Eduardo Gonza ´lez •

Francisco Antonio Comı

Etienne Muller

M E Adams

F A Gonza ´lez

Comı

Seed dispersal, germination and early seedling establishment of Populus alba L. under simulated water table declines in different substrates

Keywords: Populus alba L., Rhizopod, Water table decline, Seedling establishment, Seed dispersal, Germination, Controlled floods

Populus alba L. is an autochthonous species dominating the overstory of the floodplain forests across the Mediterranean region. In contrast to some other Populus spp., very little is known about its regeneration strategies. Poplars yearly disperse huge amounts of wind-and waterdispersed, non-dormant, short-lived tiny seeds that need the bare, open and moist substrates created by fluvial-geomorphic events to germinate and establish. To survive, the growing roots must keep pace with the falling water table and associated soil moisture zone. Using a greenhouse experimental facility, 9-day-old P. alba seedlings were subjected to five hydrological treatments (permanent saturation, drawdown rates of 1, 2.5, 5 cm day -1 and immediate drainage) in two different substrates (coarse and sandy), and their survival and growth were evaluated. Also, P. alba seed dispersal was monitored in the field, and seed germinability and longevity were tested in the laboratory. No seedlings survived the water table declines in the coarse substrate although survival was high (85%) under saturated conditions. In the sandy soil, survival was significantly greater in the permanent saturation (87%) and 1 cm day -1 (88%) treatments than in the 2.5 cm day -1 (58%), 5 cm day -1 (25%) and immediate drainage (22%) treatments. The lowest root and shoot growth rates occurred under the saturated and immediate drainage conditions. Seed dispersal lasted 6-8 weeks and peaked in mid-April, initial seed germinability was high (92%) and seed longevity was relatively long (half viability period, 30 days). The creation of nursery sites and release of controlled floods in mid-April, followed by water table declines of less than 1 cm day -1 in coarse substrates and less than 5 cm day -1 in sandy substrates are recommended for enhancing the initial establishment of P. alba seedlings.

Introduction

The family Salicaceae, and especially the genus Populus, dominates most of the floodplain forests across the northern temperate zone [START_REF] Karrenberg | The life history of Salicaceae living in the active zone of floodplains[END_REF]. The periodic recruitment of new individuals by means of either sexual or vegetative propagation is essential for the maintenance of these riparian forests [START_REF] Rood | Initial cottonwood seedling recruitment following the flood of the century on the Oldman River, Alberta, Canada[END_REF]. In particular, sexual regeneration increases genetic variation, potentially increasing population resistance to environmental change and disease [START_REF] Heinze | Genetic considerations for the restoration of riparian populations[END_REF][START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF]). Many Populus spp. are considered phreatophytes as they rely on the water table and associated soil moisture zone to survive and grow [START_REF] Smith | Water relations of riparian plants from warm desert regions[END_REF]. In addition, their regeneration strategies are intimately connected to the natural hydrograph at different spatial and temporal scales [START_REF] Cooper | Multiple pathways for woody plant establishment on floodplains at local to regional scales[END_REF]. River regulation and water reclamation projects have altered the flow patterns of most rivers worldwide [START_REF] Nilsson | Fragmentation and flow regulation of the world's large river systems[END_REF], thus disrupting the flowdependent poplar life-cycle. In particular, dams and diversions modify the timing, magnitude and frequency of peak flows, reduce sediment loads, and cause abrupt water table declines, which may lead to systemic regeneration failure and overall floodplain forest decline [START_REF] Johnson | Forest overstory vegetation and environment on the Missouri River floodplain in North Dakota[END_REF][START_REF] Rood | Abrupt riparian forest decline following river damming in southern Alberta[END_REF][START_REF] Rood | Collapse of riparian poplar forests downstream from dams in Western Prairies: probable causes and prospects for mitigation[END_REF][START_REF] Johnson | Dams and riparian forests: case study from the upper Missouri River[END_REF][START_REF] Rood | Instream flows and the decline of riparian cottonwoods along the St. Mary River, Alberta[END_REF][START_REF] Stromberg | Effects of groundwater decline on riparian vegetation of semiarid regions: the San Pedro, Arizona[END_REF][START_REF] Merritt | Riparian vegetation and channel change in response to river regulation: a comparative study of regulated and unregulated streams in the Green River Basin, USA[END_REF][START_REF] Braatne | Instream flows and the decline of riparian cottonwoods along the Yakima River, Washington, USA[END_REF].

A single mature female poplar can produce annually thousands or even millions of wind-and water-dispersed, non-dormant, short-lived tiny seeds (about 1 mm long) [START_REF] Karrenberg | Phenotypic trade-offs in the sexual reproduction of Salicaceae from flood plains[END_REF] with high viability ([90%) [START_REF] Van Splunder | Establishment of alluvial forest species in floodplains: the role of dispersal timing, germination characteristics and water level fluctuations[END_REF][START_REF] Foussadier | Initiation des successions vegetals dans les lits endigues des cours d'eau Alpins[END_REF][START_REF] Karrenberg | Phenotypic trade-offs in the sexual reproduction of Salicaceae from flood plains[END_REF]. However, the minimal energy supply of the small seeds limits their longevity to a period of only days or a few weeks [START_REF] Moss | Longevity of seed and establishment of seedlings in species of Populus[END_REF][START_REF] Muller | Conservation pendant 5 ans de graines de peupliers noirs (Populus nigra L.)[END_REF][START_REF] Johnson | Woodland expansion in the Platte River, Nebraska: patterns and causes[END_REF][START_REF] Van Splunder | Establishment of alluvial forest species in floodplains: the role of dispersal timing, germination characteristics and water level fluctuations[END_REF][START_REF] Braatne | Life history, ecology and conservation of riparian cottonwoods in North America[END_REF][START_REF] Karrenberg | Phenotypic trade-offs in the sexual reproduction of Salicaceae from flood plains[END_REF], preventing formation of longterm seed banks. Therefore, the period for seedling establishment is limited to a 'time window' defined by the period of seed dispersal and seed longevity [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF]. Besides the timing constraint, the moisture requirements for poplar recruitment are also so restrictive that they are only occasionally satisfied [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF], resulting in extensive recruitment failure, sometimes for decades [START_REF] Bradley | Plains cottonwood recruitment and survival on a prairie meandering river floodplain, Milk River, southern Alberta and northern Montana[END_REF][START_REF] Johnson | Dams and riparian forests: case study from the upper Missouri River[END_REF][START_REF] Stromberg | Fremont cottonwood-Goodding willow riparian forests: a review of their ecology, threats, and recovery potential[END_REF].

Populus germination occurs rapidly (usually within a period of 24 h) when seeds land on moist sites [START_REF] Siegel | Germination requirements of key southwestern woody riparian species[END_REF][START_REF] Van Splunder | Establishment of alluvial forest species in floodplains: the role of dispersal timing, germination characteristics and water level fluctuations[END_REF]. However, because they are shade-intolerant, seedlings cannot compete with established vegetation [START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF]. Thus, seedling colonization and early establishment rely on the fresh, open and bare new alluvial deposits formed during fluvial-geomorphic events that are exposed as floodwaters recede. Consequently, the seed dispersal period of Populus spp. strategically coincides with periods when floods are most likely to occur and when the most suitable conditions for seed germination and seedling establishment are present [START_REF] White | Pattern, process, and natural disturbance in vegetation[END_REF][START_REF] Stromberg | Fremont cottonwood-Goodding willow riparian forests: a review of their ecology, threats, and recovery potential[END_REF]). Thus, Populus spp. have a definite and predictable seed dispersal period [START_REF] Johnson | Tree recruitment and survival in rivers: influence of hydrological processes[END_REF]. However, the timing and duration of the seed release period can vary inter-annually in a range of weeks as it is basically a function of photoperiod, atmospheric temperature and precipitation [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF][START_REF] Stella | Synchrony of seed dispersal, hydrology and local climate in a semi-arid river reach in California[END_REF]. Normally, within the same species, it may begin later and be shorter at higher elevations and latitudes than at lower sites [START_REF] Barsoum | Regeneration-requirements and promotion measures[END_REF]. However, [START_REF] Stella | Synchrony of seed dispersal, hydrology and local climate in a semi-arid river reach in California[END_REF] recently found later seed release in hotter downstream sites compared to upstream sites in P. fremontii and Salix gooddingii and attributed the differences to the evolutional advantage of coordinating seed release with snowmelt spring floods at the catchment scale.

The formation of nursery sites depends on the geologic and climatic setting and the local fluvial processes [START_REF] Friedman | Extreme floods, channel change, and riparian forests along ephemeral streams[END_REF][START_REF] Cooper | Multiple pathways for woody plant establishment on floodplains at local to regional scales[END_REF]. Historically, either several years of channel narrowing followed by low peak flows, or moderate to high magnitude flooding events (3-to 15-year recurrence intervals) causing channel migration and scouring existing vegetation have been associated with successful recruitment [START_REF] Bradley | Plains cottonwood recruitment and survival on a prairie meandering river floodplain, Milk River, southern Alberta and northern Montana[END_REF][START_REF] Baker | Climatic and hydrologic effects on the regeneration of Populus angustifolia James along the Animas River, Colorado[END_REF][START_REF] Howe | On the imminent decline of Rio Grande cottonwoods in central New Mexico[END_REF][START_REF] Stromberg | Flood flows and dynamics of Sonoran riparian forests[END_REF][START_REF] Johnson | Woodland expansion in the Platte River, Nebraska: patterns and causes[END_REF][START_REF] Cordes | Factors affecting the regeneration and distribution of riparian woodlands along a northern prairie river: the Red Deer River, Alberta, Canada[END_REF][START_REF] Scott | Fluvial process and the establishment of bottomland trees[END_REF][START_REF] Scott | Flood dependency of cottonwood establishment along the Missouri River, Montana, USA[END_REF][START_REF] Friedman | Extreme floods, channel change, and riparian forests along ephemeral streams[END_REF][START_REF] Braatne | Instream flows and the decline of riparian cottonwoods along the Yakima River, Washington, USA[END_REF]). However, in many regulated reaches extensive recruitment may only occur after rare, catastrophic flood events that result in overbank flooding or lead to massive geomorphic change [START_REF] Yin | Flooding and forest succession in a modified stretch along the Upper Mississippi River[END_REF][START_REF] Rood | Initial cottonwood seedling recruitment following the flood of the century on the Oldman River, Alberta, Canada[END_REF].

In order to survive, seedlings must be positioned high enough to avoid being scoured by subsequent floods, buried by sediments or suffer from permanent anaerobic conditions; and low enough to maintain contact between the growing root and the falling water table and associated moisture zone [START_REF] Stromberg | Flood flows and dynamics of Sonoran riparian forests[END_REF][START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF][START_REF] Barsoum | Regeneration-requirements and promotion measures[END_REF]. In fact, most of the seedlings die during the first year, mainly as a consequence of erosion and sedimentation from subsequent flood events, prolonged inundation, winter ice scour, insufficient light and drought-stress [START_REF] Stromberg | Fremont cottonwood-Goodding willow riparian forests: a review of their ecology, threats, and recovery potential[END_REF][START_REF] Scott | Flood dependency of cottonwood establishment along the Missouri River, Montana, USA[END_REF][START_REF] Rood | Initial cottonwood seedling recruitment following the flood of the century on the Oldman River, Alberta, Canada[END_REF][START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF][START_REF] Johnson | Tree recruitment and survival in rivers: influence of hydrological processes[END_REF]. Then, regardless of a high sexual reproductive potential, very few or no seedlings establish at most sites in most years [START_REF] Braatne | Life history, ecology and conservation of riparian cottonwoods in North America[END_REF]) and many of the successful recruits fail to survive to maturity [START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF][START_REF] Johnson | Tree recruitment and survival in rivers: influence of hydrological processes[END_REF]. In contrast, pulses of recruitment occur in favorable years [START_REF] Rood | Initial cottonwood seedling recruitment following the flood of the century on the Oldman River, Alberta, Canada[END_REF]. Although root elongation rates for first-year poplar seedlings are reported to be less than 1.3 cm day -1 (see summary by [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF], it has been empirically demonstrated that poplar seedlings can survive drawdown rates of even 8 cm day -1 [START_REF] Mahoney | A device for studying the influence of declining water table on poplar growth and survival[END_REF]. The drawdown rate thresholds for seedling survival and overall resistance of seedlings to low water tables are determined by the soil texture, which determines the thickness of the capillary fringe, the soil stratigraphy and the extra soil moisture provided by local precipitation [START_REF] Segelquist | Establishment of Populus deltoides under simulated alluvial groundwater declines[END_REF][START_REF] Barsoum | Regeneration response of black poplar to changing river levels[END_REF][START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF]. [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF] even proposed 2.5 cm day -1 as a maximum water declining rate for the optimal growth and survival of the seedlings of many Populus species.

Partly based on the drawdown rate, Mahoney and Rood (1998) created the 'Recruitment Box Model', a graphical technique aimed at defining the ideal characteristics of the river hydrograph (magnitude, timing and rate of drawdown) for seedling establishment. The model was shown to be an effective tool for restoration of riparian poplar forests when it was successfully tested in the St. Mary River in Alberta, Canada [START_REF] Rood | Revised instream flow regulation enables cottonwood recruitment along the St[END_REF][START_REF] Rood | Managing river flows to restore floodplains forests[END_REF]) and the Truckee River in Nevada, USA [START_REF] Rood | Flows for floodplain forests: a successful riparian restoration[END_REF][START_REF] Rood | Managing river flows to restore floodplains forests[END_REF]. However, specific studies on local hydro-geomorphology, seedling growth and survival and phenological analyses are needed to calibrate and import the model to other riparian species and rivers [START_REF] Horton | Water table decline alters growth and survival of Salix gooddingii and Tamarix chinensis seedlings[END_REF][START_REF] Amlin | Comparative tolerances of riparian willows and cottonwoods to water-table decline[END_REF], as there may be differences in the minimum flow magnitudes creating nursery sites, optimum water table decline rates, seed dispersal timing and germination in those environments.

Populus alba L., the European White Poplar, is an autochthonous species that has been present in the Central Europe and Mediterranean regions, since at least the Middle or Lower Pleistocene [START_REF] Roiron | Preuve de l'indige ´nat de Populus alba L. dans le Bassin me ´diterrane ´en occidental[END_REF]. It belongs to the Section Populus, a sub-genus taxonomic group of the genus Populus that includes species typical of dry habitats [START_REF] Karrenberg | The life history of Salicaceae living in the active zone of floodplains[END_REF]. In the Iberian Peninsula (SW Europe), the species occupies the riparian areas of perennial streams and large rivers at low elevations (usually \600 m) [START_REF] Lara | La vegetacio ´n de ribera de la mitad norte espan ˜ola. Centro de Estudios Experimentales de Obras[END_REF]). In the floodplain forests of the Middle Ebro River (NE Spain), P. alba is one of the dominant overstory species. Mean P. alba basal areas of 34.2 m 2 ha -1 and stem densities of 658 stems ha -1 have been measured in gallery forests older than 50 years (Gonza ´lez et al., unpublished data). However, the species is extremely rare within active recruitment sites and forests that originated after the 1950s (Gonza ´lez et al., unpublished data), when the massive regulation works began to dramatically change the hydro-geomorphologic patterns in the Middle Ebro River [START_REF] Ollero | Channel adjustments, floodplain changes and riparian ecosystems of the Middle Ebro River: assessment and management[END_REF][START_REF] Cabezas | Hydrologic and landscape changes in the Middle Ebro River (NE Spain): implications for restoration and management[END_REF]). Although P. alba is a widespread tree species in Central and Southern Europe, Northern Africa and Central and Western Asia [START_REF] Roiron | Preuve de l'indige ´nat de Populus alba L. dans le Bassin me ´diterrane ´en occidental[END_REF]), its post-dispersive sexual regeneration strategies remain totally unstudied.

In this context, the objectives of this study were twofold: (a) to determine the 'time establishment window' of P. alba within the Middle Ebro River by examining seed dispersal, germination and longevity traits and (b) to examine the effects of groundwater decline and substrate on survival and growth of P. alba seedlings using a greenhouse device for simulating alluvial water table declines. The results should help to guide future restoration plans using controlled floods.

Methods

Seed dispersal

The seed dispersal period and seed rain intensity of P. alba within the Middle Ebro River near Zaragoza, NE Spain (41°39 0 N, 0°52 0 W) were determined for the period 2006-2008 using seed traps installed under the canopy of different white poplar stands within five different meanders along a 8-km long reach. A seed trap consisted of a 625 cm 2 wooden board hung horizontally 1 m above the soil surface by four ropes attached to nearby trees. A 400 cm 2 plastic sheet, coated with adhesive Tanglefoot Ó [START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF][START_REF] Gage | Patterns of willow seed dispersal, seed entrapment, and seedling establishment in a heavily browsed montane riparian ecosystem[END_REF], was attached to the upper surface of the board and changed every 3-4 days for seed counting. Seed dispersal phenology was monitored from late-March (before seed release) until mid-June (all fruits fallen). The initial 5 traps installed in 2006 were increased to 12 in 2007 and 2008, and remained at the same position each year. In order to evaluate the effectiveness of the trap sampling design, each year from 15 March to 15 June, the seed dispersal duration was confirmed by daily visual observations of seed release along the reach.

Seed collection

Seeds were collected in early-April 2007 from natural populations of P. alba along the reach by picking ripe catkins partially bearing opened seed capsules. The collected catkins were kept in paper bags for 24 h at ambient temperature, thus allowing the capsules to open and release their seeds [START_REF] Gladwin | Survival of plains cottonwood (Populus deltoides subsp monolifera) and saltcedar (Tamarix ramosissima) seedlings in response to flooding[END_REF]. Later, the released seeds were separated from the abundant surrounding pappus by blowing air from an air nozzle through a series of sieves. Finally, the separated seeds were frozen at -20°C and stored in the dark in sealed jars containing silica gel desiccant for 9 months until they were germinated.

Seed germination and longevity

A sub-sample of seeds from three populations of P. alba was not frozen after collection and instead was used for germination and longevity tests. The tests consisted of weekly scattering of 50 seeds in Petri dishes (three replicate dishes per population) containing wet filter paper. The germinability (i.e., percent germination) was calculated by counting seeds with both an emergent radicle and two healthy cotyledons 5 days after sowing. The seeds were kept in paper bags in the dark at ambient temperature (since the first test was performed immediately after field collection) until no further germination was registered (12 weeks later). The germinability of the three populations was averaged weekly and the mean weekly average of the 13 tests was used as the overall P. alba germination rate. Seed longevity was expressed as the G 50 (i.e., the storage time after which 50% of the seeds were still able to germinate) [START_REF] Karrenberg | The life history of Salicaceae living in the active zone of floodplains[END_REF] and calculated by adjusting a sigmoidal function to the germination curve using Sigmaplot 9.0.

Greenhouse experiment

Hydrological treatments

An experimental system based on the 'Rhizopod', a device for simulating alluvial water table declines designed by [START_REF] Mahoney | A device for studying the influence of declining water table on poplar growth and survival[END_REF], was constructed in a greenhouse to study the survival and growth of P. alba seedlings under five different groundwater decline scenarios. The five water treatments were: (1) permanent saturation for 64 days with the water table 2 cm below the substrate surface, (2) drawdown rate of 1 cm day -1 for 48 days to a water table of 50 cm below the substrate surface, (3) drawdown rate of 2.5 cm day -1 for 40 days to a water table of 100 cm below the substrate surface, (4) drawdown rate of 5 cm day -1 for 20 days to a water table of 100 cm below the substrate surface and ( 5) immediate and complete drainage (ID henceforth). The experimental facility consisted in a cluster of five water tanks (1.18 m diameter 9 1.25 m long). Each tank represented a water table decline treatment and was filled with 24 plant growth tubes that were identical in design. The plant growth tubes were made from opaque PVC piping (10.3 cm inside diameter 9 1.20 m long) capped at the bottom and cracked longitudinally. Wooden frames secured the entire assembly.

Substrates

Within each tank, 12 growth tubes were filled with a river gravel substrate (d 50 = 8.5 mm) (Substrate G-Gravel) and the remaining 12 growth tubes with a predominantly sandy substrate with some gravel (56% by weight [2 mm, 32% [63 lm, 12% \63 lm; TOC 0.35%, TN 0.05%) (Substrate S-Sand). The 24 growth tubes were arranged randomly forming a 6 9 4 rectangle within each tank. Both substrates were taken from the floodplains of the Ebro River. We used natural substrate as previous research has shown that mycorrhizal associations may be important for Populus [START_REF] Lodge | The influence of soil moisture and flooding on formation of VA-endo-and ectomycorrhizae in Populus and Salix[END_REF]) and to ensure that they were similar in texture to some riparian substrates in the potential nursery sites of the Ebro River Basin. In all, the experimental design included five water treatments (tanks) 9 two substrates (G Gravel, S Sand) 9 12 tubes (replicates) = 120 tubes. In order to settle down the substrate, the growth tubes were simultaneously watered from the bottom by filling the tanks with water collected in the Ebro River. The longitudinal cracks of the growth tubes allowed water circulation within the whole system. After settling, the tubes were refilled with dry substrate and then watered again. The operation was repeated until all the tubes were topped up with substrate and no variation in the substrate level was observed for 24 h. The substrate was kept saturated by maintaining the water level 2 cm below the sediment surface during the days preceding the scattering of seeds. Small pieces of styrofoam were spread on the water surface to control algal growth and prevent water heating.

Controlled environmental parameters

The five tanks were located in a greenhouse with controlled temperature, humidity and irradiance. A 12-h day/night cycle was maintained using a set of metal halide lamps which guaranteed an approximate minimum of 3,000 lux. Temperature averaged 22°C in the daytime and 13°C at night and ranged from 11 to 35°C. The average relative humidity was 52 and 72% (day/night, respectively), and ranged from 16 to 95%. These values are similar to mid-April to mid-June climate data at the weather station in Alfranca (Zaragoza), located less than 1,000 m from the Ebro River main channel (data provided by the Regional Weather Agency, Aragon Department of Agriculture and Alimentation). Mean diurnal light intensity was 33,485 lux. Within each treatment, the soil volumetric water content at intervals of 20 cm was monitored using time domain reflectometry (TDR) by installing a cluster of probes at 10, 30, 50, 70 and 90 cm below the substrate surface in one of the 12 sandy substrate growth tubes. TDR values were recorded twice weekly. We assumed that the gravel substrate experienced minor capillary ascension compared to the sandy substrate, as it contained no fine particles. [START_REF] Mahoney | Response of a hybrid poplar to water table decline in different substrates[END_REF] already reported a 5 cm capillary rise within a gravel substrate with a typical particle size of 10 mm (similar to the proposed in this experiment), while it reached 70 and 50 cm within a sandy and a gravel-sand mixed substrate, respectively.

Sowing and thinning

On 13 January 2008, a total of 100 seeds (12,000 seeds m -2 ) were scattered on the saturated substrate surface of each growth tube. Before, a thin layer (1 cm) of peat was added to the top substrate to provide a uniform surface on which the seeds were sown. The sowed seeds were regularly misted for the following 24 h to enhance imbibition and ensure contact between seeds and substrate, thus promoting germination and establishment. On 21 January 2008, the seedlings were thinned to the 20 healthiest individuals per growth tube. The water level was kept 2 cm below the sediment surface until 22 January 2008, when the five water treatments started (day 1). After that, the water levels were adjusted twice daily to the five proposed hydrological treatments manually by either adding or draining water from the tanks.

Plant traits measurements

Seedling survival in each growth tube was recorded twice weekly until day 53. Plant height to the shoot tip (±1 mm) and number of leaves (long axis C 2 mm) of the surviving seedlings were measured weekly. Root length and biomass measurements were destructive, and required harvest operations. Among the 12 tubes for each of the ten treatments (2 substrates 9 5 water table declines), three were harvested on day 17 (harvest 1), three on day 38 (harvest 2) and the remaining six tubes at the end of the experiment on day 56 (harvest 3). For the harvest operations, each tube was lifted from the tank, laid horizontally, gently emptied on a table and the living seedlings were separated from the substrate by carefully watering the extracted soil column. The length of the major roots was measured to the nearest mm. The seedlings were then washed before the shoots and roots were oven dried at 60°C for 72 h, and weighed to the nearest tenth of a milligram. To prevent root breaking during the tube-emptying procedure, the substrate was kept wetted for 3 days before the day of harvest by completely submerging the selected tubes in an additional tank.

Statistical analyses

The data from week 3 (harvest 1), week 6 (harvest 2) and week 8 (harvest 3) were analyzed with two-factor analysis of variance with type of substrate (2 levels) and rate of water table decline (5 levels) as fixed factors; and (1) seedling survival, (2) shoot height, (3) number of leaves, (4) root length, (5) plant biomass and (6) root to shoot biomass ratio as the dependent variables. Growth tubes were used as replicates. Tukey post hoc range tests and t tests (P \ 0.05) were performed to compare the treatments. As some measurements were destructive, the number of replicates (initially 12 tubes) decreased with the three successive harvests. In addition, through the course of the experiment, all the seedlings in some tubes died and, then, the number of cases could not remain the same for all variables. Therefore, the degrees of freedom of the ANO-VAs varied among dependent variables and dates (Table 1).

Pseudoreplication

As each water table decline rate was implemented within a single water tank, other factors could confound the influence of water table treatments on seedling survival and growth. Then, precautions were taken to minimize the potential effects of pseudoreplication in the study and ensure all the conditions (aside from the drawdown rate) were identical among tanks. Each of the two substrates was mixed homogenously before being poured into the 120 growth tubes that were then randomly assigned to each tank. The five tanks were arranged randomly in the greenhouse with homogeneous and controlled environmental parameters and placed next to each other with only enough space to allow performing measurement and harvest operations. The collected seeds were mixed before sowing and distributed randomly among the 120 growth tubes.

Results

Seed dispersal

Populus alba seed rain in 2006-2008 began in early-April, peaked in mid-April and lasted 6-8 weeks (Fig. 1). The median cumulative seed rain density varied significantly across years, with 43,526, 18,843 and 9,120 seeds m -2 Áin 2006, 2007 and 2008, respectively (P = 0.018, df = 2, v 2 = 8.031, Kruskal-Wallis test). Within each trap, seed rain density varied by an average of one order of magnitude from 1 year to another. The absolute minimum and maximum cumulative seed rain recorded in any trap in any year was 889 and 97,420 seeds m -2 , respectively. Daily seedfall differed significantly among the 3 years (P = 0.000, df = 2, v 2 = 55.433, Kruskal-Wallis test) and averaged 929, 740 and 221 seeds m -2 day -1 in 2006, 2007 and 2008, respectively. Locally in a trap, the maximum daily seedfall rate in any single measurement was 13,867 seeds m -2 day -1 .

Seed germination and longevity

Populus alba seed germinability was rather high. Almost all seeds (92%) germinated immediately after collection (Fig. 2) and most ([80%) remained viable 2 weeks after they were released from the catkins. Median seed longevity (G 50 ) was 30 days, indicating potential for survival in dry conditions well beyond 1 month. Some seeds still germinated 70 days (10 weeks) after the capsules opened.

Soil moisture changes

In the greenhouse experiment, the volumetric water content (h v ) was continuously measured in the sandy substrate after the seeds had been sown (Fig. 3). The topsoil (0-20 cm) remained saturated for at least 18 days with a 1 cm day -1 water table decrease, for 7 days with a 2.5 cm day -1 decrease and for 1 day with a 5 cm day -1 decrease. In the immediate drainage (ID) treatment, the topsoil lost saturation within the first day of water table manipulations. The drawdown rate also influenced the thickness of the capillary fringe. In the 1 and 2.5 cm day -1 treatments there was always a 20 cm layer of soil over the water table level where volumetric water content was greater than 20% (h v = 0.20 m 3 m -3 ). However, the 5 cm day -1 treatment seemed to prevent effective capillary ascension. Unexpectedly, the immediately drained substrate retained some moisture during the first weeks, especially in the lower soil layers. In addition, moisture did not always follow a steady depth gradient, probably because of the uneven presence of gravel along the substrate column. For logistical reasons, a continuous water table decline in the treatments 2.5 and 5 cm day -1 was not possible for the 8 weeks of experiment. Lower soil moisture in deep soil layers would have been expectable with lower final water table levels and might have affected seedling growth. Likewise, the 3 days of submersion before harvesting, necessary to prevent root breaking, might have slightly stimulate seedling growth.

Seedling survival

Seedling survival (Fig. 4) was lower in gravel than in sand (substrate effect; Table 1), except when the substrates were saturated (water 9 substrate interaction; Table 1). Survival was also lower with faster water level decline rates (water effect; Table 1), especially in gravel (water 9 substrate interaction; Table 1). By the end of the experiment, the only surviving seedlings in the gravel substrate were those grown under saturated conditions, but survival in that treatment was high (85%). In the sandy substrate, in contrast, survival was no lower in the 1 cm day -1 treatment than in the saturated treatment, and *20% of seedlings survived even in the 5 cm day -1 and ID treatments.

Shoot height

Seedling height (Fig. 5a) did not significantly differ among substrate types at the end of the experiment (substrate effect; Table 1), although several of the treatments had no remaining seedlings in the gravel substrate, and therefore the comparison was only across the saturated treatments. Also, the decrease in degrees of freedom due to harvests and mortality provided too little power to discern water 9 substrate interactions (Table 1). The lowest shoot heights were observed in the ID and saturation treatments (water effect; Table 1).

Number of leaves

Like shoot height, the number of leaves (Fig. 5b) was not significantly different among substrates (substrate effect; Table 1) at the end of the experiment. Similarly, widespread mortality in the gravel substrate limited the comparison among substrates to saturated conditions and reduced power to discern water 9 substrate interactions (Table 1). Again, the ID, and to a lesser extent, the seedlings subjected to the saturation treatments produced significantly less leaves than the seedlings subjected to the 1, 2.5 and 5 cm day -1 treatments (water effect; Table 1).

Root length

In the first and second harvest, root length (Fig. 6a) was lower in the drier treatments (water effect; Table 1). At the end of the experiment, roots grew significantly longer in the 1, 2.5 and 5 cm day -1 treatments than in saturation and ID conditions (water effect; Table 1). Root length was lower in saturated sand than in saturated gravel (substrate effect; Table 1), but widespread mortality in gravel made impossible to discern water 9 substrate interactions (Table 1). Some of the longest roots reached the 60 cm depth (growing rate *1 cm day -1 ).

Biomass

Although significant differences in total biomass were observed between treatments in the first and second harvest, they were difficult to interpret (Fig. 6b; Table 1) and may have been due to the difficulties of precisely washing and weighting the tiny harvested shoots and roots. However, at the final harvest, seedling biomass was greater in the 1, 2.5 and 5 cm day -1 treatments than in the ID and saturation treatments (water effect; Table 1). There were no significant differences in biomass among substrate types (substrate effect; Table 1). Like biomass, root to shoot ratios had no clear trends in the first and second harvest (Fig. 6c). However, at the end of the experiment, the seedlings subjected to permanent saturation had lower root:shoot ratio than the other four treatments (water effect; Table 1). In particular, the seedlings subjected to permanent saturation conditions allocated less biomass to roots than to shoots (root:shoot ratio \1), while the seedlings subjected to the four declining treatments concentrated more resources to the root system (root:shoot [1). In all cases, there was no effect of substrate type on total biomass and root to shoot ratios (substrate effect; Table 1), but, as mentioned before, comparisons could only be made across saturated conditions due to widespread mortality in gravel.

Discussion

Seed dispersal periods reported in the literature range in duration from 2 to 9 weeks for P. nigra L., 4 to 9 weeks for P. fremontii, 3 to 12 weeks for P. deltoides Marsh and 6 weeks for P. balsamifera L., depending on the study area (see summary by [START_REF] Guilloy-Froget | Dispersal, germination, and survival of Populus nigra L. (Salicaceae) in changing hydrologic conditions[END_REF]. A long seed dispersal period should be especially favorable for seedling establishment in a Mediterranean river such as the Ebro River, characterized by a considerable intra-and interannual flow discharge variability [START_REF] Ollero | Channel adjustments, floodplain changes and riparian ecosystems of the Middle Ebro River: assessment and management[END_REF]. Thus, the 6to 8-week seed dispersal period we observed for P. alba, which is somewhat above the average duration period of Fig. 4 Seedling survival in sandy and gravel substrates throughout the course of the experiment with five water table decline treatments. Each point represents the mean of 12 growth tubes (until day 14), 9 growth tubes (until day 35) and 6 growth tubes (until day 53). The error bars represent ±1 standard error of the mean. Letters denote significant differences between treatments at the end of the experiment (two-way ANOVA followed by post hoc t tests P \ 0.05). Background gray bands indicate the days of harvest operations Populus spp., might partly explain the historical common presence of P. alba along the Middle Ebro River (Gonza ´lez et al. unpublished data). In addition, the early spring seed release coincides with the period when floods are more likely to occur within the Middle Ebro River, as a result of rainfall and snowmelt in the bordering mountain ranges. Indeed, peak seed dispersal was close to mid-April, i.e., earlier than other Salicaceae spp. in other regions of the world [START_REF] Guilloy-Froget | Dispersal, germination, and survival of Populus nigra L. (Salicaceae) in changing hydrologic conditions[END_REF]. The timing of seed release did not vary greatly from 1 year to another, only hastened or delayed by 1 or 2 weeks. Although the photoperiod, air temperature and precipitation do not vary greatly within the Middle Ebro River (a 346 km reach), our results should be taken with caution when applying to other sections of the river and to other watersheds, as the study reach was relatively small (8 km). In this sense, future studies aimed at modeling the P. alba seed dispersal timing as a function of temperature and other climatic factors are recommended [START_REF] Stella | Synchrony of seed dispersal, hydrology and local climate in a semi-arid river reach in California[END_REF].

Mean seed rain rates in P. alba were two orders of magnitude higher than those described for P. deltoides by [START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF], who also used TanglefootÓ coated traps. This difference could be due to the fact that [START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF] installed seed traps in potential seedling establishment sites instead of directly beneath the seed sources. Nevertheless, the extremely dense seed rain reported here suggests that seed production is not a limiting factor for P. alba recruitment, even though the spatial and temporal variabilities in seed rain intensity were notable. [START_REF] Gage | Patterns of willow seed dispersal, seed entrapment, and seedling establishment in a heavily browsed montane riparian ecosystem[END_REF] also reported high inter-annual and spatial variability in seed production by six Salix spp. in the Fall River and Big Thompson River, Colorado, USA.

As expected, initial germinability of P. alba was almost complete (92%). Seed longevity (G 50 = 30 days) was longer than reported for P. nigra by [START_REF] Van Splunder | Establishment of alluvial forest species in floodplains: the role of dispersal timing, germination characteristics and water level fluctuations[END_REF] and [START_REF] Karrenberg | Phenotypic trade-offs in the sexual reproduction of Salicaceae from flood plains[END_REF] (G 50 = 15 and 23 days, respectively) but much shorter than the G 50 = 85 reported for P. deltoides by [START_REF] Fenner | Observations on seeds and seedlings of Fremont cottonwood[END_REF]. However, seeds germinate as soon as they contact a moist surface and then, weathering forces (i.e., rainwater, air humidity, dewdrops, etc.) probably substantially reduce seed longevity under field conditions [START_REF] Karrenberg | Phenotypic trade-offs in the sexual reproduction of Salicaceae from flood plains[END_REF]. Thus, the 'time window' for establishment may be determined by seed dispersal timing [START_REF] Karrenberg | The life history of Salicaceae living in the active zone of floodplains[END_REF] and local hydrological conditions [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-an integrative model[END_REF] rather than seed germinability or longevity.

The results of the greenhouse experiment show that the substrate type and water table level can influence P. alba seedling survival and growth substantially. Overall, seedlings responded best (in terms of both seedling survival and growth) to the sandy substrate and the 1 cm day -1 water table decline treatment. The absence of fine particles within the gravel substrate apparently prevented effective capillary ascension of water, resulting in widespread mortality Letters denote significant differences between treatments at the end of the experiment (two-way ANOVA followed by post hoc t tests P \ 0.05). Background gray bands indicate the days of harvest operations in the declining and ID treatments, as roots could not maintain contact with the falling water table. The highest mean root growth rates ranged from *4 mm day -1 (sand, 5 cm day -1 ) to *5 mm day -1 (sand, 1 cm day -1 ), which are similar to those reported by [START_REF] Mahoney | A device for studying the influence of declining water table on poplar growth and survival[END_REF] for the natural poplar hybrid P. deltoides 9 P. balsamifera and close to the 6 mm day -1 proposed by [START_REF] Fenner | Observations on seeds and seedlings of Fremont cottonwood[END_REF] for P. fremontii. But even the highest root elongation rates recorded in this experiment (*1 cm day -1 ) were insufficient to cope with the water table declines (C1 cm day -1 ) in gravel. However, the fact that there were no significant survival and growth differences between the substrates under saturated conditions (aside from root length) suggests that the gravel substrate per se is not limiting when enough water is available. Within the finer substrate, the capillary fringe provided the necessary soil moisture to support considerable percentage of seedlings (including in the ID treatment). By the end of the experiment, the surviving seedlings in the 2.5, 5 cm day -1 and ID treatments were rooted in soil layers with volumetric water content (h v ) \0.10 m 3 m -3 , similar to the results of [START_REF] Segelquist | Establishment of Populus deltoides under simulated alluvial groundwater declines[END_REF], who suggested that 2% soil moisture (h v = 0.02 m 3 m -3 ) is the minimum water requirement for seedling survival and growth.

It seems that both immediate drainage and permanent saturation conditions are not compatible with P. alba seedling establishment, but that the species tolerates a broad range of water table declines of up to 5 cm day -1 rather than just an optimal decline rate, at least if the substrate allows effective capillary ascension. On the one hand, although seedling survival was surprisingly not different between the 5 cm day -1 and ID treatments, poor seedling growth in the ID treatment suggests that the seedlings might have failed to survive to maturity under those conditions. On the other hand, saturation limited shoot and especially root growth, which is consistent with previous studies. In particular, [START_REF] Fenner | Observations on seeds and seedlings of Fremont cottonwood[END_REF] and [START_REF] Segelquist | Establishment of Populus deltoides under simulated alluvial groundwater declines[END_REF] described greater root elongation with declining water tables than under saturated conditions in P. fremontii and P. deltoides, respectively. [START_REF] Mahoney | Response of a hybrid poplar to water table decline in different substrates[END_REF] detected slower growth rates in P. balsamifera 9 P. deltoides hybrids growing under a constant water table and attributed that to root anaerobiosis. Surprisingly, while seedling survival decreased progressively with steeper water table declines, seedling growth (shoot height, number of leaves, biomass and root length) did not significantly differ between the 1, 2.5 and 5 cm day -1 water table treatments among surviving individuals. This paradox may reflect an increasingly positive stress response with regard to growth as the water table decline rates increased. An alternative explanation might be that only the seedlings that could achieve certain growth rates could survive the more abrupt drawdown rates.

River regulation has dramatically altered geomorphic dynamics in the Middle Ebro River. The change in geomorphic patterns has occurred sequentially in three main periods: (1) pre-regulation period until ca. 1950, with braided-meandering channel and unstable riverbanks; (2) regulation period until ca. 1980, with progressive degree of stabilization in the meandering channel and riverbanks, large reservoirs and numerous defenses built and (3) stabilization period until present time, with very reduced channel migration, restricted meandering channel and stable riverbanks [START_REF] Ollero | Channel adjustments, floodplain changes and riparian ecosystems of the Middle Ebro River: assessment and management[END_REF][START_REF] Cabezas | Hydrologic and landscape changes in the Middle Ebro River (NE Spain): implications for restoration and management[END_REF]). As a result, the area covered by barren sites (potential nursery sites) has decreased from 13.2 to 1.8% of the 10-year floodplain in the period 1946-2003[START_REF] Cabezas | Hydrologic and landscape changes in the Middle Ebro River (NE Spain): implications for restoration and management[END_REF]). Regulation has also notably decreased the frequency and magnitude of overbank floods [START_REF] Cabezas | Hydrologic and landscape changes in the Middle Ebro River (NE Spain): implications for restoration and management[END_REF]). However, the drawdown rates from April to June have not changed significantly with regard to the pre-regulation period (Fig. 7). They correspond to a median drawdown rates of 3.1, 2.6 and 2.3 cm day -1 in the pre-regulation, regulation and stabilization period, respectively (P = 0.432, df = 2, v 2 = 1.679, Kruskal-Wallis test). Apparently, the observed low recruitment of P. alba might be due to the reduction in the frequency of high floods and to the severe loss of geomorphic dynamism but not to too abrupt drawdown rates.

Many restoration projects have difficulties in providing nursery sites for phreatophyte recruitment and instead approach restoration by planting cuttings, saplings or adult stems [START_REF] Rood | Flows for floodplain forests: a successful riparian restoration[END_REF]. Nevertheless, this study shows that P. alba is a potentially valuable species for restoration based on patterns of sexual recruitment, i.e., long seed dispersal periods with peaks coinciding with early spring floods, high initial seed germinability and longevity and tolerance of early seedlings to a relatively broad range of water table decline rates. In particular, the promotion of fluvial-geomorphic events occurring in mid-April, followed by smooth water table declines of less than 1 cm day -1 in gravel substrates and less than 5 cm day -1 in sandy substrates might enhance early recruitment of P. alba seedlings. In cases where the geomorphological dynamics are highly constrained by regulation and the release of catastrophic floods is not socially admissible (e.g., the Middle Ebro River), the creation of nursery sites for P. alba might be achieved by mechanically disturbing the substrate and removing the competing vegetation on potential recruitment sites directly before seed release peaks (from late-March to early-April in the Middle Ebro River). Later, the created barren sites would need to be locally flooded to disperse the seeds and provide adequate moisture for seeds germination and seedlings establishment [START_REF] Barsoum | Regeneration-requirements and promotion measures[END_REF], and then would need to be drained according to the proposed drawdown rates. The recruitment zones would also need to be protected from grazing, as livestock has been shown to trample seedlings [START_REF] Rood | Initial cottonwood seedling recruitment following the flood of the century on the Oldman River, Alberta, Canada[END_REF] and prefer Populus spp. over other, less palatable species such as Tamarix spp. [START_REF] Stromberg | Dynamics of Fremont cottonwood (Populus fremontii) and saltcedar (Tamarix chinensis) populations along the San Pedro River, Arizona[END_REF].

However, this restoration approach may not always be effective as sexual recruitment is a complex process. Other factors such as post-colonization floods [START_REF] Johnson | Woodland expansion in the Platte River, Nebraska: patterns and causes[END_REF][START_REF] Johnson | Tree recruitment and survival in rivers: influence of hydrological processes[END_REF][START_REF] Dixon | Simulated recruitment of riparian trees and shrubs under natural and regulated flow regimes on the Wisconsin River, USA[END_REF], inter-specific competition [START_REF] Sher | Competition between native Populus deltoides and invasive Tamarix ramosissima and the implications for re-establishing flooding disturbance[END_REF][START_REF] Roelle | Establishment, growth, and early survival of woody riparian species at a Colorado gravel pit[END_REF], shade tolerance [START_REF] Sacchi | The relative roles of abiotic and biotic factors in seedling demography of Arroyo willow (Salix lasiolepis: Salicaceae)[END_REF][START_REF] Cooper | Factors controlling the establishment of Fremont Cottonwood seedlings on the Upper Green River, USA[END_REF]), short-term climatic fluctuations [START_REF] Baker | Climatic and hydrologic effects on the regeneration of Populus angustifolia James along the Animas River, Colorado[END_REF]) and herbivory (Andersen and Cooper 2000) have not been addressed in this study and may determine the fate of initially established seedlings. It would also be interesting to examine the potential of vegetative recruitment of P. alba, and its relative importance compared to sexual recruitment. Legionnet et al. (1997), [START_REF] Gom | Patterns of clonal occurrence in a mature cottonwood grove along the Oldman River, Alberta[END_REF] and [START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF] described important vegetative propagation in some Populus species, but, to our knowledge, there is no study on P. alba vegetative reproduction in the existing literature. Moreover, in the context of global warming, P. alba may increase in abundance within temperate Northern river floodplains where it is only poorly represented today. To fully understand P. alba regeneration strategies and better inform future restoration efforts, studies are needed that monitor seedling survival and growth in natural settings and that relate historical recruitment events to hydrologic regimes and geomorphological processes. In the meantime, additional greenhouse and laboratory studies may provide Fig. 7 a Monthly average discharge and b magnitude-frequency plots illustrating reduced frequency of high-discharge events in the unregulated period (1927-1957), regulation period (1957-1981) and stabilization period at the gauging station of Zaragoza, Middle Ebro River further insights into the regeneration strategies of this riparian tree species.

Fig. 2

 2 Fig. 2 Percent germination of seeds stored for increasing lengths of time. Each point represents the mean of three natural populations. The error bars represent ±1 standard error of the mean. Each population mean was obtained from three replicate Petri dishes with 50 seeds each. The line denotes the adjusted sigmoidal function to the germination curve

Fig. 5 a

 5 Fig.5a Plant height to the shoot tip (±1 mm) (cm) and b number of leaves (long axis C2 mm) of the seedlings growing in sandy and gravel substrates throughout the course of the experiment with five water table decline treatments. Each point represents the mean of surviving seedlings in the growth tubes at each date. The error bars represent ±1 standard error of the mean. Letters denote significant differences between treatments at the end of the experiment (two-way ANOVA followed by post hoc t tests P \ 0.05). Background gray bands indicate the days of harvest operations

Fig. 6 a

 6 Fig. 6 a Root length (cm), b total plant biomass (mg) and c root to shoot ratios (mg/mg) at harvest on days 17, 38 and 56. Vertical bars represent the mean of each plant measurement. The error bars represent ±1 standard error of the mean. Lowercase letters denote significant differences between treatments (two-way ANOVA followed by Tukey post hoc range tests P \ 0.05) (S sand, G gravel)

Acknowledgments The authors are grateful to C. Aniento, A. Barcos, B. Bueno, M.L. Dehesa, A. Frutos, B. Gallardo, M. Garcı ´a, M. Gonza ´lez, D. Jime ´nez, L. Mancebo, L. Miguel, T. Pascual, S. Presa, M. Trabucchi and A. Villarroya for their assistance in the field surveys, rhizopods construction, seed collection, sorting, harvest and careful measurements of the seedlings. We also express our gratitude to J.L. Arru ´e and D. Moret, who assessed the designing and manufacturing of the TDR probes; and to the Professor C.S. Crawford and two anonymous reviewers who provided helpful comments on manuscript drafts. A. Vallejo, J.A. Pescador and all the members of the Field Research Support Unit, Estacio ´n Experimental de Aula Dei, CSIC provided essential logistic support. This research was funded by the Department of the Environmental Science, Technology and University, Aragon Government (Research group E-61 on Ecological Restoration), and Ministry of Science and Innovation of Spain, MICINN (CGL2008-05153-C02-01/BOS). The first author was funded by the former Ministry of Education and Science of Spain, MEC.

Note that the degrees of freedom (df) of each analysis decreased with each harvest and differed among variables as a result of destructive harvests and seedling mortality. In addition, the df of the W 9 S interaction declined when all replicates for some water


M Ormaza
email: ormaza@ipcms.unistra.fr

P Abufager

B Verlhac

N Bachellier

M.-L Bocquet

N Lorente

L Limot
email: limot@ipcms.unistra.fr

Controlled spin switching in a metallocene molecular junction

Paseo Manuel de Lardizabal 4, 20018 Donostia-San Sebastián, Spain (Dated: June 15, 2017) The active control of a molecular spin represents one of the main challenges in molecular spintronics. Up to now spin manipulation has been achieved through the modification of the molecular structure either by chemical doping or by external stimuli. However, the spin state of a molecule when adsorbed on a surface depends primarily on the interaction between its localized orbitals with the electronic states of the substrate. Here we change the effective spin of a single molecule by controllably modifying the molecule/metal interface using a low-temperature scanning tunneling microscope. A nickelocene molecule reversibly switches from a spin 1 to 1/2 when varying the electrode-electrode distance in a single molecular junction from tunnel to contact regime. This switching is experimentally evidenced by inelastic and elastic spin-flip mechanisms observed in reproducible conductance measurements and understood using first principle calculations. Our work demonstrates the active control over the spin state of single molecule devices through interface manipulation.

The ability to modify the spin of a molecule in a controlled and reversible way is essential to develop novel spin-based technologies [1]. Although the spin of surface-supported single molecules is mainly determined by molecule/surface interactions [2][3][4][5], it can be modified by external parameters such as the mechanical [6] or the chemical modification of the molecule [7][8][9][10][11][12], the application of electric fields [13][14][15], light or temperature [16,17]. The effect of approaching the metallic tip of a scanning tunneling microscope (STM) to the molecule is a way to alter the local environment of the molecule [18,19]. It can result in structural relaxations [20], or even modify the molecular configuration through the application of bias pulses [21], ultimately changing the spin state of the molecule. However, determining the factors leading the spin modification of a molecule when adsorbed on a surface or when coupled between metallic electrodes is a complex process in which different subtle contributions need to be considered.

Scanning tunneling spectroscopy (STS) is a useful tool to determine the spin of surface-supported magnetic single objects by exploring the zero-bias anomalies produced by different spin-flip scattering mechanisms [22,23]. While the Kondo screening involves the elastic spin-flip scattering of conduction electrons with the magnetic impurity below a critical temperature T K [START_REF] Hewson | The Kondo Problem to Heavy Fermions[END_REF], the magnetic anisotropy of single objects leads to an inelastic scattering of these electrons. In fact, the magnetic anisotropy of surface-supported objects -atoms or molecules-has been attracting a growing interest in relation to ultra-dense storage technology [START_REF] Gambardella | [END_REF]26] and quantum computing [27,[START_REF] Gatteschi | Molecular Nanomagnets[END_REF]. From a practical viewpoint, a uniaxial magnetic anisotropy energy of the form DS 2 z provides stable magnetic states into which information can be encoded and may, moreover, be externally controlled [12,21,[START_REF] Hirjibehedin | [END_REF][30][31][32][33][34][35][36]. The general picture to address is that single objects have the potential for both magnetic anisotropy and the Kondo effect [37], the outcome of their interplay depending on the object's spin [23,[38][39][40] and on the relative weight of k B T K versus D [41,42].

Elastic and inelastic tunneling spectra can be used to probe the above mentioned interplay in an environment that can be characterized with atomic-scale precision. In recent experiments [12,[33][34][35], different surface topologies were used to tune the Kondo exchange interaction between the spin and the underlying metal, while spin control was possible through hydrogen doping of transition metal atoms. Here, we control the spin of a single metallocene molecule in a junction using a STM. We attach a S = 1 molecule with an easy-axis magnetic anisotropy (D > 0), nickelocene [Ni(C 5 H 5 ) 2 , noted Nc hereafter], to the tip apex of a STM and form an atomically precise contact with a Cu(100) surface. The molecular spin switches from 1 to 1 2 when we go from the tunnel to the contact transport regime, as monitored by the two-order of magnitude change in the differential conductance near the Fermi level. This abrupt difference comes from the different nature of the spin-flip scattering that governs the conductance around the Fermi energy of the junction in the two regimes. Our findings are corroborated by density functional theory (DFT)

calculations.

An image of the Cu(100) surface after a molecular deposition is shown in Fig. 1(a), in which isolated Nc decorate the surface terraces. The molecules show a ring-shaped pattern, indicating that one cyclopentadienyl ring (C 5 H 5 , noted Cp hereafter) is exposed to vacuum, i.e., the long molecular axis of Nc is perpendicular to the Cu(100) surface [43].

We found that in order to contact nickelocene between both electrodes, surface and tip, it is more stable to first transfer the molecule from the metallic surface to the tip -details of the molecule transfer to the metallic tip can be found in [36]. Prior to molecular transfer, care was taken to select a monoatomically sharp tip apex [44]. Information may be gathered on the status of Nc at the tip apex by acquiring counter-images [45], which consist in imaging single atoms on the surface with the molecular tip. The typical molecular pattern observed in the counter images of Cu adatoms is presented in Fig. 1 We then engineered a molecular junction by bringing the Nc-tip into contact with Cu(100). For an increased control over the molecular junction, the contact was always performed atop a copper surface atom thanks to the use of atomically-resolved images [Inset of Fig. 2 (b)]. These images were routinely acquired by scanning the molecular tip while in contact with the surface [47,48], which may be taken as an indication of the robustness of a Nc-tip to external strain. The right panel of Fig. 2 (a) presents the conductance (G) versus tip displacement (z) curve acquired at a fixed bias of -2 mV with a Nc-tip vertically displaced towards the surface. The tip is here moved from its initial tunneling position z = -2.1 Å(G = 2 × 10 -4 in units 2e 2 /h) up to z = 0 (G = 0.04) where an abrupt increase of G by more than a factor 10 (G = 0.7) reveals the transition between the tunneling and the contact regime (indicated by a dashed line). Notice that the exact values of the conductance as well as of the contact position under the same conditions present some tip dependancy. As we show below, the sudden change in G is exclusively driven by a spin switch of nickelocene.

To elucidate this finding, we present in the left panel of Fig. 2 (a) a two-dimensionnal intensity plot of a series of dI/dV spectra acquired at varying z. For completeness, Fig. 2 (b) presents the spectra for decreasing tip-surface distances. The spectra reveal the presence of spin-flip scattering events, whose nature may be reversibly controlled via z. Within the tunneling regime, the spectra exhibit inelastic spin-flip excitations as anticipated in Fig. 1(d). We note an enhancement of the differential conductance at voltages corresponding to the excitation threshold, which is increasingly pronounced as the contact point is approached (z > -0.5 Å). Recent studies have pointed out that this enhancement could reflect many-body effects including Kondo-like phenomena [23,39,40]. However, this scenario also requires a sizable reduction of the magnetic anisotropy energy, and therefore of the excitation threshold [12,33], at variance with our experimental observations. The enhancement here rather reflects a stationary population of excited states [23,[49][50][51] due to the increasingly large currents flowing through the junction when approaching contact. This so-called spin pumping can be expected to be significant due to the efficiency of the spin-flip excitations in Nc produced by tunneling electrons [36], or more generally, in molecules [52][53][54].

The most striking result is found once the molecular contact to the surface is established (z > 0). As we anticipated above, a sudden increase in the conductance occurs at zero bias [Fig. 2 (a)], which we ascribe to the presence of a Kondo resonance. As shown in Fig. 2 (a), a sharp peak appears in the dI/dV , while the inelastic excitation thresholds are lost. The emergence of a spin-1 Kondo effect in the presence or in the absence of positive magnetic anisotropy is in principle possible [41,42] -the former case being unlikely here as the resonance does not split apart [31]. However, we find that the system actually behaves as a spin 1/2-system. The peak is perfectly fitted by a Frota function (see Supplementary Material), which is close to the exact line shape expected for a spin-1/2 Kondo system [55]. From the fit, we find that the peak is centered at ϵ K = (0.0 ± 0.1) meV for all studied tips up to the highest tip excursion explored (0.8 Å). The line width, therefore T K , increases nearly exponentially with z [Fig. 2(c)], similarly to other junctions comprising a single Kondo impurity [56][57][58]. To further confirm the spin-1/2 nature of the Kondo effect, we recall that the resonance amplitude (noted σ) should be a universal function of the normalized temperature T /T K . For a quantitative analysis we therefore fit the curve in Fig. 2 (d) to the function σ = [1 + (T /T K ) 2 (2 1/s -1)] -s (in units of 2e 2 /h) [59] and extract s = (0.29 ± 0.02), in remarkable agreement with the spin-1/2 Kondo effect of semiconductor quantum dots [60]. The amplitude of the resonance is close to the unitary limit and indicates a complete Kondo screening [61]. Our findings therefore strongly support the emergence of a spin-1/2 Kondo effect where there is no magnetic anisotropy. To elucidate its origin, here below, we show through DFT calculations that Nc switches its spin from 1 to 1/2 upon contact with the surface. From Fig. 3 (f) we can obtain the ratio between the transmission probabilities for the different spin channels (spin-up T ↑ , spin-down T ↓ ), which is related to the spin polarization efficiency of the molecular junction. We find a -74% spin polarization for the junction in the tunneling regime and -55% in contact, where the spin polarization is

T ↑ -T ↓ T ↓ +T ↑ .
The decreased value at contact is in agreement with the trend found in a previous theoretical study [62]. objects modify the molecular magnetism of these molecules.

I. SUPPLEMENTARY MATERIAL

In the supplementary material we find details about the experimental methods, sample preparation, STM/STS measurements and Kondo fitting procedure. Details on the theoretical results, including calculation details, structural details of the molecule and the comparison of the PDOS results obtained using two different methods (VASP and

  Fig. 1(a)]. The pattern reveals that the tip is terminated by a Cp ring of a tilted Nc molecule, the tilt angle exhibiting some tip dependency. To confirm this assignment, we mimicked the molecular tip through DFT calculations by considering a Nc molecule adsorbed on a Cu atom on Cu(100) [inset of Fig. 1(c), see Supplementary Material]. The molecule is undeformed, tilted by 13 • with respect to the surface normal and linked through two C atoms to the Cu atom [46]. The corresponding simulated image in Fig. 1(c) is in good agreement with the experimental counter-image of Fig. 1(b).The top panel of Fig.1(d) presents the dI/dV spectrum acquired with a Nc-tip above Cu(100). For comparison, we have included the dI/dV spectrum acquired with a metallic tip above a Nc molecule on Cu(100). Both spectra were acquired with a lock-in amplifier using a frequency of 716 Hz and an amplitude of 150 µV rms; the metallic tip was verified to have a flat electronic structure in the bias range presented. The steps, symmetric with respect to zero bias, that are visible in the dI/dV correspond to the manifestation of efficient spin-flip excitations within the molecule[36].These occur between the ground state |S = 1, M = 0⟩ and the doubly degenerate |S = 1, M = ±1⟩ excited states of the molecule, the threshold energy observed corresponding to the longitudinal magnetic anisotropy energy, D. The bottom panel shows the numerical derivative of the dI/dV spectra in order to facilitate the identification of the threshold values. For Nc on the surface we find D = (3.2 ± 0.1) meV, while for Nc on the tip the magnetic anisotropy grows to D = (3.7 ± 0.3) meV. This reflects changes in the local ligand field induced by the different adsorption configuration[21,32,36].

  FIG. 1. (a) STM image acquired with a metallic tip (5.3 × 5.3 nm 2 , -15 mV, 20 pA). (b) Counter image of the Cu atom obtained with a Nc-terminated tip (1.7 × 1.3 nm 2 , -15 mV, 20 pA). (c) Calculated relaxed configuration of a Nc molecule on top of a Cu adatom on Cu(100), together with the simulated STM image. Atom colors: Cu (yellow), C (grey), H (white) and Ni (green). (d) dI/dV spectra and their derivative acquired with a Cu-tip over a Nc molecule (solid blue line) and with a Nc-terminated tip over the Cu(100) surface (solid red line). Feedback loop opened at 100 pA and -15 mV.

FIG. 2 .

 2 FIG. 2. (a)Left pannel: 2D intensity plot of dI/dV spectra acquired with a Nc-tip at increasing z. The intensity of the spectra have been normalized by the opening conductance value (at -30 mV) of the spectrum at z=0 Å. As a guideline to identify the differences, three characteristic spectra (white dashed lines) are superimposed. Right pannel: G-versus-z curve measured at -2 mV for a Nc-tip approaching the Cu(100) surface. The boundary between the tunnel and the contact regime occurs at z=0 pm and is indicated with a dashed line. (b) Individual dI/dV spectra in the tunnel (bottom panel) and contact (top panel) regimes for several z. Note that right after contact, the width of the Kondo resonance is smaller than the inelastic excitation threshold. Inset: Contact image of the Cu(100) surface using a Nc-terminated tip (2 × 1.4 nm 2 ; 300 pA, 30 mV). The cross indicates the contact point. (c) Evolution of TK with z. The Kondo temperature is 4.5 K or lower at z = 0, and 63.4 K at z = 1 Å. (d) Evolution of the resonance maximum σ with T /TK, where T = 2.4 K is the working temperature. σ and TK are extracted from the Frota fits (see Supplementary Material).

The

  Fig 3 (c), the distance between the two Cu atomic planes (noted d) is 12.17 Å and corresponds to the tunneling regime [see Fig. 1(c)] described previously. Figure 3 (d) shows the molecular junction at d = 11.14 Å and corresponds to the transition between the tunneling and contact regimes. The transition point has been assigned to the point in which a change in the behavior of structural parameters is observed, and has been confirmed by the theoretical transmission probabilities [Fig. 3 (f)]. The molecule is distorted at the transition, but still bonded to the Cu atom through two C atoms. Finally, Fig. 3 (e) corresponds to d = 9.66 Å and is representative of the molecular junction in the contact regime. The molecule exhibits Cp rings that are parallel to both metallic planes and the Cu atom is coordinated to five C atoms. Figure 3 (b) quantifies the structural changes with d. As shown, at the transition point the distances between the Cu and Ni atom with respect to the tip electrode, noted d 1 and d 1 + d 2 respectively, is maximum, as well as the tilt difference between the Cp rings, noted ∆Θ. The distance between the Ni atom and the surface-electrode (noted d 3 ) instead decreases continuously with d. Experimentally, we have seen that in the tunneling regime despite the slightly different initial orientations that the molecule might have on the tip, similar results are obtained in the dI/dV spectra for different molecular tips when going to contact. This indicates that the Nc molecule tends to adopt always the same configuration, with parallel Cp rings, when contacted between the tip and the surface.

Figure 3 (Fig. 4 (

 34 Figure 3 (g) presents how the magnetization of the Nc molecule, the Ni atom and the Cu atom change with d. In the tunneling regime, the calculated magnetic moment for Nc is 1.7µ B , the Ni atom carrying 1µ B . At the transition, these values decrease to 1µ B and 0.7µ B , respectively, and afterwards, at contact, stabilize around 0.75µ B for Nc and 0.5µ B for the Ni atom. The Cu atom remains non-magnetic during the contact process. The initial magnetic moment of the molecule is halved when contacted between the two electrodes, meaning that the molecular spin changes from S = 1 to S = 1/2. Such a spin switch is in agreement with the spectroscopic fingerprints highlighted experimentally. The change on the molecular charge with respect to the gas phase from tunneling (-0.12e -) to contact (+0.07e -) is not enough to explain such a change on the magnetization. To scrutinize the effect of the deformation of the molecule on the molecular magnetization, the magnetization of the isolated Nc molecule in the same geometrical configuration as it shows in tunnel, at the transition and in contact was computed. No relevant difference was observed for the different configurations, indicating that the magnetization change is neither driven by the molecule-sustrate charge transfer nor the deformation suffered by the molecule. Fig.4 (a) presents the spin-resolved transmission for the tunnel (thick line) and contact (thin line) regimes. The transmission of Fig. 4 (a) implies that the transport is mainly due to the hybridization of surface electronic states with the frontier molecular orbitals, which are d xz -and d yz -based molecular orbitals [36]. To get a deeper understanding of the different features observed in the transmission function, the density of states projected (PDOS) onto the C(2p) and Ni(3d) atomic orbitals are shown in Fig. 4 (b). The clear correspondence between spin up and spin down peaks on the transmission function and the PDOS [Fig. 4 (b)] makes it possible to assign both peaks to the transmission through the spin-polarized degenerate frontier orbitals. The comparison between contact and tunnel results shows that the peaks associated to the frontier-orbital in both spin up and spin down channels shift towards the Fermi level in the contact regime, reducing the exchange splitting energy due to the screening of the intra-orbital Coulomb repulsion (U ). This is due to the enhancement of the molecule-electrode interaction that also induces a more pronounced broadening of the molecular levels. Such increase of the hybridization (Γ) reduces the charge in the majority spin and increases the minority spin occupation. Our analysis then shows that both effects, namely, the increase of Γ and the

FIG. 3 .FIG. 4 .

 34 FIG. 3. (a) Structure of the calculated molecular junction, including the main relevant distance parameters. (b) The panels show how the structural parameters d1, d2, d3, ∆Θ change as a function of d. The structure of the molecular junction for: (c) the tunneling regime (d=12.17 Å), (d) the transition between the tunneling and the contact regimes (d=11.14 Å), and (e) the contact regime (d=9.66 Å). (f) Total transmission (black line) and the transmission per spin channel (red and blue lines) at the Fermi level for the explored configurations. (g) Magnetization of: Nc (black), Ni (red), Cu atom (green) as a function of d. Full symbols correspond to SIESTA (Mulliken) results and open symbols to VASP (Bader) results.

ACKNOWLEDGMENTS

This work has been supported by the Agence Nationale de la Recherche (Grant No. ANR-13-BS10-0016, ANR-11-LABX-0058 NIE, ANR-10-LABX-0026 CSC) the ANPCyT project PICT Bicentenario No. 1962, the CONICET project PIP 0667, and the UNR project PID ING235. We acknowledge computer time provided by the CCT-Rosario Computational Center and the Computational Simulation Center (CSC) for Technological Applications, members of the High Performance Computing National System (SNCAD, MincyT-Argentina). Financial support from MINECO MAT2015-66888-C3-2-R and FEDER funds is also gratefully acknowledged.