by the seed lot and the species identity, suggesting that these traits are subject to intra and interspecific variability, respectively. Alpine region and laboratory had a lower average effect and more vari ability as shown by their CI. The germination uncertainty index showed the opposite pattern, with a stronger effect of laboratory and alpine region. The phylogenetic signal in the response to all germination parameters (Fig. 4) was significantly positive for the final germination proportion and the mean germination time. For the germination uncertainty index, phylogenetic signal over lapped with zero, indicating a weak effect of phylogeny. The results for the germination uncertainty index indicate that it might have been more sensitive to the frequency of germination scoring, which was different in different laboratories. Detailed values of phylogenetic signal for all model interactions are avail able in the results folder of the GitHub repository (see Data Availability statement).

FAMD ordination

The first FAMD axis explained 17% of the variation (Fig. 5). The quantitative variables with the largest contribution to this first axis were germination temperature, germination uncertainty, GA 3 and alternating temperatures. Among the qualitative factors, the strict alpine/generalist categories contributed to axis 1. In summary, the horizontal axis separated (i, left) generalist species with a positive response to alternating temperatures and more germination uncertainty from (ii, right) strict alpine species with a preference for warmer temperatures and a positive response to GA 3 . Axis 2 explained 12% of the variability. The main con tributing variables were embryo : seed, mean germination time, seed mass and scarification; and the main categorical factors were physical dormancy and morphophysiological dormancy. This axis separated (iii, bottom) morphophysiologically dormant species with longer mean time to germination from (iv, top) physically dormant species that responded to scarification and had heavier, less endospermic seeds.

Discussion

Our meta analysis of primary germination data shows that alpine seeds tend to germinate when, after weeks of exposure to condi tions that mimic winter, they experience mid summer tempera tures. The germination ecology of strict alpine species is characterised by: (1) physiological seed dormancy; (2) a strong need for cold stratification or GA 3 to alleviate dormancy; (3) warm cued germination; (4) a positive response to alternating Ó Ó temperatures, although not so prominent as in generalist species;

(5) a positive response to light; (6) slow and relatively syn chronous germination; and (7) an interplay with seed and embryo size, with smaller and more endospermic seeds being more responsive to stratification, warmth, alternating tempera tures and light. Here we discuss the ecological significance of Ó Ó these responses and how they define the seed germination spec trum of alpine plants.

Seed dormancy

Our dataset showed that seed dormancy is frequent across alpine species and plays an important role in postponing plant recruit ment to favourable times. The frequency of dormancy classes dis played by strict alpine and generalist species was similar, suggesting that this is a general trait of cold adapted mountain species. Seeds with the two most frequent dormancy classes, that is physiological and morphophysiological, require a stratification period during which certain conditions (e.g. cold and wet, although other combinations are possible) are met over a period of months (Baskin & Baskin, 2014;Rosbakh et al., 2020a). Indeed, our results show that cold stratification consistently pro motes germination, while the phytohormone gibberellic acid (GA 3 ) works as a substitute of cold stratification to overcome physiological dormancy. Additionally, we found some species with physical dormancy. This dormancy type can be broken by mechanical scarification via freezing-thawing cycles in spring or extreme temperature fluctuations during summer, but it is also capable of responding to seasonal cycles of temperature and humidity [START_REF] Van Assche | Seasonal cycles in the germination capacity of buried seeds of some Leguminosae (Fabaceae)[END_REF].

Together, these results confirm that overwintering is essential for promoting seed germination in alpine species, concurring with previous local studies [START_REF] Cavieres | Seed germination response to cold stratification period and thermal regime in Phacelia secunda (Hydrophyllaceae)[END_REF]Sch€ utz, 2002;[START_REF] Gim Enez-Benavides | Seed germination of high mountain Mediterranean species: altitudinal, interpopulation and interannual variability[END_REF][START_REF] Shimono | Comparisons of germination traits of alpine plants between fellfield and snowbed habitats[END_REF][START_REF] Sommerville | Can seed characteristics or species distribution be used to predict the stratification requirements of herbs in the Australian Alps?[END_REF][START_REF] Garc Ia-Fern Andez | Effects of the duration of cold stratification on early life stages of the Mediterranean alpine plant Silene ciliata[END_REF][START_REF] Hoyle | Seed germination strategies: an evolutionary trajectory independent of vegetative functional traits[END_REF]Fern andez Pascual et al., 2017a;[START_REF] Cavieres | Assessing the importance of coldstratification for seed germination in alpine plant species of the High-Andes of central Chile[END_REF]. As global climate change is causing major changes in snow cover duration [START_REF] Beniston | Is snow in the Alps receding or disappearing?[END_REF][START_REF] Gobiet | 21st century climate change in the European Alps a review[END_REF], it could disrupt natural cold stratification cycles in alpine environments, compromising population viability and indirectly favouring species with no stratification requirement [START_REF] Sommerville | Can seed characteristics or species distribution be used to predict the stratification requirements of herbs in the Australian Alps?[END_REF]. This risk will be partly alleviated by the plasticity of physiological seed dormancy in response to the seed maturation temperature [START_REF] Andez-Pascual | A local dormancy cline is related to the seed maturation environment, population genetic composition and climate[END_REF]Carta et al., 2016b). 'Seed memory' via maternal effects facilitates acclimatisation to changing environments, and can reduce the stratification require ments of seeds produced in a warmer climate [START_REF] Ó Ó Fern Andez-Pascual | Seeds of future past: climate change and the thermal memory of plant reproductive traits[END_REF].

Thermal regulation of germination

We found a general increase in seed germination with higher incubation temperatures. This effect was more pronounced in strict alpine compared with generalist species, confirming previ ous local studies [START_REF] Walder | Temperature and drought drive differences in germination responses between congeneric species along altitudinal gradients[END_REF]Fern andez Pas cual et al., 2017a). It was also more pronounced in small seeded and endospermic species. Small seeded species have a lower pro portion of seedling survival under drought ( Ó Ó , 1994), but may require warmer germination tempera tures than large seeded species, especially in seasonal climates [START_REF] Ar Ene | Temperature but not moisture response of germination shows phylogenetic constraints while both interact with seed mass and lifespan[END_REF]. Considering that risk reduction is an impor tant selective pressure for the evolution of germination traits [START_REF] Venable | The selective interactions of dispersal, dormancy, and seed size as adaptations for reducing risk in variable environments[END_REF], a plausible explanation for these pat terns is that alpine plants received selection pressure from damag ing spring frosts and evolved a frost avoidance mechanism by germinating at high temperatures [START_REF] Taschler | Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary[END_REF][START_REF] Marcante | Frost as a limiting factor for recruitment and establishment of early development stages in an alpine glacier foreland?[END_REF]Rosbakh et al., 2020b). Moreover, strict alpine species may be less subjected to drought damage during establishment compared with generalist species: as elevation increases, plants face less drought risk because precipitation increases (except in Mediterranean and semiarid mountains), but at the same time there is a higher probability of early or late sea son frosts (K€ orner, 2003;[START_REF] Van Der Schrier | European Alpine moisture variability for 1800 2003[END_REF][START_REF] Beniston | Environmental change in mountains and uplands[END_REF].

Westoby

Together, these results indicate that germination patterns in alpine species are driven by an interplay of seed size and germina tion temperature to escape either frost or drought stress: small seeds are being selected for fast germination at warm tempera tures, in order to escape unfavourable early spring frost; while large seeds are being selected for slower germination under cooler conditions, which should maximise emergence during snowmelt (when water availability is highest) giving seedlings adequate time to develop a root system to cope with desiccation risks in sum mer. Supporting this view, seedlings of alpine pioneer species have a low frost resistance [START_REF] Marcante | Frost as a limiting factor for recruitment and establishment of early development stages in an alpine glacier foreland?[END_REF], yet can tolerate heat up to 40-50°C [START_REF] Marcante | Heat tolerance of early developmental stages of glacier foreland species in the growth chamber and in the field[END_REF].

In our analysis, increasing temperatures also decreased germi nation uncertainty, showing that germination synchrony is a plas tic trait driven by temperature. While asynchronous germination is thought to be a form of adaptation to unpredictable environ ments [START_REF] Wagner | Divergent norms of reaction to temperature in germination characteristics among populations of the arctic-alpine annual, Koenigia islandica[END_REF][START_REF] Simons | Modes of response to environmental change and the elusive empirical evidence for bet hedging[END_REF], our results indicate that asynchronous germination occurs when tempera tures are relatively cool, likely as a bet hedging strategy against the risk of early spring frost. Conversely, a fast and synchronised germination at warm conditions (i.e. in late spring) may be another important strategy that increases alpine seedling survival by avoiding drier conditions in summer. The extreme germina tion uncertainty syndrome, that is staggering germination so that emergence occurs both before and after winter, is known in alpine species (K€ orner, 2003;[START_REF] Hoyle | Seed germination strategies: an evolutionary trajectory independent of vegetative functional traits[END_REF][START_REF] Satyanti | Temperature variability drives withinspecies variation in germination strategy and establishment characteristics of an alpine herb[END_REF]. If autumn seedlings grow faster than spring seedlings [START_REF] Satyanti | Temperature variability drives withinspecies variation in germination strategy and establishment characteristics of an alpine herb[END_REF], this will probably have cascading effects on individual fitness along the life of the plant [START_REF] Donohue | Niche construction through germination cueing: life-history responses to timing of germination in Arabidopsis thaliana[END_REF][START_REF] Donohue | Germination, postgermination adaptation, and species ecological ranges[END_REF].

Alternating temperatures and light

We also found a positive germination response to alternating temperatures and light, conditions that mimic the environment in the upper soil and certain microsites during the snow free sea son [START_REF] Billings | The ecology of arctic and alpine plants[END_REF]K€ orner, 2003;[START_REF] Mondoni | Climate warming could shift the timing of seed germination in alpine plants[END_REF]. Alternating temperatures had a slightly lower effect on strict alpine species, compared with generalist species, but still had some effect. This partly contradicts the findings of [START_REF] Liu | Effect of diurnal fluctuating versus constant temperatures on germination of 445 species from the eastern Tibet Plateau[END_REF], who found no response to temperature fluctuations for species distributed only at high elevations of the Qinghai Tibet Plateau. Liu and co workers argued that, in that area, high tem perature fluctuations may occur throughout the year, and there fore fluctuating temperature alone may not be a reliable indicator of suitable conditions for seedling establishment and growth [START_REF] Liu | Effect of diurnal fluctuating versus constant temperatures on germination of 445 species from the eastern Tibet Plateau[END_REF]. When analysed here at the global level, alternating temperatures generally increased germination of alpine seeds, which corresponds well with the fact that most alpine regions have a strong day/night climatic variation during the growing sea son. It is also not surprising that we detected a negative interac tion between seed mass and the germination response to light and alternating temperature regimes, as large seeds can afford to germinate at greater depths (where they are more protected from the alpine environment) and still reach the soil surface [START_REF] Bond | Seed size and seedling emergence: an allometric relationship and some ecological implications[END_REF][START_REF] Pons | Seed responses to light[END_REF].

The alpine seed germination spectrum

Using the whole set of traits compiled in this study we can visu alise the seed germination spectrum of alpine plants. At one end of the spectrum, strict alpine species tend to germinate slowly and synchronously, require warm temperatures and light to ger minate, and need stratification or GA 3 to alleviate physiological dormancy. At the other end, generalist species tend to germinate fast, show a bet hedging strategy (i.e. high germination uncer tainty) and germination traits that suggested fast germination after specific cues (e.g. scarification or alternating temperatures). The dormancy classes without a physiological component (non dormant, physical, and morphological) appear to be associated with generalist species. This gradient of regenerative strategies mirrors prominent gradients of fast or slow population dynamics [START_REF] Silvertown | A demographic interpretation of Grime's triangle[END_REF][START_REF] Nobis | Adult age of vascular plant species along an elevational land-use and climate gradient[END_REF], fast or slow developing leaf traits [START_REF] Wright | The worldwide leaf economics spectrum[END_REF], or large sets of plant ecological features [START_REF] Grime | Evidence for the existence of three primary strategies in plants and its relevance to ecological and evolutionary theory[END_REF]. 'Fast' regeneration can be understood as a plastic and opportunistic strategy to cope with frequently disturbed habitats such as avalanche ways, steep erod ing slopes, regressing glaciers, and riverbeds [START_REF] Pierce | Disturbance is the principal a-scale filter determining niche differentiation, coexistence and biodiversity in an alpine community[END_REF][START_REF] Gentili | Geomorphological disturbance affects ecological driving forces and plant turnover along an altitudinal stress gradient on alpine slopes[END_REF]. The 'slow' regeneration of strict alpine species, conversely, seems to be a more specialised syndrome and might be the result of convergent evolution acting on those species that are truly restricted to the alpine vegetation belt.

A general assumption of seed trait ecology is that seed mass and embryo : seed ratios are relatively conserved across related species [START_REF] Moles | A brief history of seed size[END_REF][START_REF] Vandelook | Relative embryo length as an adaptation to habitat and life cycle in Apiaceae[END_REF]. In our alpine dataset, both traits showed no differences between strict alpine and generalist species. This was in line with the ambiguity of rela tionships between seed mass and elevation that have been found for alpine species, including examples of negative [START_REF] Wang | Geographic variation in seed traits within and among fortytwo species of Rhododendron (Ericaceae) on the Tibetan plateau: relationships with altitude, habitat, plant height, and phylogeny[END_REF], positive [START_REF] Pluess | Seed weight increases with altitude in the Swiss Alps between related species but not among populations of individual species[END_REF] or absent [START_REF] Vandelook | Relative embryo length as an adaptation to habitat and life cycle in Apiaceae[END_REF][START_REF] Bauk | Are seed mass and seedling size and shape related to altitude? Evidence in Gymnocalycium monvillei (Cactaceae)[END_REF] correlations. The similarity in embryo : seed ratio between generalist and strict alpine species also confirmed the lack of correlation between embryo : seed ratio and elevation found in the Apiaceae [START_REF] Vandelook | Relative embryo length as an adaptation to habitat and life cycle in Apiaceae[END_REF]. By contrast with the supposed phylogenetic conservatism of these morphological traits, physiological germination responses are understood to be more plastic traits that can quickly respond to Ó Ó new situations [START_REF] Ó Ó Fern Andez-Pascual | Seeds of future past: climate change and the thermal memory of plant reproductive traits[END_REF]. While phyloge netic signal in seed germination is generally found, especially in highly selective environments (Carta et al., 2016a;[START_REF] Ar Ene | Temperature but not moisture response of germination shows phylogenetic constraints while both interact with seed mass and lifespan[END_REF]Fern andez Pascual et al., 2017b), germination patterns can also be shared between phylogenetically distant species (Vande look et al., 2019) and have low or absent phylogenetic signal [START_REF] Rosbakh | Initial temperature of seed germination as related to species occurrence along a temperature gradient[END_REF][START_REF] Fang | Seed germination of Caragana species from different regions is strongly driven by environmental cues and not phylogenetic signals[END_REF]Fern andez Pas cual et al., 2017a). The phylogenetic signal in germination responses that we found here indicates that evolutionary history cannot be neglected when studying seed germination patterns. Furthermore, we detected that seed embryo size influences ger mination responses to environmental cues, with smaller and more endospermic seeds being more responsive to warmth, light and alternating temperatures. Previous studies have shown that nonendospermic seeds are smaller and are likely to be nondor mant at the time of dispersal and thus germinate more quickly than alpine endospermic seeds [START_REF] Sommerville | Can seed characteristics or species distribution be used to predict the stratification requirements of herbs in the Australian Alps?[END_REF][START_REF] Hoyle | Seed germination strategies: an evolutionary trajectory independent of vegetative functional traits[END_REF][START_REF] Satyanti | Temperature variability drives withinspecies variation in germination strategy and establishment characteristics of an alpine herb[END_REF]. Together, these results indicate that seed morphology and physiology are connected and may be subjected to co adaptation. This connection, intertwined with their shared phylogenetic history, may constrict the potential variability of the seed ecological spectrum.

Future directions

Our first global assessment of the seed germination spectrum in relation to seed morphology supports the existence of trade offs between sets of regenerative traits [START_REF] Saatkamp | A research agenda for seed-trait functional ecology[END_REF], such as the relationship we found between seed/embryo size, germination speed and germination responses to temperature and light. Future studies will need to consider, as we did, evolutionary his tory and seed morphology as intrinsic constraints of the seed eco logical spectrum; while the seed germination spectrum we described here needs to be explored at the community level [START_REF] Enez-Alfaro | Seed germination traits can contribute better to plant community ecology[END_REF].

This study also highlights the general lack of seed germination studies on tropical alpine species. Although there is an increasing interest in the ecology of plants from tropical alpine regions, we could not find seed germination data to include in our meta anal ysis. Compared with temperate alpine ecosystems, tropical alpine environments are relatively aseasonal and most plants have been reported as flowering throughout the year [START_REF] Gehrke | Staying cool: preadaptation to temperate climates required for colonising tropical alpine-like environments[END_REF]. Rather than being exposed to snow in winter and high tempera tures in summer, tropical alpine plants face a year round stress from night time frost (K€ orner, 2003). These different selective pressures suggest that the cold adapted germination syndrome of temperate alpine seeds may not be prevalent in the tropical alpine flora. That means that our study is valid for temperate alpine regions, where alpine habitats mainly occur [START_REF] Testolin | Global distribution and bioclimatic characterization of alpine biomes[END_REF], but not necessarily for tropical alpine systems. We thus recommend future research to focus on the seed ecology of high mountain tropical areas.

Another aspect that calls for future efforts is a finer study of germination cues. To produce this meta analysis, we had to assume some simplifications in order to merge disparate datasets: we considered coarse cues that are routinely recorded in germina tion tests, like the presence/absence of light, alternating tempera tures or cold stratification. New research should aim to decompose these binary cues into finer quantitative variables which provide a more precise environmental signalling, such as the red : far red ratio [START_REF] Jankowska-Blaszczuk | Impact of red: far red ratios on germination of temperate forest herbs in relation to shade tolerance, seed mass and persistence in the soil[END_REF], the length of the photoperiod [START_REF] Black | Photoperiodic control of germination in seed of birch (Betula pubescens Ehrh.)[END_REF], the ampli tude of the diurnal thermal oscillations [START_REF] Andez-Pascual | Simulating the germination response to diurnally alternating temperatures under climate change scenarios: comparative studies on Carex diandra seeds[END_REF] or the length and temperature of cold stratification [START_REF] Pritchard | Development of a thermal time model for the quantification of dormancy loss in Aesculus hippocastanum seeds[END_REF]. In addition, new field research is neces sary to assess germination phenology in response to temperature and water availability on the upper soil layer.

Conclusions

This study demonstrates how alpine plants from different regions of the world respond similarly to germination cues that support successful regeneration: cold stratification, warm and alternating temperatures and light. These cues, particularly cold stratification and warm temperatures, are generally associated with cold adapted herbaceous species in a broad sense (Fenner & Thomp son, 2005;Baskin & Baskin, 2014;[START_REF] Rosbakh | Initial temperature of seed germination as related to species occurrence along a temperature gradient[END_REF]. Nonetheless, our results show that the response to stratification and temperature is more prominent in strict alpine species when comparing them with generalist plants that can grow opportunis tically in the alpine belt. This suggests an evolutionary conver gence of germination patterns in those species truly adapted to alpine ecosystems. We can conclude that, while alpine plants do not show a unique germination syndrome, they do show a more extreme or specialised version of the cold adapted temperate ger mination syndrome.

  Fig.1(a) Seed dormancy classes, (b) seed mass and (c) embryo to seed ratio in the subsets of strict alpine and generalist species analysed in this study. Strict species are mostly restricted to the alpine belt, generalist species can be found in the alpine belt but also below the treeline. Seed dormancy classes are abbreviated as follows: MD (morphological), MPD (morphophysiological), ND (nondormant), PD (physiological), PY (physical). Whiskers in the boxplots (b, c) extend to the most extreme data point which is no more than 1.5 times the length of the box away from the box.

Fig. 2

 2 Fig.2Effect of the germination environment on (a) final germination proportions, (b) mean germination time and (c) germination uncertainty index; according to the MCMC meta analysis of primary data. Dots indicate the posterior mean of the effect size, and whiskers its 95% credible interval. The line of zero effect is shown. When the credible intervals overlap with the zero effect line, the effect is not significant. Some effects that overlapped with zero (i.e. had no effect) and whose credible intervals were excessively wide are not shown for clarity's sake. The figure shows first the main effect, then the effects for the strict alpine and generalist groups, and then the interaction with seed mass and embryo : seed. A negative interaction (e.g. seed mass and light, in the case of the germination proportion) indicates that, as the interacting factor increases (e.g. seed mass) the response to the germination cue (e.g. light) decreases, in comparison with the main effect. GA3, gibberellic acid.

Fig. 3

 3 Fig.3Effect of the random factors on (a) final germination proportions, (b) mean germination time and (c) germination uncertainty index; according to the MCMC meta analysis of primary data. Dots indicate the posterior mean of the effect size, and whiskers its 95% credible interval. The line of zero effect is shown. GA3, gibberellic acid.

Fig. 4

 4 Fig.4Phylogenetic signal in the response to the germination environment. Dots indicate the posterior mean of lambda, and whiskers its 95% credible interval. When lambda 0, related taxa are no more similar than expected by chance; when lambda 1, the trait is evolving following a constant variance random walk or Brownian motion model. GA3, gibberellic acid; FGP, final germination proportion; MGT, mean germination time; UNC, germination uncertainty index.

Fig. 5

 5 Fig. 5 Factorial analysis of mixed data (FAMD) ordination of the alpine seed germination spectrum. Each square is a species. Labels indicate the contribution of the variables to the axes. (a, b) Quantitative traits. (c, d) Qualitative traits. GA3, gibberellic acid; MGT, mean germination time; UNC, germination uncertainty index. Seed dormancy classes are abbreviated as follows: MD (morphological), MPD (morphophysiological), ND (nondormant), PD (physiological), PY (physical). Strict species are mostly restricted to the alpine belt, generalist species can be found in the alpine belt but also below the treeline.

Table 1

 1 Description of the datasets contributing primary data to this meta analysis. The number of germination records, number of species and number of strict alpine species are given.

	Source	Coverage	Records Species Strict
	V. F. Briceno, unpublished	Chilean	236	25	5
		Andes
	Bu et al. (2007, 2008)	Qinghai	1766	242	68
		Tibet
	Cavieres & Arroyo (2000),	Chilean	804	31	6
	Cavieres & Sierra Almeida	Andes
	(2018)
	ENSCOBASE	Europe	510	124	75
	Fern andez Pascual et al.	Cantabrian	792	22	14
	(2017a)	Mts
	Liu et al. (2013)	Qinghai	465	151	42
		Tibet
	A. Mondoni, unpublished	Apennines	32	10	7
	Mondoni et al. (2009, 2012)	European	506	101	74
		Alps
	S. Rosbakh, unpublished	Caucasus	263	18	9
	Rosbakh & Poschlod (2015)	European	971	21	15
		Alps
	A. Satyanti, unpublished	Australian	632	91	41
		Alps
	Sommerville et al. (2013)	Australian	1395	13	3
		Alps
	Tudela Isanta et al. (2018)	European	376	47	29
		Alps
	Venn (2007), Venn & Morgan	Australian	1051	15	5
	(2009)	Alps

Acknowledgements

EFP received financial support from the Government of Asturias and the FP7 -Marie Curie -COFUND programme of the European Commission (Grant 'Clar ın' ACB17 19). BJ A and EFP were funded by the Spanish Research Agency (PID2019 108636GA/AEI/10.13039/501100011033). EM was supported by the Future Leaders Fellowship -Diversity and Livelihoods, of the Royal Botanic Gardens, Kew. Authors thank Amelia Martyn Yenson and Catherine A. Offord for germination and dormancy data for 19 Australian alpine species. This research was supported by NGSeeds, the Next Generation Seed Ecology, Evolution and Data Science virtual working group.

Data availability

The original data, R code for the analysis and creation of the manuscript can be accessed at the GitHub repository https:// github.com/efernandezpascual/alpineseeds. A version of record of the repository can be found at https://doi.org/10.5281/zenodo. 4270344.

Author contributions

BJ A conceived the original idea. EFP, AC, AM, LC, SR, SV, ASatyanti, LG, VFB, FV, HB, KS, PP, KL and AN contributed data. EFP assembled and managed the database. AC and EFP analysed data. EFP led manuscript writing, with contributions from BJ A, AC, AM, LC, SR, SV, ASatyanti, LG, VFB, FV, EM and ASaatkamp. All authors revised the manuscript and approved the final version.

Ó Ó


Olivier De Almeida
email: olivier.dealmeida@mines-albi.fr

Emeline Bessard

Gérard Bernhart

DSC Investigation of the influence of carbon content on peek crystallisation and stability

Keywords: PEEK, Carbon fibers, Crystallisation, Differential Scanning Calorimetry

Introduction

Properties of semi-crystalline thermoplastics are known to be heavily dependent on the degree of crystallinity and therefore on the cooling conditions. This influence of cooling rate on properties has been observed in structural thermoplastic composites, but the influence of carbon fibers on the crystallisation behavior remains poorly known. Indeed, apart from the role of carbon fibers on nucleation that has already been related in literature, its influence on the entire crystallization process is still poorly understood [START_REF] Gao | Cooling rate influences in carbon fibre/PEEK composites. Part 1. Crystallinity and interface adhesion[END_REF].

Among the semi-crystalline thermoplastics used as composite matrix, PEEK is of major interest due to its high glass transition temperature and mechanical performances. Therefore, assessing the influence of carbon fibers on PEEK crystallization is required in prospect of composite performance optimization.

Neat PEEK exhibits an unusual crystallisation behavior resulting in the apparition of two distinct melting peaks on endothermic thermograms [START_REF] Cebe | Non-isothermal crystallisation of poly(etheretherketone) aromatic polymer composite[END_REF][START_REF] Lee | Double-melting behavior of poly(ether ether ketone)[END_REF][START_REF] Bessard | Experimental and modelling of isothermal crystallization of PEEK matrix and C/PEEK composites with application of parallel Avrami model[END_REF]. The origin of this secondary melting peak is still discussed but recent studies tend to show that it would refer to an imperfect crystal structure and/or a low thickness lamellar structure [START_REF] Cebe | Non-isothermal crystallisation of poly(etheretherketone) aromatic polymer composite[END_REF][START_REF] Blundell | Spherulitic morphology of the matrix of thermoplastic peek carbon-fiber aromatic polymer composites[END_REF][START_REF] Marand | Influence of structural and topological constraints on the crystallization and melting behavior of polymers. 2. Poly(arylene ether ether ketone)[END_REF]. This phase is however always taken into account when measuring the degree of crystallinity thanks to Differential Scanning Calorimetry (DSC) traces.

The aim of this study is therefore to assess the influence of "long" carbon fibers on PEEK crystallisation [START_REF] Bessard | Matériaux composites structuraux à base de PEEK élaborés par thermocompression dynamique: relation procédé -propriétés[END_REF]. The crystallisation kinetics, heat of fusion and DSC scans are analyzed and the influence of carbon content on neat PEEK stability and crystallisation behavior is discussed.

Materials

Polymer used in this study is a Poly-Ether-Ether-Ketone (PEEK) provided as coarse powder by Victrex (grade 150). This polymer is a common grade used as matrix in structural composites. Carbon fibers are HR fibers removed from a quadriaxial Non-Crimp Fabric (NCF). The removed carbon yarns have been cut in order to obtain 5mm long carbon fibers.

Experimental procedures

Aluminum sealed pans have been prepared with 3 to 5mg of PEEK powder and different carbon fiber contents. By controlling the carbon weight ratio, DSC samples of "C/PEEK composites" were thus obtained with 18 to 72%wt of carbon fibers. It is actually assumed in this study that this annealing stage favors the blend homogenization and that the material in the DSC pans can be considered as representative composite materials. DSC measurements have been performed with a Perkin Elmer DSC 8000 apparatus. All the tests have been started by applying a 5min isothermal annealing stage at 400°C so as to perform crystallisation from a nuclei-free melt.

From this molten state, two different cooling conditions have been achieved. Isothermal crystallisation have been realized at 305°C or 310°C during 15min. A constant cooling ramp of 150°C.min -1 was used to cool the samples from melt before and after the isothermal step. Non-isothermal crystallisation has also been performed during 20°C.min -1 cooling ramps. Whatever crystallisation condition used, the melting endotherms were measured during 10°C.min -1 heating scans.

DSC INVESTIGATION OF THE INFLUENCE OF CARBON CONTENT ON PEEK CRYSTALLISATION AND STABILITY

O. De Almeida 1* , E. Bessard 1 , G. Bernhart 1 1 Université Toulouse, Institut Clément Ader, Mines Albi, Campus Jarlard, 81013 Albi Cedex 09, France

Many different results have been collected on the crystallisation exotherms and melting endotherms: crystallisation and melting temperatures, heat of fusion and degree of crystallinity, crystallisation kinetics. The transformation rate was assessed from the crystallization heat flux by using the partial area method, and the mass fraction crystallinity was determined from melting DSC traces in considering a reference enthalpy of fusion of 130 J.g -1 [23].

Influence of carbon fibers ratio

Concerning non-isothermal crystallisation, Fig 1 shows the crystallisation kinetic for different carbon ratios when subjected to a cooling ramp of 20°C.min -1 . The curves exhibit a typical sigmoidal shape which point of inflection corresponds to the maximum crystallisation kinetic. No sudden change is discernible on the sigmoidal curves, which reveal occurrence of a continuous crystallization process whatever the carbon fiber content.

For all different materials, crystallisation occurs within 2.5 minutes, but crystallisation starts earlier for samples filled with carbon fibers, which confirms the nucleation effect of the fibers surface. This crystallization precocity is all the more true that the fiber content is high.

However, the half-time crystallization kinetic (i.e. the kinetic for a relative crystallinity of 0.5) does not seem to be dependent on the carbon fibers ratio. This constant maximum crystallisation kinetic suggests that the development of the crystalline structure is not affected by the carbon fibers, except for the nucleation phase.

As already reported in the literature, PEEK has a particular crystallisation behavior characterized by the apparition of a secondary melting peak on the DSC traces. This secondary melting peak appears when samples are crystallized at low cooling rates (<10°C.min -1 ) or when samples are isothermally crystallized.

Fig. 2 and Fig. 3 show the melting DSC curves respectively resulting from isothermal (305°C) and non-isothermal crystallizations (-20°C.min -1 ) for different carbon fiber ratios. The double melting peak is clearly observable in Fig 2 for neat PEEK and, as expected, only one peak is visible for samples of neat PEEK cooled at 20°C.min -1 (Fig. 3).

The DSC traces recorded for carbon fibers composites shows that fibers do not modify the conditions of appearance of a secondary melting peak. Whatever the carbon fiber content, isothermally cooled samples exhibit two melting peaks and samples cooled at 20°C.min -1 only display one peak. Moreover, the temperature of the melting peaks remains constant with increasing the carbon fibers content.

The fact that carbon fibers do not modify both the maximum crystallization kinetics and melting temperatures but only the nucleation process suggest that the appearance of a double melting peak only depends on the crystal growth mechanisms.

Nevertheless, despite its nucleation ability, the presence of carbon fibers is not beneficial for total crystallinity. As plotted in Fig 4, the higher the carbon fiber content is, the lower is the PEEK degree of crystallinity. As a consequence, according to the literature, increasing the fiber content in composite structural parts may lead to a drop of the matrix properties. However, the decrease of the degree of crystallinity is less than 7 points and the loss in matrix properties may be sensible.

Influence of ageing on crystallization

Carbon/PEEK composites are commonly processed by autoclave or in a hot press that are rough processes for high temperature polymer matrices. Indeed, during the processing cycle, impregnation of carbon fibers requires to maintain PEEK matrix at high temperature and pressure during several tens of minutes. Due to the organic nature of PEEK polymer, degradation may occur and must be taken into account when optimizing processing conditions.

In order to evaluate the influence of ageing on the crystallisation behavior of PEEK and Carbon/PEEK composites, heating and cooling cycles have thus been achieved to samples with various carbon fiber ratios.

First, DSC samples with 47 and 59%wt of carbon fibers have been heated up and maintained during 2h at 400°C. After this ageing period, the samples have been isothermally crystallized at 310°C for 15 min using the method previously described. The crystallization kinetic has then been determined from the curves by applying the partial area method.

As shown in Fig. 5, ageing of Carbon/PEEK samples results in a variation of the crystallization behavior that show evidence of degradation. Indeed, after ageing, the sigmoidal shape is modified, and in particular crystallization duration increase with ageing and with the carbon fiber content. Moreover, ageing results in a noticeable decrease of the maximum crystallisation kinetic (slope at the inflection point) but also on a decrease of the transformed fraction at maximum crystallization kinetic.

Secondly, in order to characterize the evolution of degradation effects, short ageing cycles have been achieved to samples with various carbon fibers ratios. One cycle consisted in a 5 minutes long isothermal step at 400°C followed by a cooling phase at 20°C.min -1 and by a heating phase at 10°C.min -1 .

The melting temperature and degree of crystallinity have been systematically determined during the melting phase. Fig. 6 shows the evolution of the melting temperature and degree of crystallinity with cycles for neat PEEK and Carbon/PEEK. Neat PEEK exhibits a quite good stability after 20 ageing cycles. Its melting temperature and degree of crystallinity remains almost constant although the curves show a progressive decreasing trend through cycling. In opposition, ageing cycles induce a significant decrease of the melting temperature and degree of crystallinity in carbon/PEEK composites. It can also be noticed that, despite the fluctuations due to measurement accuracy, the effects of degradation are gradual with a linear decrease of the physical properties. This influence is particularly marked for composites with high fiber ratios. Indeed, after only 8 cycles, i.e. for a total isotherm duration of 40min, the composite with 72%wt of carbon exhibits a decrease of 3,5°C of the melting temperature and of 2,5 points of the mass degree of crystallinity.

These evolutions with ageing can be explained by the results of Day et al. Indeed, they showed that PEEK degradation is the consequence of crosslinking development between polymer chains and that air environment may increase the degradation rate [START_REF] Day | Thermal Degradation of Poly(aryl-Ether-Ether-Ketone) (PEEK): A Differential Scanning Calorimetry Study[END_REF][START_REF] Day | Thermal-Degradation of Poly(aryl-ether-ether-Ketone): Experimental Evaluation of Crosslinking Reactions[END_REF]. The increase of chains entanglement due to crosslinking may then make chains folding and arrangement more complex during crystallization, which consequence would be lower melting temperature and degree of crystallinity.

Thermal ageing has low influence on neat PEEK but such a rapid degradation is surprising in PEEK composites since it occurs in a neutral environment (DSC oven). Therefore it can be assumed that the carbon surface is responsible of degradation initiation and that the degradation rate is all the more rapid that the carbon fiber content is high. The sizing on carbon fiber surface that contains oxygen atoms may be responsible of this degradation process.

Conclusions

Performing DSC scans on PEEK matrix filled with controlled carbon fiber ratios has allowed to characterize PEEK crystallization behavior with accuracy. The results show that carbons fibers, apart from crystallization initiation and nucleation that decrease crystallization duration, do not modify the global crystallization process. In particular, the sigmoidal shape of crystallization kinetics remains constant as well as the maximum kinetic at inflection point. Surprisingly, carbon fibers however induce a lower degree of crystallinity, variation that cannot be explained by the unique change in nucleation. Moreover, if the presence of carbon fibers has limited effect on PEEK crystallization, it strongly influences PEEK stability. Indeed, after a few minutes at high temperature, it has been observed that PEEK matrix exhibits lower melting temperature and crystallinity that can be related to crosslinking development between the polymer chains.

Finally, this study shows that particular attention must be paid to the matrix evolution when processing Carbon/PEEK composites at high temperature. Indeed, the effects of carbon fibers on PEEK stability may have been minimized due to the neutral environment in DSC. Larger degradation effects can thus be expected in air environment.

Fig. 1 .Fig. 3 . 1 Fig. 4 .Fig. 5 .Fig. 6 .

 131456 Fig.1. Influence of carbon fiber content on nonisothermal crystallisation kinetic (20°C.min -1 cooling)