Lucy Alford
email: l.alford.02@cantab.net

Kévin Tougeron

Jean-Sébastien Pierre

Francoise Burel

Joan Van Baaren

Françoise Burel

The effect of landscape complexity and microclimate on the thermal tolerance of a pest insect

Keywords: Landscape influences insect thermal tolerance Agro-ecosystems, biological control, cereal aphids, climate change, cold tolerance, landscape ecology

Introduction

The earth is presently experiencing rapid changes in both climatic conditions and landscape structure. Over the past century the climate has warmed by approximately 0.6°C and an increased incidence of extreme climatic events such as droughts and heat and cold waves is predicted [START_REF] Easterling | Maximum and minimum temperature trends for the globe[END_REF], 2000, Karl et al., 2000, Walther et al., 2002;IPCC, 2013). Since many species can tolerate only a restricted range of environmental conditions, climate change is expected to have . significant consequences for the phenology, life history and distribution of species, which will ultimately impact the severity and timing of pest outbreaks and ecosystem functioning [START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF]. In addition to climatic changes, much of Europe has experienced large scale agricultural intensification over the past 50 years, resulting in the alteration of landscape patterns such as an increase in mean field size, the disappearance of semi-natural habitats and a change in farming practices combined with a dramatic increase in chemical inputs [START_REF] Roschewitz | The effects of landscape complexity on arable weed species diversity in organic and conventional farming[END_REF]. This intensification has led to a considerable increase in agricultural productivity but also to a simplification of agricultural landscapes [START_REF] Roschewitz | The effects of landscape complexity on arable weed species diversity in organic and conventional farming[END_REF], a drastic loss of biodiversity [START_REF] Krebs | The second silent spring?[END_REF][START_REF] Tilman | Agricultural sustainability and intensive production practices[END_REF], and degradation of ecosystem services [START_REF] Roschewitz | The effects of landscape complexity on arable weed species diversity in organic and conventional farming[END_REF].

As human population density continues to rise, agricultural productivity must increase to meet demand [START_REF] Tilman | Global environmental impacts of agricultural expansion: the need for sustainable and efficient practices[END_REF] which necessitates an improvement or at least maintenance in the efficacy of crop protection against agricultural pests. Much research has focused on the effect of climatic conditions on the thermal tolerance and physiological plasticity of agriculturally important insects so that we may better understand and predict the potential impacts of climate change on agroecosystems (e.g. [START_REF] Collier | Possible impact of global warming on cabbage root fly (Delia radicum) activity in the UK[END_REF][START_REF] Chen | Variation in cold hardiness of Liriomyza huidobrensis (Diptera: Agromyzidae) along latitudinal gradients[END_REF][START_REF] Le Lann | Thermal tolerance of sympatric hymenopteran parasitoid species: does it match seasonal activity[END_REF], Alford et al., 2012b[START_REF] Piyaphongkul | Can tropical insects stand the heat? A case study with the brown planthopper Nilaparvata lugens (Stal)[END_REF]. However, given that landscape changes are known to exacerbate the impact of global climate change [START_REF] Delattre | Interactive effects of landscape and weather on dispersal[END_REF][START_REF] Dixon | Tritrophic interaction influenced by warming and tillage: A field study on winter wheat, aphids and parasitoids[END_REF] the effect of landscape complexity on insect thermal tolerance has been under studied [START_REF] Tougeron | Comparing thermal tolerance across contrasting landscapes: first steps towards understanding how landscape management could modify ectotherm thermal tolerance[END_REF]. If agroecosystem functioning is to be maintained into the future, it is imperative that the impacts of landscape complexity and climate on agro-ecosystems are no longer studied in isolation. This is especially true if we are to understand, develop, and successfully implement landscape management techniques to reduce the impacts of climate change and associated extreme weather events on crucially important organisms (e.g. organisms that provide a valuable ecosystem service such as natural pest control). Furthermore, through understanding the effect of landscape complexity on . local microclimate and insect thermal tolerance, we may elucidate which components of the microclimate (e.g. mean temperature, temperature extremes, humidity etc.) are most important in determining insect thermal tolerance.

Using cereal fields of North-West France as a study system, the present study investigates variation in microclimate and insect thermal tolerance from contrasting simple and complex landscapes.

Although landscape composition can impact the local microclimate, the relationship is a complex one involving many factors (vegetation type, cover, topography etc.), often leading to confusing and sometimes contradictory results with some studies suggesting a warming effect of landscape intensification [START_REF] Chen | Microclimate in forest ecosystem and landscape ecology -Variations in local climate can be used to monitor and compare the effects of different management regimes[END_REF][START_REF] Quénol | Microclimate in forest ecosystem and landscape ecology[END_REF][START_REF] Suggitt | Habitat microclimates drive fine-scale variation in extreme temperatures[END_REF], whilst another suggest a cooling effect [START_REF] Argent | The influence of a plant canopy on shelter effect[END_REF]. As such, meteorological data will be recorded directly from the study area to elucidate the exact relationship between landscape intensification and microclimate.

The focus species of the current study are the cereal aphids (Hemiptera: Aphididae) Sitobion avenae (Fabricius) common name the English grain aphid, Metopolophium dirhodum (Walker) common name the rose grain aphid, and Rhopalosiphum padi (L.) common name the bird cherry oat aphid. All three species are major pests of commercially important cereal crops in temperate climates [START_REF] Fereres | Behavioural aspects influencing plant virus transmission by homopteran insects[END_REF][START_REF] Dedryver | The conflicting relationships between aphids and men: A review of aphid damage and control strategies[END_REF]. These cereal aphids possess a complex lifecycle with a series of asexual and sexual generations produced over the course of a year [START_REF] Moran | The evolution of aphid life cycles[END_REF]. At the onset of winter, the production of sexual morphs is triggered and sexual reproduction occurs. This holocyclic (sexual) lifecycle results in the production of very cold hardy eggs that overwinter [START_REF] Strathdee | Cold-hardiness of overwintering aphid eggs[END_REF]. An alternative anholocyclic (asexual) lifecycle also exists which resulted from a stable genetic mutation affecting the photoperiodic switch, resulting in a failure to produce sexual forms in autumn [START_REF] Moran | The evolution of aphid life cycles[END_REF][START_REF] Dixon | Tritrophic interaction influenced by warming and tillage: A field study on winter wheat, aphids and parasitoids[END_REF]. As such, asexual, parthenogenetic reproduction may occur all year . round, resulting in the existence of genetically distinct anholocyclic clones. These clones have been shown to vary in insecticide resistance and thermal tolerance, with implications for the clonal types that come to dominate within a population [START_REF] Fenton | Micro-evolutionary change in relation to insecticide resistance in the peach-potato aphid, Myzus persicae[END_REF]Alford et al., 2012b).

Insects inhabiting higher latitudes generally exist in climates cooler than their thermal optima [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF] and this is true of the cereal aphids of temperate climates. As such, global warming could act to increase the fitness of temperate insects [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF]. Instead, it is the unpredictable occurrence of extreme environmental events (e.g. cold waves) that could prove a greater challenge to insects [START_REF] Godfray | The population dynamic consequences of phenological asynchrony between parasitoids and their hosts[END_REF][START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF] and thus be more important in dictating insect survival and persistence. The importance of increasing extreme weather events places increased importance on the study of insect thermal tolerance. Here we report on laboratory experiments designed to investigate the cold tolerance of these three aphid species, as determined by the critical thermal minima (CT min ), originating from wheat fields varying in agricultural intensification regime. The following hypotheses were tested: i) Local climate will differ between simple and complex landscapes. ii) Aphid cold tolerance will vary with landscape complexity in accordance with the local microclimate. iii) Differences in aphid thermal tolerance between landscapes will be less marked in spring than winter because exposure to stressful low temperatures will be less frequent in spring months.

Materials and methods

Meteorological data

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Meteorological data in the study area was recorded using established BWS200 weather stations (Campbell Scientific France) during two sampling sessions; one in winter (13th January to 10th March 2014) and one in spring (17th March to 12th May 2014). One weather station per type of landscape was used to record air temperature and relative humidity (using CS215 probes accurate at 0.3°C and 2% respectively), and wind speed (using Wind Sentry anemometer accurate at 0.5m.s -1 ) at a sampling rate of once per hour for the duration of the sampling period. Weather stations were placed on flat ground at a height of 1 meter and at a minimum distance of 15 meters from the hedge boundary.

Aphid sampling and rearing

Laboratory cultures of S. avenae, M. dirhodum and R. padi were established using aphids collected in the field between the 13th January to 10th March 2014 (representing a winter population) and the 17th March to 12th May 2014 (representing a spring population). In both sampling seasons, aphids were collected on winter wheat and triticale fields in the Long Term Ecological Research (LTER) site Armorique (48 ° 36 'N, 1 ° 32' W) (http://osur.univ-rennes1.fr/zaarmorique/) located in Brittany, north-western France. Aphids were sampled from healthy plants to minimize any potential effect of host plant quality on aphid fitness. Sampling fields were selected to represent two contrasting landscape types: complex and simple. The landscape types were selected from a landscape intensification gradient previously established as part of the long-running project Farmland (farmlandbiodiversity.org). Complex landscapes are characterized by high diversity, high hedgerow density (>3200m), small field sizes (<0.93ha) and the presence of grassland areas (>45%) and, due to these characters, are considered as fine grain heterogeneous landscapes. In contrast, simple landscapes represent intensively farmed homogenous landscapes . characterized by low diversity, large field sizes (>2.70ha) and few semi-natural elements (grassland density <20% and low hedgerow density <550m).

In winter 2014, a total of 27 fields within these contrasting landscape types were prospected for the presence of aphids. A total of 5 fields were found to contain aphids: 3 fields in the simple landscape and 2 fields in the complex landscape. In spring 2014, a total of 6 fields were sampled, with 3 fields in the simple landscape and 3 fields in the complex landscape for consistency with winter sampling. Due to sampling in agricultural landscapes, the selected fields were dependent upon the crop regime of the farmer and the presence of aphids. As a consequence, the fields sampled in winter and spring were not the same. Sampling was performed by 2 people during a 30 minute period and as many plants as possible were examined for living aphids. All living aphid adults and nymphs were collected and returned to the laboratory. Sampling occurred approximately three times per week, weather permitting, over the sampling period to produce a laboratory culture representative of the sampled fields.

All collected aphids were returned to the laboratory and identified as one of three species: S. avenae, M. dirhodum and R. padi. Aphids were subsequently separated by species and field and a culture established for each species x field combination. During the winter sampling period, R. padi and M. dirhodum were successfully collected in all 5 sample fields. Sitobion avenae was collected in only 2 of the fields: 1 in each landscape type. During the spring sampling period, S. avenae was successfully collected in all 6 sampled fields. Metopolophium dirhodum was collected in only 2 of the fields: 1 in the complex and 1 in the simple landscape. Rhopalosiphum padi was not found in any of the sampled fields in spring. This resulted in a total of 20 species x field combinations. The lack of certain species in some . fields represents a source of confounding effects which will be discussed under the heading of statistical analysis.

Each culture was reared on winter wheat, Triticum aestivum, 'Renan' cultivar grown in vermiculite within Plexiglas cages (50 × 50 × 50 cm) and housed within a controlled environment room at 20±1°C and LD 16 : 8 h photoperiod. Fresh wheat was added to the cultures on a twice weekly basis. Before field collected aphids were added to the cultures, an initial quarantine period was carried out to ensure that aphids were not host to parasitoid wasps. For this, field collected aphids were housed in microcages (L = 16 cm, Ø = 4 cm) comprising T. aestivum grown in vermiculite for a period of 10 days. Following this quarantine period, all aphids within the microcages which had not turned into an 'aphid mummy' (a dead aphid containing an immature parasitoid pupa) were transferred to the relevant culture. Winter field populations of aphids are markedly reduced in comparison to spring populations. However, at least 30 founding field-collected aphids were used to establish cultures for each species x field combination, with cultures subsequently allowed to build up to large enough numbers for use in experiments. Lab rearing duration was approximately 3-4 weeks before use in experiments.

To obtain apterous adults synchronized in age for use in experiments, reproductive adults were taken from the stock culture and were allowed to reproduce for 24 h in microcages.

Resultant nymphs were allowed to develop into pre-reproductive adults for use in experiments.

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Determination of low temperature activity thresholds (CTmin)

The low temperature at which an insect is rendered immobile, known as the critical thermal minima (CT min ), was determined for pre-reproductive adults of the three aphid species.

CT min was chosen as the measure of cold tolerance since such non-lethal thresholds are considered to be of more ecological relevance than lethal thresholds [START_REF] Macdonald | Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster[END_REF], because survival at unfavorable temperatures is of little importance if the insect is rendered inactive and thus unable to find food, mate or escape predation [START_REF] Mellanby | Low temperature and insect activity[END_REF][START_REF] Bale | Insect cold hardiness -freezing and supercooling -an ecophysiological perspective[END_REF].

CT min was measured using a glass column, as described by [START_REF] Powell | Effect of long-term and rapid cold hardening on the cold torpor temperature of an aphid[END_REF]; a modified design from apparatus previously used by Weber and Diggins (1990) and [START_REF] Huey | A method for rapid measurement of heat or cold resistance of small insects[END_REF]. The glass column (350 × 50 mm) was connected to a programmable alcohol bath (Haake F3, Thermo Electron Corp., Karlsruhe, Baden-Württemberg, Germany), enabling the circulation of alcohol fluid around the outer chamber and thus fine control over the air temperature experienced within the inner column. Previous work has shown that air temperature is consistent along the length of the column [START_REF] Powell | Effect of long-term and rapid cold hardening on the cold torpor temperature of an aphid[END_REF]. In addition, due to the relatively small body size of the test insects, it is concluded that the air temperature of the column approximates the body temperature of the insects [START_REF] Huey | A method for rapid measurement of heat or cold resistance of small insects[END_REF].

Consequently, a single thermocouple was placed against the glass surface of the inner column to monitor air temperature during experimentation.

In all experiments, approximately 10-15 adult aphids of one species from one field were inserted into the bottom of the column pre-set to the culture temperature of 20°C. The column was subsequently closed with a sponge stopper to reduce air flow and maintain a stable thermal environment within the inner column. Following a 10 min acclimatization period, the . programmable alcohol bath was set to decrease the temperature of the column from 20°C to -10°C at a rate of 0.5°C min -1 .

During the cooling phase, the CT min was determined as the temperature at which an individual aphid was no longer able to cling to the vertical column and subsequently fell to the bottom of the column. The CT min temperature was recorded manually from the thermocouple display reading to an accuracy of 0.1°C. The procedure was repeated to obtain CT min values for 50 individuals of each species × field combination.

Statistical analysis

Meteorological data differences between landscapes were tested using an ANOVA for repeated measures and a Tukey HSD post-hoc test to compare data among landscapes for both seasons using the R software (R Core Team, 2013). The meteorological variable in question was considered as a fixed effect, and recording time was included as a random effect to control for correlation between meteorological data at recording times t and t-1. Meteorological data differences between seasons were tested using a Welch's two sample ttest.

CT min data were analyzed using a Cox regression / proportional hazard model which belongs to the class of generalized linear models. It applies to survival data which are typically not normally distributed (exponential, Weibull, Gamma, etc.). The model has the advantage of being "semi parametric" in the sense that it does not require any knowledge of the underlying distribution. The only hypothesis is that the factors and covariates act multiplicatively on the . death rate   t  . The original data (death time) are then transformed to a death rate, and subsequently regressed on the factors and covariates by the equation

    0 1 exp , p ii i t t X         
where   0 t  is the so called "baseline hazard", i X is the i th covariate or factor among p and i  regression coefficients. Here, we take advantage of the confusion between time and temperature to treat the temperature of fall as a death time. Cox models give access to all classic models of variance (deviance) analysis, covariance and regression. When positive, the coefficients i  indicate an increase of the death rate and thus a negative effect on survival, and the opposite when they are negative [START_REF] Cox | Regression models and life tables[END_REF][START_REF] Fox | Cox proportional-hazards regression for survival data: appendix to an R and S-PLUS companion to applied regression[END_REF][START_REF] Collett | Modelling Survival Data in Medical Research[END_REF]. The replication of fields in each environment was treated as a frailty factor (random factor) [START_REF] Therneau | Modelling Survival Data, Extending the Cox Model[END_REF]. Frailty function defines a factor as random. Where there is no replication, the model is only estimable when this random effect is assumed to be additive. The within error, associated to . the cross levels Species x Landscape x exp does not need any distribution assumption as the Cox model is distribution free. This lack of replication is especially true in winter, and for S.

avenae originating from the simple landscape, where there is only one replicate. Thus, the additive frailty term is estimated necessarily as equal to what is observed in the complex landscape. A confounding effect Sitobion x replication A x complex landscape and Sitobion x replication C x simple landscape is theoretically possible; a fact we have to keep in mind when discussing the results.

Results

Meteorological data

For both seasons, complex landscapes were on average colder than simple landscapes.

Temperature amplitude and standard deviations revealed complex landscapes to be less variable in temperature than simple landscapes. In winter, complex landscapes showed higher relative humidity than simple landscapes, and a lower wind speed. Results further revealed greater variation between complex and simple landscapes in winter than in spring for the meteorological factors measured (Table 1). The number of days in which temperatures dropped below zero were 1 and 4 for complex and simple landscapes respectively in winter, and 0 and 4 respectively in spring.

The effect of landscape complexity on the CT min of winter aphids

For aphids sampled in winter, there was a significant effect of aphid species (χ 2 2 = 139.29, P < 0.001) and landscape complexity (χ 2 1 = 19.56, P < 0.001) on the rate of drop-off (CT min ) from a vertical column (Fig. 1). No interaction effect was observed between aphid species . and landscape complexity (χ 2 2 = 3.62, P = 0.164). The  coefficients were negative for R.

padi and positive for S. avenae, and both proved significantly different from M. dirhodum considered as the baseline ( 0  , default option in coxph). Thus, for the species effect, the greatest cold tolerance, when measured as CT min induced drop-off from the vertical column, was observed for R. padi. Sitobion avenae displayed the lowest cold tolerance and dropped from the column at warmer temperatures, with M. dirhodum proving intermediate of the two species. A significant effect of landscape complexity was observed for R. padi (χ 2 1 = 3.91, P = 0.048), M. dirhodum (χ 2 1 = 11.54, P < 0.001) and S. avenae (χ 2 1 = 4.41, P = 0.036). The  coefficients were negative for the complex landscape, as compared to the simple landscape considered as the baseline, meaning that aphids remain attached for longer and accordingly at lower temperatures when originating from complex landscapes. Thus, for all three species, aphids from complex landscapes displayed a reduced rate of CT min induced drop-off from the vertical column indicative of a greater cold tolerance than aphids from simple landscapes.

The frailty effect caused by replications within the same environment was always very low and non-significant. It is pointed out that this effect is poorly known for Sitobion avenae due to the lack of replicates within landscapes. Although this effect is coherent with that observed for the two other species, we cannot exclude a sampling bias in this case.

The effect of landscape complexity on the CT min of spring aphids

For aphids sampled in spring, there was a significant effect of aphid species (χ 2 1 = 16.60, P < 0.001) and a weak effect of landscape complexity (χ 2 1 = 4.14, P = 0.042) on the rate of CT min induced drop-off from a vertical column (Fig. 2). No interaction effect was observed between aphid species and landscape complexity (χ 2 1 = 1.757, P = 0.185). The greatest cold tolerance .

was displayed by M. dirhodum and the lowest cold tolerance by S. avenae. When analyzing the data separately for each species, a significant effect of landscape complexity was found for S. avenae (χ 2 1 = 10.32, P = 0.001) with CT min induced drop-off from a vertical column occurring at a faster rate for individuals originating from simple landscapes. The effect of landscape complexity proved non-significant for M. dirhodum (χ 2 1 = 0.003, P = 0.954).

Discussion

Effects of landscape complexity on microclimate

Different components of the landscape such as topography, vegetation type and cover directly impact the microclimate [START_REF] Chen | Microclimate in forest ecosystem and landscape ecology -Variations in local climate can be used to monitor and compare the effects of different management regimes[END_REF]. Although landscape composition is known to affect the microclimate, the relationship is complex, often leading to confusing and sometimes contradictory results [START_REF] Argent | The influence of a plant canopy on shelter effect[END_REF][START_REF] Chen | Microclimate in forest ecosystem and landscape ecology -Variations in local climate can be used to monitor and compare the effects of different management regimes[END_REF][START_REF] Quénol | Microclimate in forest ecosystem and landscape ecology[END_REF][START_REF] Suggitt | Habitat microclimates drive fine-scale variation in extreme temperatures[END_REF]. In the current study, simple landscapes (homogenous landscapes characterized by low diversity, large field sizes and few semi-natural elements) were, on average, warmer than more complex landscapes (fine grain heterogeneous landscapes characterized by high species diversity, high hedgerow density, small field sizes and the presence of grassland areas) in both winter and spring. This supports hypothesis 1 that local climate will differ between simple and complex landscapes. However, whilst warmer with regard to mean temperature, simple landscapes displayed an increased range of temperature extremes.

As a landscape becomes progressively more open, it is exposed to increasing amounts of daytime short radiation, acting to raise local temperatures [START_REF] Chen | Microclimate in forest ecosystem and landscape ecology -Variations in local climate can be used to monitor and compare the effects of different management regimes[END_REF][START_REF] Suggitt | Habitat microclimates drive fine-scale variation in extreme temperatures[END_REF]). Furthermore, the increased hedgerow density characteristic of complex landscapes provides a windbreak function, causing a reduction in local wind speed and retention of denser, cooler air [START_REF] Quénol | Microclimate in forest ecosystem and landscape ecology[END_REF], acting to lower mean local temperatures. This windbreak effect and reduction in wind speed further functions to raise relative humidity, as observed in the current study during winter sampling. As the season changes from winter to spring, increased vegetation growth across all landscape types results in a buffering of temperatures. Such buffering of temperature leads to a reduction in variation between simple and complex landscapes, although the complex landscape still remains significantly colder, and the simple landscape with greater extremes. Meteorological data collected in the current study therefore support the idea that landscape intensification and homogenization could act to raise local temperatures, whilst increasing temperature variations and extremes, and reducing relative humidity.

Seasonal aphid population composition

The general decline in dominance of R. padi from winter to spring, with a concurrent increase to dominance of S. avenae by spring is a commonly observed pattern in the population composition of cereal aphids [START_REF] Andrade | Winter host exploitation influences fitness traits in a parasitoid[END_REF][START_REF] Andrade | Spatiotemporal variations in aphid-parasitoid relative abundance patterns and food webs in agricultural ecosystems[END_REF][START_REF] Alford | Could behaviour and not physiological thermal tolerance determine winter survival of aphids in cereal fields?[END_REF]. In the current study, during winter sampling, R. padi and M. dirhodum were present in all sampled fields and S. avenae in 2 of the 5 sampled fields. However, by spring, R. padi was no longer present. Sitobion avenae had increased to dominance and was found in all spring sampled fields, whilst M. dirhodum was present in only 2 of the 6 sampled fields. This observation is further supported by the significant inter-species variation in thermal tolerance observed in the current study, with R. padi displaying the lowest temperatures of CT min and S. avenae the highest. It is known that R. padi prefers cool, humid conditions, whilst S. avenae has a

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preference for warm and dry conditions [START_REF] Honek | Plant density and abundance of cereal aphids (Hom., Aphidina)[END_REF][START_REF] Leather | Factors determining the pest status of the bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), in Europe: a study and review[END_REF][START_REF] Jarošík | Comparison of field population growths of three cereal aphid species on winter wheat[END_REF]. The ability of R. padi to remain active and attached to the substrate at colder temperatures than its counterparts could therefore provide the species with a fitness advantage in winter. This theory is supported by recent research which revealed behavioral variations that could contribute to the dominance of R. padi in winter [START_REF] Alford | Could behaviour and not physiological thermal tolerance determine winter survival of aphids in cereal fields?[END_REF], offering an explanation to the observed variation in aphid sampling between seasons.

Effects of landscape complexity on aphid thermal tolerance

Hypothesis 2 states that aphid thermal tolerance will vary with landscape complexity in accordance with the local climate. With the microclimate data collected in the current study, two opposing sub-hypotheses are proposed. Firstly, aphids from simple landscapes could possess an enhanced thermal tolerance in response to greater temperature variations (representing a more thermally stressful environment); a pattern recently observed for the parasitoid wasps of cereal aphids [START_REF] Tougeron | Comparing thermal tolerance across contrasting landscapes: first steps towards understanding how landscape management could modify ectotherm thermal tolerance[END_REF]. Secondly, aphids from complex landscapes could possess an enhanced thermal tolerance in response to colder mean temperatures. Interestingly, the current study supported the latter sub-hypothesis, with the aphid species from complex landscapes being more cold tolerant. Since the response to the landscape is observed at the guild level, it suggests that the strength of external filtering (Violle et al., 2012) constrains traits to a narrow range within a guild exposed to the same environmental conditions [START_REF] Diaz | Vive la difference: plant functional diversity matters to ecosystem processes[END_REF]White, 2008;[START_REF] Aparicio | Fragmentation and comparative genetic structure of four Mediterranean woody species: complex interactions between life history traits and the landscape context[END_REF].

When exposed to unfavorable low temperatures, survival is dependent upon a number of factors including the extremes of temperature, the rate of cooling, the duration of exposure and the extent to which the temperature fluctuates around freezing [START_REF] Sinclair | Insects at low temperatures: an ecological perspective[END_REF]. For many insects, and arthropods more generally, temperature extremes are considered to be of . greater importance in determining thermal resistance than temperature means (Paaijmans et al., 2003;[START_REF] Bahrndorff | Geographic variation for climatic stress resistance traits in the springtail Orchesella cincta[END_REF][START_REF] Estay | The role of temperature variability on insect performance and population dynamics in a warming world[END_REF][START_REF] Lawson | Environmental variation and population responses to global change[END_REF]. However, whilst research evidence highlights the importance of temperature extremes in determining thermal tolerance, the relationship is perhaps not that simple, with any environmental unpredictability acting to undermine the strength of the environmental cue received by the insect [START_REF] Deere | Phenotypic plasticity of thermal tolerances in five oribatid mite species from sub-Antarctic Marion Island[END_REF]. For this reason, plasticity is predicted to be more common in environments that are predictably variable [START_REF] Deere | Phenotypic plasticity of thermal tolerances in five oribatid mite species from sub-Antarctic Marion Island[END_REF]. As such, the increased temperature variation and enhanced extremes around a warmer mean recorded in simple landscapes may provide an unpredictable cue to the aphids. In contrast, the colder mean temperatures with reduced temperature extremes recorded in complex landscapes could provide a more reliable cue, thus eliciting a greater physiological response and acting to increase aphid cold tolerance.

The relationship between temperature variation and thermal tolerance is therefore a complex one, with low temperature exposure being far more multifaceted than simply a mean temperature. However, whilst researchers are increasingly factoring in temperature extremes and fluctuations into models in pursuit of enhancing our understanding of insect thermal tolerance, particularly in the face of global climate change [START_REF] Estay | The role of temperature variability on insect performance and population dynamics in a warming world[END_REF], Vasseur et al., 2014), we cannot rule out the possibility that insects may respond to different aspects of the temperature exposure. A recent study investigating the thermal tolerance of Aphidius (Hymenoptera: Braconidae) parasitoids along a landscape intensification gradient found that parasitoids originating from intensive 'simple' landscapes were significantly more cold tolerant than those originating from more natural 'complex' landscapes [START_REF] Tougeron | Comparing thermal tolerance across contrasting landscapes: first steps towards understanding how landscape management could modify ectotherm thermal tolerance[END_REF]; the reverse pattern to what was observed for their aphid host in the current study. As temperature exposure is multifaceted, so too are the mechanisms involved in conferring cold .

tolerance, ranging from behavioral to molecular [START_REF] Bale | Insects and low temperatures: from molecular biology to distributions and abundance[END_REF][START_REF] Sulmon | Abiotic stressors and stress responses: What commonalities appear between species across biological organization levels?[END_REF], and strategies concerned with withstanding freezing or avoiding freezing altogether [START_REF] Bale | Insects at low-temperature -a predictable relationship[END_REF].

As such, insects may utilize different mechanisms and respond to different environmental triggers. For example, whilst increased temperature variation depressed the lower limits of the fruit fly Drosophila melanogaster (Diptera: Drosophilidae) and the moth Helicoverpa armigera (Lepidoptera: Noctuidae), the same pattern was not observed for the aphid Acyrthosiphon pisum (Hemiptera: Aphididae) [START_REF] Estay | The role of temperature variability on insect performance and population dynamics in a warming world[END_REF]. Instead, the lower limit of A. pisum was raised following increased temperature fluctuations (variance of temperature around the mean), therefore acting to reduce cold tolerance, with more importance placed on a constant mean temperature [START_REF] Estay | The role of temperature variability on insect performance and population dynamics in a warming world[END_REF].

The work of [START_REF] Estay | The role of temperature variability on insect performance and population dynamics in a warming world[END_REF], in conjunction with the current study, suggests that, although temperature extremes are important in determining thermal resistance for many insects, temperature means could be of greater importance in determining aphid thermal resistance.

Aphids are phloem feedings insects, dependent upon a host plant. Subsequently, any engagement in behavioral thermoregulation e.g. seeking out more thermally suitable microhabitat could result in lost feeding opportunity and be detrimental to the individual.

Furthermore, the aphid phloem sap diet is high in cryoprotectant sugars and thus continuation of feeding at unfavorable low temperatures acts to enhance cold tolerance [START_REF] Danks | Modes of seasonal adaptation in insects .1. Winter survival[END_REF][START_REF] Sømme | Adaptations to low-temperature in high-altitude insects from Mount Kenya[END_REF]. As such, an aphid may withstand rather than evade unfavorable cold temperatures, remaining fixed to the host plant. For this reason, aphids may be more sensitive to mean temperatures, displaying enhanced thermal tolerance in response to smaller temperature variations around a lower mean temperature, as recorded in complex landscapes.

.

Seasonal variation in aphid thermal tolerance

The effect of landscape intensification on aphid thermal tolerance was observed in both winter and spring, although the relationship was more pronounced in winter. This offers support to hypothesis 3 that differences in aphid thermal tolerance between landscapes will be less marked in spring than winter because exposure to stressful low temperatures will be less frequent in spring months. This finding further suggests that seasonal thermal tolerance of aphids is conferred via phenotypic plasticity as opposed to genetic adaptation. Aphids, as with all organisms, can respond to variation in environmental temperature via phenotypic plasticity [START_REF] Addo-Bediako | Thermal tolerance, climatic variability and latitude[END_REF][START_REF] Ayrinhac | Cold adaptation in geographical populations of Drosophila melanogaster: phenotypic plasticity is more important than genetic variability[END_REF] and display a high degree of plasticity in their inherent thermal tolerance [START_REF] Powell | Intergenerational acclimation in aphid overwintering[END_REF]Alford et al., 2012a,b).

Natural selection is unlikely to favor unnecessary thermal tolerance and, as such, any acquired cold tolerance should be lost on cessation of winter temperatures. In the study system, mean temperatures increased from winter to spring. In conjunction with this temperature increase, variation in microclimate with landscape complexity became less pronounced. As such, variation in aphid thermal tolerance with landscape complexity should also become less pronounced in spring, as was observed in the current study.

Conclusion

The current study suggests that landscape intensification could act to raise local temperatures, whilst increasing temperature variations and extremes. Indeed, future land use changes could prove as important in dictating local microclimate as global climate change [START_REF] Pyke | Habitat loss confounds climate change impacts[END_REF][START_REF] Oliver | Interactions between climate change and land use change on biodiversity: attribution problems, risks, and opportunities[END_REF]. In the current study, the thermal tolerance of cereal aphids was shown to vary with landscape complexity, with aphids from more natural 'complex'

. landscapes proving significantly more cold tolerant than those originating from intensively farmed, homogenous 'simple' landscapes. Future land use changes could therefore have profound implications for the thermal tolerance and adaptability of insects. Furthermore, the study highlights that all insect species may not respond in a similar manner to thermal conditions and changes to microhabitat and microclimate. If true, this could prove particularly problematic if the response of pest insects and their biological control agents are in anyway decoupled, as suggested in the current study and the study by Tougeron et al.

(2016) on aphid parasitoids. This is an important consideration when faced with land use decisions or landscape manipulation strategies, particularly with the aim of maintaining agroecosystem function into the future.

.



  The program used was coxph in the package survival of R version 3.03 (R Core Team, 2013). Analysis of deviance tests are issued from the 2 approximation of the deviance likelihood ratio. The overall model tested was the following: Surv (CT min ) = intercept + Species* Landscape + frailty(exp) + error. Here, Surv (Survival) indicates the death rate by degrees Celsius, intercept is checked, Species indicates the aphid species, Landscape indicates the type of landscape, and exp represents the replication of fields within the landscape type. In Cox model analysis, the

Fig. 1 .

 1 Fig. 1. Cumulative percentage drop-off of 'winter' aphids (Rhopalosiphum padi, Metopolophium

Fig. 2 .

 2 Fig. 2. Cumulative percentage drop-off of 'spring' aphids (Metopolophium dirhodum and

Acknowledgments

The authors gratefully acknowledge Mathilde Méheut and Charlotte Alford for assistance in the field, to Stephanie Llopis, Herve Amat, Jean-Luc Foulon, Thierry Fontaine and Fouad Nassur for technical assistance, and to Alban Thomas, Valérie Bonnardot and Hervé Quénol from COSTEL lab for their help in collecting meteorological data. This work was funded by a Marie Curie Intra-European Fellowship for the project 'Climland' (FP7-PEOPLE-2012-IEF-326943) awarded to L. Alford, F. Burel and J. van Baaren.

Disclosure

The authors have no conflict of interesting to declare.

. Tscharntke, T., Klein, A.M., Kruess, A., Steffan-Dewenter, I. and Thies, C. (2005) Landscape perspectives on agricultural intensification and biodiversity-ecosystem service management. Ecology Letters, 8, 857-874.

Vasseur


Federica Gilardelli
email: gila.gilardelli@alice.it

Chiara Vergani
email: chiara.vergani@hotmail.com

Rodolfo Gentili
email: rodolfo.gentili@unimib.it

Anne Bonis
email: anne.bonis@univ-rennes1.fr

Pierre Chanteloup
email: chanteloup.pierre@laposte.net

Sandra Citterio
email: sandra.citterio@unimib.it

Enrico A Chiaradia
email: enrico.chiaradia@unimi.it

Gilardelli Federica

Vergani Chiara

† Gentili

Chanteloup Pierre

Citterio Sandra

Root Characteristics of Herbaceous Species for Topsoil Stabilization in Restoration Projects

Keywords: Bromus erectus, Anthyllis vulneraria, Stachys recta, root reinforcement, topsoil stability

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Landscape in limestone quarries affected by long-lasting extractive activities is often deeply altered and covered with only sparse vegetation. This pattern is especially true at the foot of quarry fronts where waste materials (mainly consisting of stone fragments) are dumped in inactive areas [START_REF] Gentili | Plant species patterns and restoration perspectives in the highly disturbed environment of the Carrara marble quarries (Apuan Alps, Italy)[END_REF][START_REF] Gilardelli | Ecological filtering and plant traits variation across quarry geomorphological surfaces: implication for restoration[END_REF]. Dump deposits usually deter the revegetation process and can remain bare for long periods of time because of their undesirable chemical and physical properties (Gilardelli et al., 2016b).

The question of surface stabilization after extractive activities has been previously considered in literature. Since the mid-1990s, the application of geomorphic principles to land reclamation after surface mining [START_REF] Bugosh | Restoring erosional features in the desert. New landform design software and automated machine guidance combine in award-winning reclamation project[END_REF] has been reported in the literature, mostly from Australia, Canada and the USA, as a suitable tool to control soil erosion in restoration project [START_REF] Martín-Duque | Restoring earth surface processes through landform design. A 13-year monitoring of a geomorphic reclamation model for quarries on slopes[END_REF][START_REF] Espigares | Performance of vegetation in reclaimed slopes affected by soil erosion[END_REF]. In general terms, the geomorphological approach represents the best way to manage landscape reconstruction because it aims to reproduce the final step of the erosion/deposition process, when no more soil movement happens. At the same time, this approach is more difficult to be applied in practice because the complexity of the shape that would be achieved [START_REF] Hancock | The design of post-mining landscapes using geomorphic principles[END_REF].

Traditional restoration strategies, instead, suggest to divide the slope in inclined surfaces separated by benches. This approach has the limit to generate high erosion rates and shallow soil stability problems, that must be taken into account during the restoration process.

The presence of vegetation can mitigate the erosion phenomena and improve the surface stability of banks. Various authors have in fact demonstrated that the root systems of herbaceous, shrub and tree species play a key role in both improving slope stability [START_REF] Mafian | Evaluation of root theories and root strength properties in slope stability[END_REF][START_REF] Simon | Quantifying the mechanical and hydrologic effects of riparian vegetation on streambank stability[END_REF][START_REF] Rickson | The use of vegetation for erosion control and environmental protection[END_REF][START_REF] Tosi | Root tensile strength relationships and their slope stability implications of three shrub species in the Northern Apennines (Italy)[END_REF][START_REF] Burylo | Soil reinforcement by roots of six dominant species on eroded mountains marly slopes (Southern Alps, France)[END_REF] and controlling erosion and biogeochemical cycles [START_REF] Gyssels | Impact of plant roots on the resistance of soils to erosion by water: a review[END_REF][START_REF] Baets | Root characteristics of representative Mediterranean plant species and their erosion-reducing potential during concentrated runoff[END_REF][START_REF] Carbutt | Plant-soil interactions in lower-upper montane systems and their implications in a warming world: a case study from the Maloti-Drakensberg Park, southern Africa[END_REF].

. Roots may influence the soil hydrological features by forming macropores within the soil and controlling the infiltration rate and movement of water; thus, the roots drive the soil moisture content [START_REF] Ziegler | Influence of revegetation efforts on hydrologic response and erosion, Kaho"Olawe Island, Hawai"i[END_REF][START_REF] Bernini | Operatore nei cantieri d"ingegneria naturalistica[END_REF][START_REF] Vergani | Soil permeability, aggregate stability, and root growth: a pot experiment from a soil bioengineering perspective[END_REF]. Likewise, roots improve the soil structure and, thereafter, enhance the stability of soil aggregates: this effect is of upmost importance to limit the soil erosion and to allow the establishment of a permanent vegetation cover [START_REF] Burri | Revegetation measures improve soil aggregate stability: a case study of landslide area in Central Switzerland[END_REF][START_REF] Podwojewski | How tree encroachment and soil properties affect soil aggregate stability in an eroded grassland in South Africa[END_REF].

Roots also affect the soil mechanical properties by forming a binding network within the soil layer and by anchoring the superficial unstable soil layer to the deeper, more stable layer or to the bedrock [START_REF] Waldron | The Shear Resistance of Root-Permeated Homogeneous and Stratified Soil[END_REF][START_REF] Schmidt | The variability of root cohesion as an influence on shallow landslide susceptibility in the Oregon Coast Range[END_REF][START_REF] Gyssels | Impact of plant roots on the resistance of soils to erosion by water: a review[END_REF]. The way vegetation contributes to the soil stability depends on the development and structure of the root system and, accordingly, varies with the plant morphogenetic features and soil structure [START_REF] Bischetti | Root choesion of forest species in the Italian Alps[END_REF], as well as plant species and root size [START_REF] Waldron | The Shear Resistance of Root-Permeated Homogeneous and Stratified Soil[END_REF][START_REF] Norris | Slope stability and erosion control: ecotechnological solutions[END_REF].

The three main mechanisms by which roots can mechanically reinforce the soil (generally defined as "root reinforcement") have been defined by [START_REF] Giadrossich | Mechanical interactions between neighbouring roots during pullout tests[END_REF]. The first, and most efficient, is the basal reinforcement that roots provide when they cross the basal slip surface of a shallow landslide and reach the underlying stable layer. When roots do not reach the failure plane, their contribution to stability is reduced, which depends on the dimensions of the potential shallow landslide (Schwarz et al., 2010a). In this case, roots stabilize the soil through the so-called lateral reinforcement, the mobilization of root tensile strength and the compression force at the lateral surface of the landslide (tension crack). The third stabilizing mechanism is the stiffening of the soil material due to the roots in the sliding mass: this process is relevant when there is a strong interaction of neighbouring root systems.

While [START_REF] Gilardelli | Ecological filtering and plant traits variation across quarry geomorphological surfaces: implication for restoration[END_REF] suggested that many common herbaceous species with fast growth rates and specific traits may be suitable candidates for substrate stabilization in quarry areas, their effectiveness has not yet been fully investigated from the slope stability point of view. The contributions of roots to the control of landslides, riverbank stabilization and bioengineering interventions have indeed been evaluated mostly for tree and shrub species, and information concerning native herbaceous species is limited [START_REF] Simon | Quantifying the mechanical and hydrologic effects of riparian vegetation on streambank stability[END_REF][START_REF] Mattia | Biotechnical characteristics of root systems of typical Mediterranean species[END_REF][START_REF] Baets | Root tensile strength and root distribution of typical Mediterranean plant species and their contribution to soil shear strength[END_REF]. To our knowledge, no studies have investigated the role of herbaceous species on soil stabilization for the restoration of the highly disturbed environment of quarry areas. Knowledge of the root system characteristics of herbaceous species is crucial for identifying pioneer species that may effectively enhance revegetation processes and promote topsoil stabilization. The advantage of using herbaceous species rather than shrub and wood species, is that they grow and establish rather quick and hence can cover these soils quickly, favouring natural succession. It is well known the competence of herbaceous plants on shrubs and planted seedlings, which stops ecological succession (Moreno de las [START_REF] Moreno De Las Heras | Vegetation succession in reclaimed coal-mining slopes in a Mediterranean-dry environment[END_REF]. On the contrary shrub and wood species are subject to high mortality ratio due to stress or disturbance such as plant transplanting, water lack, disease, insects, slope stability, etc. (Martínez-Garza et al., 2011;[START_REF] Anton | Survival and growth of planted seedlings of three native tree species in urban forest restoration in Wellington, New Zealand[END_REF][START_REF] Gilardelli | Ecological filtering and plant traits variation across quarry geomorphological surfaces: implication for restoration[END_REF]. During restoration actions, herbaceous species can be established at relatively lower economic cost (Gilardelli et al., 2016b). The principal objective of the present study is to evaluate the ability to promote the superficial soil stabilization by three herbaceous species (Bromus erectus Huds., Stachys recta L. and Anthyllis vulneraria L.), which show different types of root systems. The applied objective is to promote the superficial soil stabilization of quarry dump deposits after their abandonment on the limestone quarries of the Botticino extractive basin (Lombardy, Italy).

In particular, we aim to answer the following questions: how do the differences in root characteristics of the three studied herbaceous species affect the soil reinforcement? and is the root reinforcement provided by the selected species effective for topsoil stabilization in restoration projects? Thus, we provide useful information to select the most suitable species for quarry restoration.

Materials and methods

Study area and selected species

The study area is located in the Botticino basin, in a hilly area (180-650 m a.s.l.) of the Brescia Province (Lombardy). This area represents the second most important extractive basin in Italy, containing over a hundred working quarries. The area is known worldwide for the extraction of the famous "Botticino Marble" limestone. The climate is continental, with cold and dry winters (mean annual temperature: 13.5°C) and rainy springs and autumns (mean annual precipitation: 1026.13 mm). The local lithology is composed of limestone and karst rocks of the "Corna" formation, with calcareous and breccia facies (Servizio Geologico d"Italia, 2008). The vegetation that grows in the surroundings of the quarries is mainly copse woodlands dominated by Quercus pubescens Willd; Ostrya carpinifolia Scop. and Fraxinus ornus L. are abundant. However, these native woodlands might be locally replaced by planted woodlands dominated by Castanea sativa Mill.

Most restoration projects for the quarries of the Botticino extractive basin include landform remodelling and soil preparation before the revegetation phase. Generally, a homogeneous topsoil with an average thickness from 50 cm to 1 m is created by using waste material deriving from local quarry activities. The target vegetation types during restoration refer to In this study, the restored area was characterized by terraces where the flat surface was at a maximum of 5° slope between steep slopes up to 45° (Fig. 1).

To evaluate the contribution of the vegetation to the topsoil stabilization, we selected three herbaceous species with different types of root systems: A. vulneraria, B. erectus, and S. recta. All of them usually grow in arid and dry environments, including in the dump deposits of the Botticino extractive basin, where they characterize different phases of the spontaneous revegetation process (Table 1).

Anthyllis vulneraria (Fabaceae) is a perennial, short basal herb (H scap) of 8-40 cm that prefers arid and dry environments and basic or neutral-basic substrates, even nutrient-poor substrates, such as arid grasslands [START_REF] Pignatti | Flora d"Italia. Edagricolae[END_REF][START_REF] Pignatti | Biondicazione attraverso le piante vascolari. Valori di indicazione secondo Ellenberg (Zeigerwerte) per le specie della Flora d"Italia[END_REF]. Its root system consists of one main tap root and several thinner lateral roots, with some mycorrhizae. Within the Botticino extractive basin, it is usually absent during the initial and intermediate phases of recolonization; however, it is present in the last stage. This species is characteristic of the local grasslands and may be used as a reference for the success of restoration (Gilardelli et al., 2016a).

Bromus erectus (Poaceae) is a perennial tussock (H caesp) of 40-60 cm that develops in arid and dry environments on nutrient-poor calcareous substrates, such as arid grasslands [START_REF] Pignatti | Flora d"Italia. Edagricolae[END_REF][START_REF] Pignatti | Biondicazione attraverso le piante vascolari. Valori di indicazione secondo Ellenberg (Zeigerwerte) per le specie della Flora d"Italia[END_REF]. The root system is fasciculated and composed of thick, dense and fibrous primary roots. Within the Botticino extractive basin, it is usually present at later phases of recolonization, but it is a typical species of the local grasslands that can be used as a reference for the success of restoration efforts (Gilardelli et al., 2016a).

Stachys recta (Lamiaceae) is an erect, leafy, perennial herb (H scap) of 20-40 cm. It is found in arid and dry environments and highly basic substrates, even in nutrient-poor ones, such as rocks, heaps of stones and arid grasslands [START_REF] Pignatti | Flora d"Italia. Edagricolae[END_REF][START_REF] Pignatti | Biondicazione attraverso le piante vascolari. Valori di indicazione secondo Ellenberg (Zeigerwerte) per le specie della Flora d"Italia[END_REF]. The root system is characterised by a main tap root and several lateral roots. Within the Botticino extractive basin, it is present during all the phases of plant recolonization after extraction, especially on recent dump deposits abandoned in the previous 1-10 years (Gilardelli et al., 2016a).

Modelling the stability of reconstructed slope

In civil engineering, the classical methods for slope stability analysis are based on the concept of "limit equilibrium" [START_REF] Lu | Hillslope hydrology and stability[END_REF]. A slope is stable if the shear stresses acting along a mass of soil over a sliding surface are less than the shear strengths (i.e., resisting forces). Otherwise, the slope collapses. The factor of Safety, FoS, defines this equilibrium as the following ratio:

(1) where  f is the shear strength, and  d is the shear stress. The shear strength behaviour of a slope is commonly modelled by the Mohr Coulomb failure criterion:

(2) Eq. 2 means that the effective normal stress, ', due, in first approximation, to the weight of the mobilized volume of soil generates shear strength through the soil. The effective cohesion, c", and the internal friction angle, ", are two parameters that describe the geomechanical properties of soils (i.e., how they react to force solicitations).

The shear stress, instead, is related to the component of soil weight acting parallel to the sliding surface and is the driving force that induces the sliding phenomena.

In a two dimensional representation of the slope, defining the geometry of the sliding surface is the first step of the stability analysis. In most practical cases, the potential failure plan is circular. Then, the sliding area is divided into vertical slices of the same width, and the force analysis is applied (Fig. 2). Among the different methods currently available to consider for forces acting on the sliding volumes, the "ordinary method" [START_REF] Fellenius | Erdstatische Berchnungen[END_REF] provides the most conservative factor of safety and, for that reason, can be used for a preliminary analysis under the design process [START_REF] Burman | A Comparative Study of Slope Stability Analysis Using Traditional Limit Equilibrium Method and Finite Element Method[END_REF].

The factor of safety (FoS) is then evaluated as follows:

(3)

Where, l n is the width of the slice, W n is the weight of the slice,  w is the volumetric weight of the water, h is the local elevation of the water table with respect to the sliding surface,  is the local angle of the sliding surface, m is the total number of slices, and n is a counter.

The presence of vegetation is included in eq. 3 by the introduction of an additive term called "root reinforcement", c r (originally named "root cohesion"; see [START_REF] Waldron | The Shear Resistance of Root-Permeated Homogeneous and Stratified Soil[END_REF][START_REF] Wu | Investigation of landslides on prince of Wales Island, Alaska[END_REF][START_REF] Wu | Strength of tree roots and landslides on Prince of Wales Island, Alaska[END_REF]. Root reinforcement can be estimated analysing the morphology and the mechanical properties of the root elements as explained in the following section.

Root reinforcement evaluation

Root reinforcement (C r ) can be estimated by the application of several models taking into account mainly 1) the root distribution along the soil profile and 2) the ultimate resisting force obtained before the rupture of each root [START_REF] Wu | Investigation of landslides on prince of Wales Island, Alaska[END_REF][START_REF] Waldron | The Shear Resistance of Root-Permeated Homogeneous and Stratified Soil[END_REF][START_REF] Pollen | Estimating the mechanical effects of riparian vegetation on streambank stability using a fiber bundle model[END_REF]Schwarz et al., 2010b).

In this study, we chose the Fiber Bundle Model, FBM [START_REF] Pollen | Estimating the mechanical effects of riparian vegetation on streambank stability using a fiber bundle model[END_REF] because it is considered a reference estimator of the mechanical contribution of plant roots [START_REF] Docker | Quantifying root-reinforcement of river bank soils by four Australian tree species[END_REF][START_REF] Bischetti | Root choesion of forest species in the Italian Alps[END_REF][START_REF] Mickovski | Mechanical reinforcement of soil by willow roots: Impacts of root properties and root failure mechanism[END_REF][START_REF] Ji | Effect of spatial variation of tree root characteristics on slope stability. A case study on Black Locust (Robinia pseudoacacia) and Arborvitae (Platycladus orientalis) stands on the Loess Plateau, China[END_REF][START_REF] Mao | Modelling root demography in heterogeneous mountain forests and applications for slope stability analysis[END_REF].

In addition, this model can be implemented in a simple way and only requires a small number of parameters to be calculated.

In the most simplified condition, when all the roots are assumed to break at the same time, according to [START_REF] Wu | Investigation of landslides on prince of Wales Island, Alaska[END_REF] and [START_REF] Waldron | The Shear Resistance of Root-Permeated Homogeneous and Stratified Soil[END_REF][START_REF] Waldron | Soil reinforcement by roots: calculation of increased soil shear resistance from root properties[END_REF], the C r factor can then be determined as follows:

(6)

where N is the total number of roots crossing the shear reference area a, and F i is the maximum tensile force of each root. Note that F i varies according to mechanical properties and diameter of each single root.

The condition of simultaneous breakage of all roots is poorly realistic, and the FBM approach [START_REF] Pollen | Estimating the mechanical effects of riparian vegetation on streambank stability using a fiber bundle model[END_REF] was introduced to account for the progressive failure of roots. The method considers different increasing steps of load and removes from the list of the intact roots, those roots that have an ultimate stress at failure lower than the applied loading force.

At each time, certain roots fail as their resistance threshold value is reached; the applied load is then redistributed to the remaining intact roots at the following time step, causing further root ruptures until the remaining root bundle reaches an equilibrium. At this point, an increment of load is applied, and the process repeats until the whole root bundle has broken.

The root reinforcement is exerted by roots that cross both the lateral and the basal surfaces of the sliding volume, which gives the lateral, c r_l , and the basal, c r_b , root reinforcement, respectively.

Due to the relatively shallow root systems of the considered herbaceous species (most of the roots are found in the first 30 cm of soil, Fig. 3), we consider here only the contribution of the lateral reinforcement and null basal reinforcement [START_REF] Schwarz | Spatial characterization of root reinforcement at stand scale: Theory and case study[END_REF].

Sampling strategy to evaluate the root reinforcement

The root distribution and the root tensile force data, required to apply the FBM, were obtained for the three selected species, whose root systems are shown in Fig. 3.

. For each species, we collected 25 individuals for the root mechanical properties determination and 10 individuals with a soil parallelepiped that encompassed the majority of the root system (30 depth x 15 x 15 cm) for the root distribution determination, as 75% of the root biomass is commonly located in the first 30 cm of soil [START_REF] Jackson | A global analysis of root distribution for terrestrial biomes[END_REF].

All sampling was conducted between July and August 2012 to avoid any effect of temporal variability. Before collection, we also counted or measured a) the number of stems, b) the number of rosettes, c) the number of mycorrhizae, c) the height of the stems, d) the number of leaves in each stem (for B. erectus and S. recta)/rosette (for A. vulneraria), e) the maximum depth of the root system (cm) and f) the maximum lateral extension of the root system. Individuals of A. vulneraria were collected from dump deposits of a working quarry (coordinates Gauss-Boaga: 1607763-5047960; altitude: 641 m a.s.l.; aspect: 69°; slope: 26°) characterized by high stoniness (50%, with stones of 1-60 cm). The vegetation at this location consisted of ruderal species that are typical of the pioneer phases of revegetation process, with a low vegetation cover (20%) consisting of only shrub (13%) and herb (7%) layers. Individuals of B. erectus and S. recta were sampled at similar dump deposits (coordinates: 1607763-5047960; altitude: 219 m a.s.l.; aspect: 212°; slope: 41°), with low stoniness (10%, with stones of 1-30 cm), high rockiness (50%) and low vegetation cover (20%) consisting of only shrub (8%) and herb (12%) layers.

Tensile strength tests

We gently separated the collected roots from the soil by hand. We repeatedly washed the roots with jets of water and stored them in a 15% alcohol solution to prevent the growth of mould and microbial degradation [START_REF] Mattia | Biotechnical characteristics of root systems of typical Mediterranean species[END_REF]. As soon as possible, we conducted Considering that the root tensile strength increases from 8% to 20% when the displacement rate is increased rapidly from 10 to 400 mm min -1 [START_REF] Cofie | Test speed and other factors affecting the measurements of tree root properties used in soil reinforcement models[END_REF], we used a constant speed of 10 mm min -1 , according to previous studies [START_REF] Mattia | Biotechnical characteristics of root systems of typical Mediterranean species[END_REF]. We did not include the results for samples that broke in close proximity to the clamps (50 tests, i.e., 11.88% of the total tests) because these breaks were most likely associated with the presence of damage to the root structure [START_REF] Cofie | Test speed and other factors affecting the measurements of tree root properties used in soil reinforcement models[END_REF][START_REF] Mattia | Biotechnical characteristics of root systems of typical Mediterranean species[END_REF][START_REF] Baets | Root tensile strength and root distribution of typical Mediterranean plant species and their contribution to soil shear strength[END_REF].

Data were acquired at 200 Hz and then processed using the Texture Exponent 32 software (Stable Micro Systems, Vienna Court, UK).

The relationship between root tensile force F (N) and root diameter d (mm) can be expressed by a power law: F = a d b [START_REF] Vergani | Variability in tensile resistance of roots in Alpine forest tree species[END_REF].

We used the analysis of covariance (ANCOVA), with the root diameter as the covariant factor, to test the differences among the tensile forces between S. recta and A. vulneraria, as these roots have a broader range of diameters.

Before running the ANCOVA, we log-transformed the data. To test the normality of the data at the 1% significance level, we performed the Kolmogorov-Smirnov test (ks-test).

The tensile force of B. erectus was compared with the other species by considering the values in the classes of diameter < 0.5 mm and between 0.5 and 1 mm. As these data were not normally or log-normally distributed, a non-parametric test (Mann Whitney test) was used to compare the median value of tensile strength in each diameter class.

The mechanical properties of the selected species have also been expressed in terms of the tensile strength (Tr, Mpa) -diameter (d, mm) relationship: Tr = a d b , to compare the results with the available literature (Table 3). The tensile strength can be obtained by dividing the root tensile force, F, by the root diameter, d.

All the analyses were performed with the open source software R (R Core Team, 2014).

Root diameters distribution characterization

We cleaned and photographed the root system of each plant using a white sheet as background. In order to reduce the distortion error of the different root pictures we rectified and scaled the pictures with a Geographic Information System, GIS (QGIS), using a graph paper square of 10 x 10 mm as a background reference (Fig. 3). The same software was used to digitize the roots. We recorded information about root diameters and their position along the explored soil profile. In particular, the diameter of roots at 5, 10 and 15 cm from the middle axes of the plant were used to evaluate the lateral root reinforcement.

We calculated the Root Area Ratio (RAR), which is defined as the ratio between the cross sectional area of all the roots intersecting the lateral surface and the area of the soil, for each species at each distance. This parameter provides a measure of the root density in the soil [START_REF] Bischetti | Root strength and root area ratio of forest species in Lombardy (Northern Italy)[END_REF].

Application to a test case

To assess the effective contribution to the slope stability of the three species studied, we performed a stability analysis applied to a simplified topographic profile that reproduces the condition showed in Fig. 1. The "ordinary method of slices" model [START_REF] Fellenius | Erdstatische Berchnungen[END_REF] was used to predict the stability of the slope under different conditions (see par. 2.2).

The analysed condition is represented by a bank of 45° slope and 5 m height. The soil properties were internal friction factor, ", equal to 39.2°, the soil cohesion, c", equal to 0 kPa under the hypothesis of soil near to saturation along the sliding surface and soil density equal to 1579 kg/m 3 .

Because the actual sliding surface was unknown, different centres and radii of the circular sliding surface were tested.

Different vegetation coverages were considered: 1) bare soil and 2) three monospecific stands of A. vulneraria, B. erectus and S. recta with a density of 100 plants/m 2 uniformly distributed over the surface.

Results

Root mechanical properties

The diameters of the tested roots as well as the number of valid tests are reported in Table 2.

The root tensile force -diameter relationships for the three species are reported in Fig. 4.

The root tensile forces of A. vulneraria and S. recta increased with the root diameters according to the power law curve (Fig. 4). Their root tensile forces were not found to be significantly different at the 0.5 level of significance (ANCOVA, p = 0.064). For B. erectus, all the root diameters were smaller than 1 mm; thus, even if the power law was still significant, extrapolating the values for higher diameter classes was misleading and illogical, as the range of diameters of this species" root system is extremely limited. B. erectus tensile force was significantly higher than the tensile forces of S. recta and A. vulneraria if we consider the diameter class 0-0.5 mm (Mann Whitney test, p < 0.001), while there were no significant differences for the diameter class 0.5-1 mm (p > 0.1).

Root diameters distribution

At a distance of 5 cm from the plant axis, B. erectus showed the highest number of roots (Fig. 5), closely followed by S. recta, whilst the lowest values were calculated for A. vulneraria.

The fasciculate root system of B. erectus was mostly constituted by fine roots between 0.1 and 1.5 mm in diameter. S. recta was characterized by a large primary root (diameter >3 mm), and by secondary thinner roots with diameters between 0.1-2 mm. The root system of A. vulneraria was, in most cases, characterized by a tap root with few and thin lateral roots with diameters between 0.1 and 1 mm.

When further from the plant axis, i.e., from 10 cm to 15 cm from the plant axis (Table 4), S. recta had the highest number of roots because its root system is wider compared with the architecture of the other two species. This species is also the one that showed the highest number of roots with diameters higher than 1 mm, while the root system of the other two species mainly comprised roots smaller than 1 mm in diameter.

The Root Area Ratio (RAR, Table 3 and4), was the highest for Bromus erectus at a distance of 5 cm and the highest for Stachys recta at distances of 10 and 15 cm.

Root reinforcement evaluation

At a 5 cm distance from the plant axis, B. erectus yielded the highest values of root reinforcement (0.00221 MPa) and S. recta the second highest (0.00213 MPa); in contrast, the degree of soil reinforcement provided by A. vulneraria was particularly low (0.00043 MPa).

Considering a larger distance from the plant stem (10 and 15 cm), the reinforcement provided by A. vulneraria and B. erectus was negligible, while S. recta was still able to apply a certain degree of reinforcement (Table 4). It is important to note that the high value of reinforcement given by A. vulneraria at a distance of 15 cm (Table 4) was due to an outlier value of a single plant that had the tap root (diameter = 5 mm) intersecting the lateral area at 15 cm. The mean reinforcement value excluding this outlier would be zero.

Results from stability analysis

Under the condition of bare soil (i.e., without vegetation) and applying eq. 3, the results of the stability analysis are represented in Fig. 6A. Hypothetical sliding surfaces explore most of the bank section and reached a maximum depth of 18 m; however, instability conditions were concentrated in the most superficial part of the slope. The maximum depth of the unstable surface was between 0.06 m to 1.60 m, and the most unstable conditions were represented by the lowest FoS values that were obtained with the most superficial sliding surface (Fig. 6B). This result is in agreement with field evidence where soil movements were superficial (Fig. 1). The most unstable condition (FoS = 0.8) was, indeed, obtained with a maximum depth of 0.09 m.

By including the effect of vegetation, we considered that root reinforcement was bringing an additive cohesion to the soil. In the first case, we considered a uniform distribution of A. vulneraria plants with a distance between each plant of 10 cm (100 plants/m 2 ). This value is consistent with the density range reported for numerous herbaceous/grass species used in greening projects (Dunnet & Hitchmough, 2004) In this case, c r was set to 0.43 kPa. This low reinforcement induces slow stability to the slope but the number of unstable sliding surfaces decreases by 37%, and the lowest FoS increases to 0.87.

If a uniform distribution of B. erectus was considered with the same distance between plants, as in case of A. vulneraria, the average root reinforcement was set to 2.21 kPa. The results of the stability analysis are shown in Fig. 6A. The number of unstable surfaces decreased significantly from 60 to 13 and the minimum FoS was equal to 0.94.

Finally, the effect of S. recta was considered. Under the same hypothesis as B. erectus and A. vulneraria, the root reinforcement was set to 2.13 kPa. The stability analysis showed a low number of critical sliding surfaces (13), and the lowest FoS was 0.94.

Discussion

The identification of the most suitable species for quarry restoration projects is crucial to ensure the success of human interventions on fully exploited dump deposits, especially where the overlap of "soil layers" with different characteristics may frustrate restoration efforts due to the inhibition of vegetation establishment (Gilardelli et al., 2016b).

Our results highlight that the root reinforcement of perennial herbaceous species, chosen among species that can be naturally found in quarry areas at different phases of spontaneous revegetation processes, B. erectus, S. recta and A. vulneraria in this study, is discriminating for their ability to stabilize the topsoil. In particular, the value of lateral reinforcement provided by roots on superficial "soil" layer stability could give useful indications for restoration purposes. The parameters of the root tensile strength -diameter (Tr -D) relationship were in the same range reported by other studies on different herbaceous species (Table 3). In this study, we choose to present our data in terms of a force (F) -diameter relationship instead of a tensile strength (Tr)-diameter relationship to avoid amplifying the uncertainty involved in the determination of diameters [START_REF] Vergani | Variability in tensile resistance of roots in Alpine forest tree species[END_REF], which can be relevant especially for fine and very fine roots.

The values of root reinforcement found for the considered species are also in the range reported in the literature for herbaceous species.

Our results showed that the fasciculate root system of B. erectus yielded the highest values of root reinforcement at 5 cm from the plant stem, due to the high number of fine roots (diameter mostly between 0.1 and 1.0 mm). The same is true for S. recta, which provides root reinforcement values close to B. erectus at the 5 cm distance.

Nevertheless, it is also important to consider the spatial distribution of root reinforcement because it was not spatially homogeneous and varied depending on the root system architecture and on the plant distributions [START_REF] Schwarz | Spatial characterization of root reinforcement at stand scale: Theory and case study[END_REF]. At longer distances from the plant axis, the species that provided the highest reinforcement values was S. recta because its root system is wider than those root systems of the other considered species. The use of a simple stability model was useful to understand the effect of the analysed species on slope stability.

Considering all the simulations, the effect of the vegetation is remarkable in all the cases. A. vulneraria induced stability on the most superficial landslides. Both B. erectus and S. recta gave the highest stabilization effect by reducing both the number of unstable surfaces and increasing the lowest FoS. At the same time, the effect of stabilization of the selected species is limited to the superficial soil while deeper layers must be stabilized in a proper way (gabion wall, stone or concrete wall, stepped boulders; see [START_REF] Morgan | Slope stabilization and erosion control. A bioengineering approach[END_REF].

The data provided here give the first important quantitative information to improve the planning of quarry restoration efforts. Future research should be focused on the performance of development of different species in the field (coverage density by time) and the use of different methods of seeding and superficial soil protection.

It is however important to keep in mind that the capacity of the species to promote soil stability is not the only criteria to be considered in mining restoration. Also the other ecosystem services of the vegetation must be considered, and in particular is important to promote species which allow the starting of a revegetation processes without hindering the succession process towards more mature forms of vegetation complexes / plant communities (Moreno de las [START_REF] Moreno De Las Heras | Vegetation succession in reclaimed coal-mining slopes in a Mediterranean-dry environment[END_REF].

This study contributes to the knowledge of root reinforcement of herbaceous species that can be used in restoration projects of dump deposits of quarry areas to avoid problems of topsoil stability after the phase of soil preparation. The aim is the enhancement of vegetation establishment. Species-specific data are fundamental because the use of species with different types of root systems in restoration projects, inspired by natural revegetation dynamics, could ensure a more effective control of superficial mass movements. The use of species such as B. erectus and S. recta, with a density of 100 plants/ m 2 , has been demonstrated to guarantee the stability of superficial layers in the short and long term.

.

  Quercus pubescens and Ostrya carpinifolia woodlands (Quercetalia pubescentis) as well as to natural/semi-natural arid grassland (Festuco-Brometalia community) for open stands. In the work of Giladelli et al. (2016b) several methods or revegetation applied in the Botticino extractive basin are described in detail: a) spontaneous succession (passive method); b) seeding of hayseed; c) seeding of commercial seed mixtures.

  421 tensile force tests on 15-cm-long root samples after we measured the diameter at the midpoint and at the endpoints. We analysed 35 root samples of A. vulneraria, 262 samples of B. erectus and 124 samples of S. recta. Differences in the number of analysed samples depended on the different root architectures of the three species; when possible, roots were chosen according to the proportion of roots in each root diameter class. We performed these tests using the Stable Micro System TA Hd Plus apparatus (load cell of 500 N, noise of 0.01%) at the CIRA Laboratory of the University of Milan; two non-serrated clamps specifically developed by the Institute of Agricultural Hydraulics of the University of Milan were used.

Fig. 1 .

 1 Fig. 1. The bank on a quarry at the Botticino basin

Fig. 2 .

 2 Fig. 2. Illustration of the ordinary method of slices for a finite slope (slope geometry, position

Table 1 .

 1 Characteristics of the selected species

	Species	Family	Biological form Height (cm)	Preferred substrate*	Type of root	Presence within the Botticino extractive basin**
					basic or neutral-basic	one main tap root and several
			short basal herb
					substrates, even very nutrient-	thinner lateral roots (only
	A. vulneraria	Fabaceae	(H scap -scapose	8-40	poor substrates, such as arid	secondary), with some	Presence at late succession stage, usually absent during the initial
			hemicryptophyte)		grasslands	mycorrhizae	and intermediate phases of revegetation process;
							typical of the local grasslands Surrounded the quarry and can be
			tussock (H caesp -		calcareous substrates that are	fasciculated and composed	used as a reference for the success of restoration efforts
	B. erectus	Poaceae	caespitose	40-60	nutrient-poor, such as arid	of thick, dense and fibrous
			hemicryptophyte)		grassland	primary roots
					highly basic substrates, even
			erect, leafy herb			a main tap root and several
					nutrient-poor ones, such as		present during all the phases of revegetation process, especially on
	S. recta	Lamiaceae	(H scap -scapose	20-40		lateral roots (secondary and
					rocks, heaps of stones and arid		dump deposits abandoned in the previous 1-10 years
			hemicryptophyte)			tertiary)
					grassland
				Data from: *Pignatti, 1982 and Pignatti et al., 2005; **Gilardelli et al., 2016a

Table 2 .

 2 Parameters (a and b values) and R 2 values for the relationships among root tensile strength and root diameter

			A. vulneraria	B. erectus	S. recta
	MORPHOLOGY
	Number of stems	Mean	8	64*	6
		St.dev	6	48*	5
	Number of rosettes	Mean	5	0	0
		St.dev	4	0	0
	Number of mycorrhizae	Mean	49	0	0
		St.dev	58	0	0
	Height of the stems (cm)	Mean	6.00	54.01	50.03
		St.dev	2.55	10.35	11.91
	Number of leaves in each stem/rosette	Mean	6	2	59
		St.dev	3	0	34
	ROOT SYSTEM
	Maximum depth (cm)	Mean	16.62	26.82	24.48
		St.dev	5.83	10.83	11.21
	Maximum lateral extension (cm)	Mean	13.85	20.04	17.62
		St.dev	5.26	3.66	3.30
	Number of valid tensile tests		28/35	236/262	107/124
	Root length (cm)	Mean	16.59	19.20	17.81
		St.dev	3.66	4.85	3.28
	Mean diameter of all roots (mm)	Mean	0.98	0.29	1.24
		St.dev	1.13	0.10	1.30
	Mean diameter of tested roots (mm)	Mean	0.86	0.29	1.02
		St.dev	0.98	0.10	1.10
	Max. diameter of tested roots (mm)		3.50	0.84	5.30
	Min. diameter of tested roots (mm)		0.05	0.07	0.07
	*included 41 (± 27) sterile shoots

Table 3 .

 3 Literature data for root tensile strength, RAR, and root cohesion of herbaceous species; the results of the present study are shown in bold. * RVR was reported for A. vulneraria, B. erectus and S. recta. Legend for authors: A:[START_REF] Schiechtl | Bioengineering for land reclamation and conservation[END_REF] B: Waldron, 1977; C: Wu, 1984; D: Mattia et al., 2005; E: de Baets et al., 2008.

	Species	D (mm)	a	B	Tr	mean (MPa)	RAR* (%)	Root cohesion (MPa)	Author
								0.005-
	Ammophila spp.							0.010	B
	Ammophila spp.	<0.3					0.015-0.15		C
	Anthyllis
	vulneraria	0.05-3.50 11.88 -0.81		55.19	0.0000275	0.00221	-
	Atriplex halimus 1.9 (0.8)	73	0.6		57.2 (23.1)			D
	Avenula
	bromoides	0.15-0.32 4.77 -1.52			0.0033		E
	Brachypodium
	retusum	0.10-1.45 45.05 -0.61					E
	Bromus erectus 0.07-0.84 33.30 -0.61		81.41	0.000132	0.00043	-
	Helictotrichon
	filifolium	0.34-1.22 14.51 -1.08			0.0046		E
	Herbaceous
	vegetation	<10					0.02-0.08		A
							0.002-
	Hordeum vulgare	<0.5					0.008		B
								0.001-
	Hordeum vulgare							0.0025	B
								0.244-
	Juncus acutus	0.18-1.10 23.23 -0.89				0.304	E
	Limonium
	supinum	0.34-3.90 33.82 -0.85			0.00125		E
	Lygeum spartum 0.26-2.72 19.28 -0.68			0.002575		E
	Lygeum spartum	1.3-1.7	60.7	1.3		37.8 (12.5)			D
	Phragmites
	australis	0.10-7.91 34.29 -0.78					E
	Piptatherum
	miliaceum	0.10-0.64 11.49 -1.77					E
	Plantago
	albicans	0.21-2.55 16.75 -0.52					E
	Stachys recta	0.07-5.30 15.27 -0.71		30.99	0.000104	0.00213	-
	Stipa tenacissima 0.43-1.34 24.34 -0.61					E

Table 4 .

 4 Mean values and standard deviations of lateral reinforcement and RAR at the three considered distances

	Species	Distance (

cm) Mean cr lat (kPa)

							Standard
				Standard			Deviation
				Deviation	N	Mean RAR	RAR
	A. vulneraria	5	0.43	0.88	10	2.7535E-05	3.91459E-05
		10	0.189555556	0.263845	10	1.51477E-05	2.1245E-05
		15	0.4	1.25	10	9.98229E-06	3.15668E-05
	B. erectus	5	2.21	2.32	10	1.32E-04	6.55432E-05
		10	0.08	0.09	10	4.51364E-06	6.11906E-06
		15	0	0	10	0	0
	S. recta	5	2.13	2.89	10	1.04E-04	8.70E-05
		10	0.76	1.60	10	4.31E-05	8.57E-05
		15	0.42	1.02	10	1.13E-05	2.62E-05

Acknowledgments

The present work was funded by the Province of Brescia (Lombardy, Itlay). Authors are grateful to Dr Pierangelo Barossi (Province of Brescia), Prof. Gian Battista Bischetti (University of Milano), Dr. Lorenzo Fongaro (DiSTAM -University of Milano), Prof. Mara Lucisano (DiSTAM -University of Milano) and Prof. Sergio Sgorbati (University of Milano-Bicocca) for their technical support to this research.