Thierry Rabilloud
email: thierry.rabilloud@cea.fr

How to use 2D gel electrophoresis in plant proteomics

Keywords: two-dimensional electrophoresis, proteomics, blotting, post-translational modifications

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Introduction

At the beginning of the 21st century, it is fashionable to describe 2D gel-based proteomics as an outdated and poorly efficient technique. This feeling is mainly due to the fascination of many scientists in proteomics for big lists, and in this case the welldocumented undersampling of 2D gel-based proteomics is dearly resented, as well as its almost complete inability to analyze transmembrane proteins [START_REF] Santoni | Membrane proteins and proteomics: Un amour impossible?[END_REF][START_REF] Rabilloud | Membrane proteins and proteomics: love is possible, but so difficult[END_REF], despite of lot of effort devoted to this particular issue (e.g. in [START_REF] Santoni | Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels[END_REF][START_REF] Santoni | Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties[END_REF] for plant samples). This undersampling has been documented mostly on animal samples [START_REF] Petrak | Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins[END_REF][START_REF] Wang | Generally detected proteins in comparative proteomics--a matter of cellular stress response?[END_REF], but it is obvious that it will take place on plant samples as well.

In fact, all proteomic techniques undersample complex samples by a ratio of 1:10 (1000 proteins analyzed on 2D gels among the 10,000 present in a cell, 20,000 peptides analyzed by shotgun proteomics among the 200,000 derived from a cell sample).

However, as 2D gel-based proteomics is the only setup that analyses complete proteins, the consequences of undersampling are clear and heavy. If a protein is not seen, it is lost for ever. In addition, the undersampling rules in 2D gel-based proteomics are well known. Hydrophobic proteins, high molecular weight proteins and rare proteins do not show up.

In shotgun techniques, the undersampling rules are also known. However, they apply to peptides, and how they translate in terms of protein identification and even more in terms of protein quantification is complex, and linked to the complexity of the protein inference problem [START_REF] Nesvizhskii | Interpretation of shotgun proteomic data: the protein inference problem[END_REF][START_REF] Li | A bayesian approach to protein inference problem in shotgun proteomics[END_REF][START_REF] Grobei | Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function[END_REF] .

On top of this strong undersampling problem comes the often unrecognized fact that 2D gel-based proteomics is a low yield process [START_REF] Choe | Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis[END_REF][START_REF] Zhou | A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure[END_REF] and the fact that plant samples are among the most difficult samples to prepare for an analysis by 2D gels [START_REF] Saravanan | A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues[END_REF], due to the variety and amounts of interfering, non-protein compounds present in many plant cell types and tissues.

All these negative sides explain the trend from 2D gel-based proteomics to shotgun proteomics, as exemplified by the study of cadmium response in plant. Gel-based in 2006 [START_REF] Sarry | The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses[END_REF], shotgun-based in 2011 [START_REF] Villiers | Investigating the plant response to cadmium exposure by proteomic and metabolomic approaches[END_REF].

However, 2D gel-based proteomics is still very popular in plant proteomics, as shown by recent publications in the field (e.g. [START_REF] Tanou | Proteomic signatures uncover hydrogen peroxide and nitric oxide cross-talk signaling network in citrus plants[END_REF][START_REF] Catusse | Proteomics reveals potential biomarkers of seed vigor in sugarbeet[END_REF], but among more than 150 publications in 2011). There are several built-in reasons for this sustained interest in this technique, which are named reproducibility, robustness, efficiency, ability to analyze intact proteins, adequacy to study post-translational modifications, easy interface with powerful biochemical techniques. These different points will be developed below.

Reproducibility and robustness

It is not an overstatement to write that 2D gel-based proteomics is still, and by far, the most reproducible and robust proteomic setup [START_REF] Hackett | Science, marketing and wishful thinking in quantitative proteomics[END_REF]. A further proof of this statement lies in the much higher expectations from proteomic journals, in terms of sample numbers and experimental power, applied to 2D gel-based proteomics compared to shotgun proteomics [START_REF] Celis | Gel-based proteomics: what does MCP expect?[END_REF][START_REF] Wilkins | Guidelines for the next 10 years of proteomics[END_REF]. Still today, what is common practice in 2D gel-based proteomics, i.e. quantitative comparison of two or more biological conditions with several individual biological replicates for each condition, is still rarely found in publications using shotgun proteomics.

This reproducibility is further testified by the interlaboratory reproducibility of 2D maps [START_REF] Corbett | Positional Reproducibility of Protein Spots in 2-Dimensional Polyacrylamide-Gel Electrophoresis Using Immobilized Ph Gradient Isoelectric-Focusing in the First Dimension -an Interlaboratory Comparison[END_REF][START_REF] Blomberg | Interlaboratory Reproducibility of Yeast Protein-Patterns Analyzed by Immobilized Ph Gradient 2-Dimensional Gel-Electrophoresis[END_REF], while recent test of shotgun techniques on the same sample (yeast) revealed a much higher standard deviation between laboratories [START_REF] Paulovich | Interlaboratory study characterizing a yeast performance standard for benchmarking LC-MS platform performance[END_REF][START_REF] Tabb | Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry[END_REF] Ironically enough, the high robustness of 2D gel-based proteomics comes in part from the terrible reproducibility of isoelectric focusing in its early days. Put bluntly, only the gels run simultaneously were comparable. This led the proteomic pioneers to develop devices to run several gels in parallel [24, [5]. When immobilized pH gradients were finally introduced in 2D gel electrophoresis with solid protocols [START_REF] Gorg | Methodology of Ipg-Dalt for the Analysis of Cell Lysates and Tissue Proteins[END_REF], the reproducibility and robustness of the technique made a quantum leap that is still enjoyed today.

Moreover, this parallelization of the technique is also a very strong advantage when multiple comparisons, and not only binary ones, must be performed [START_REF] Natera | Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis[END_REF].

Efficiency of 2D gel-based proteomics

Nowadays, Edman sequencing for protein identification has been almost completely replaced by mass spectrometry-based protein identification, which is much more sensitive and much faster. However, mass spectrometers are expensive and delicate instruments, and the machine time is often considered as precious.

In this frame, shotgun proteomic techniques often represent a waste of machine time. In many comparative experiments, which represent most of the proteomic experiments, most of the proteins do not change between the various conditions tested. Thus, the mass spectrometer is analyzing again and again a very high number of peptides from proteins that have no interest in the biological question studied.

Oppositely, 2D gel-based proteomics makes an optimal use of mass spectrometer time, because most of what is carried out within the mass spectrometer in shotgun protemics is carried out upfront at the 2D gel stage in 2D gel-based proteomics. In fact, the modern gel staining techniques [START_REF] Miller | Protein stains for proteornic applications: Which, when, why?[END_REF][START_REF] Patton | A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics[END_REF][START_REF] Minden | Difference gel electrophoresis[END_REF], allow to perform a sensitive and linear detection of the protein spots on the gels. Then, image analysis [START_REF] Bandow | Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies--COPD biomarker discovery study[END_REF][START_REF] Stessl | Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data[END_REF] coupled with statistical analysis [START_REF] Karp | Experimental and statistical considerations to avoid false conclusions in proteomics studies using differential in-gel electrophoresis[END_REF], allow to perform a quantitative analysis of all protein spots and to determine which spots are worth identifying. These spots are then the only ones that need to be processed by the mass spectrometer.

When financial resources are scarce, which is often the case in academic research, this allows to build a "hub and spokes" model, where a central hub hosting the mass spectrometry platform is able to serve very efficiently several biology-oriented laboratories, each using 2D gel electrophoresis and image analysis to select the relevant proteins within their respective research projects.

With plant proteomics usually less funded than medicine-oriented proteomics, and this situation even harder for the proteomics of traditional crops, this efficiency factor cannot be neglected in the research landscape.

Interface with biochemical techniques

Within this section, the focus will be shifted from classical, quantitative proteomics, where the name of the game is to find differences in expression on total extracts, to more focused proteomics experiments where the interest is centered on subclasses of proteins. In many cases, the protein subclasses are defined by a common biochemical feature, and quite often this biochemical feature is a post-translational modification, for which an affinity reagent exist.

When such an affinity reagent exists, two types of strategies can be devised to perform proteomic studies targeted for the proteins recognized by the affinity reagent.

In the first strategy, the affinity reagent is used as a pre-proteomic preparative tool, aiming at selecting the proteins or peptides of interest in the complex starting extract.

The selected proteins/peptides are then analyzed by a classical proteomic setup. This strategy is the only one that can be used with shotgun proteomics, but it can also be used with 2D gel-based proteomics as well.

In the second strategy, the affinity reagent is used as an analytical reagent, after the separation stage in the proteomic setup, in order to detect selectively the proteins of interest. This strategy works extremely well with 2D gel-based proteomics, thanks to the easy interface provided by the blotting process between 2D gels and affinity reagents. Thus, the final performance of the whole process will largely depend on the efficiency of the affinity reagent under the preparative and analytical setups, and it must be kept in mind that for several reasons, the affinity reagent is always used in a large excess over the biological extract.

Then two major cases can be distinguished: i) the affinity reagent is not a protein typical examples of that configuration are represented by boronic acid for sugars and either IMAC or metal oxides for phosphate. These non-proteinaceous reagents are best used in the preparative mode, and work wonderfully with shotgun proteomics [START_REF] Thingholm | Analytical strategies for phosphoproteomics[END_REF].

ii) the affinity reagent is a protein In this case, the suitability of the reagent as a preparative tool will depend not only on its selectivity, but also on the existence of mild elution conditions that are able to elute all the analytes without eluting out the affinity reagent.

Lectins represent such a happy case, and have used as a preparative reagent in 2D gelbased proteomics [START_REF] Robotti | Acute-phase proteins investigation based on lectins affinity capture prior to 2-DE separation: application to serum from multiple sclerosis patients[END_REF] and in shotgun proteomics [START_REF] Ligat | Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins[END_REF][START_REF] Catala | Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis[END_REF][START_REF] Zhang | Combining various strategies to increase the coverage of the plant cell wall glycoproteome[END_REF].

They have also been used as a post-2D gel selective detection reagent [START_REF] Komatsu | Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths[END_REF] Conversely, antibodies represent a case where elution without polluting the extract with antibodies is very difficult. They are therefore much better used as analytical reagents on blots rather than preparative reagents. Thus, 2D gel-based proteomics is a very efficient strategy to use in conjunction with antibodies, and this strategy has been used to identify several post-translational modifications, such as citrullinated proteins [START_REF] Ishigami | Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease[END_REF], carbonylated proteins [START_REF] Job | Patterns of protein oxidation in Arabidopsis seeds and during germination[END_REF], proteins containing nitrotyrosine [START_REF] Castegna | Proteomic identification of nitrated proteins in Alzheimer's disease brain[END_REF][START_REF] Kanski | Proteomic identification of 3nitrotyrosine-containing rat cardiac proteins: effects of biological aging[END_REF] or hydroxynonenal adducts [START_REF] Perluigi | Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis[END_REF][START_REF] Reed | Proteomic identification of HNE-bound proteins in early Alzheimer disease: Insights into the role of lipid peroxidation in the progression of AD[END_REF].

Although very successful, this strategy has a major caveat, i.e. the fact that a minor and modified spot detected with the antibody may comigrate with a major, unrelated spot which will be the one identified by the downstream mass spectrometry process, resulting in a major mistake. The severity of this problem increases with spot crowding and thus with the complexity of the biological extract. There is however an easy counter, at least for acidic and neutral proteins, which is the use of narrow range pH gradients that greatly decrease the probability of comigration. Such narrow pH range gels have been used under a variety of circumstances [START_REF] Parker | Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination[END_REF][START_REF] Westbrook | Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms[END_REF][START_REF] Sarma | A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum[END_REF][START_REF] Zhao | Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways[END_REF], and have proven very efficient, especially when used in conjunction with prefractionation techniques [START_REF] Righetti | How to bring the "unseen" proteome to the limelight via electrophoretic pre-fractionation techniques[END_REF].

Analysis of intact proteins and of post-translational modifications

The more proteomics progresses, the more it stresses the importance of posttranslational modifications in biology (e.g. in [START_REF] Rajjou | Proteomics and posttranslational proteomics of seed dormancy and germination[END_REF][START_REF] Arc | Reboot the system thanks to protein post-translational modifications and proteome diversity: How quiescent seeds restart their metabolism to prepare seedling establishment[END_REF] ). However, not all post-translational modifications can be easily studied through the use of an affinity reagent, and some, as methylations, are not easily detected except on abundant proteins [START_REF] Santoni | Methylation of aquaporins in plant plasma membrane[END_REF].

For the study of these difficult, and sometimes unknown, modifications, 2D gel-based proteomics is a platform of choice, either through selective and transient labelling [START_REF] Fratelli | Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes[END_REF], or by using the fact that many modifications induce a pH shift and thus a separation of the modified protein from the bulk of the unmodified one. This can be used in the field of protein cleavage [START_REF] Lee | Identification of caspase-3 degradome by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionizationtime of flight analysis[END_REF][START_REF] Jang | Mining of caspase-7 substrates using a degradomic approach[END_REF] , but also as a preparative tool to isolate the modified spot, thereby leading to an easier identification of the modifications. Such an approach has been used for the identification of thiol oxidations [START_REF] Rabilloud | Proteomics analysis of cellular response to oxidative stress -Evidence for in vivo overoxidation of peroxiredoxins at their active site[END_REF][START_REF] Weber | Oxidative stress triggers thiol oxidation in the glyceraldehyde-3-phosphate dehydrogenase of Staphylococcus aureus[END_REF][START_REF] Hwang | Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions[END_REF].

More generally, the separating ability of 2D gels, coupled with their ability to be used as a micropreparative tool [START_REF] Hanash | Highly Resolving 2-Dimensional Gels for Protein Sequencing[END_REF], now allow to set very comprehensive maps of protein modifications [START_REF] John | Complete sequencing and oxidative modification of manganese superoxide in medulloblastoma cells[END_REF][START_REF] Seo | Strategy for comprehensive identification of post-cellular proteins, including low abundant modifications: glyceraldehyde-3phosphate dehydrogenase[END_REF]. In this area, the use of ultra narrow pH gradient [START_REF] Westbrook | Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms[END_REF], with their almost infinite resolving power [START_REF] Bosisio | Electrophoretic and chromatographic techniques for the differential diagnosis of a haemoglobin abnormality: Hb E heterozygosity[END_REF][START_REF] Cossu | Resolution of G gamma and A gamma foetal haemoglobin tetramers in immobilized pH gradients[END_REF] should be extremely useful, by providing a very thorough deconvolution of the protein spots into individual protein species.

Conclusions

Nowadays, 2D gel-based proteomics is usually selected on the basis of its robustness and relatively low cost, among the proteomic techniques. It can be reasonably anticipated, however, that the uses directed toward the identification of post-translational modifications, and especially the determination of the real combination of modifications present on a single protein species, will develop considerably in the future.


Régis Céréghino
email: cereghin@cict.fr

Beat Oertli

Marcello Bazzanti

Cristina Coccia

Arthur Compin

Jeremy Biggs

Nicolas Bressi

Patrick Grillas

Andrew Hull

Thomas Kalettka

Olivier Scher

A Compin Ecolab

Biological traits of European pond macroinvertebrates

Keywords: Biogeographie regions, Community functions, Database, Freshwater biodiversity, Fuzzy coding

Whilst biological traits of river macroinvertebrates show unimodal responses to geographie changes in habitat conditions in Europe, we still do not know whether spatial turnover of species result in distinct combinations of biological traits for pond

allocated lesser energy to reproduction and dispersal, and organisms were short lived with high diversity of feeding groups. These characteristics suggest higher resilience. The main difference between ponds in the Alpine and Atlantic regions was their elevation. Alpine conditions necessitate specifie adaptations related to rapid temperature fluctuations, and low nutrient concentrations. Even if our samples did not cover the full range of pond conditions across Europe, our analyses suggest that changes in community composition have important impacts on pond ecosystem functions. Consistent information on a larger set of ponds across Europe would be much needed, but their low accessibility (unpublished data and/or not disclosed by authors) remains problematic. There is still, therefore, a pressing need for the incorporation ofhigh

Introduction

Ponds can be defined as small standing freshwater bodies about 1 m 2 to a few hectares in surface area, and from a few centimetres to a few metres in depth [START_REF] Oertli | Conservation and monitoring of pond biodiversity: introduction[END_REF]. Recent studies have shown that ponds in Europe are just as important as rivers and lakes in supporting a wide range of aquatic biodiversity [START_REF] Williams | Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in Southem England[END_REF][START_REF] Davies | Comparative biodiversity of aquatic habitats in the European agricultural landscape[END_REF]. Information collected at sites across Europe which represent a geographical distribution of biodiversity found in the Atlantic, Central and Mediterranean regions, illustrated that ali types of freshwater systems (rivers, lakes, and ponds) support unique species not found in other water body types (Céréghino et al., 2008a), but that collectively, ponds host more species, and more rare species, than streams, rivers or lakes [START_REF] Williams | Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in Southem England[END_REF]. Until recently, ponds have been mostly ignored by freshwater biologists or regarded as smaller versions of larger lakes, and much research aimed at assessing the biodiversity that ponds support [START_REF] Oertli | Pond Conservation in Europe[END_REF]. Ponds are threatened by human activity (agriculture, industry, and urbanization), though these ecosystems have obvions ecological functions [START_REF] Chase | Connectivity, scaledependence, and the productivity--diversity relationship[END_REF][START_REF] Hansson | Con:flicting demands on wetland ecosystem services: nutrient retention, biodiversity or both?[END_REF] and recognized social and economie uses [START_REF] Chapman | Wetlands of East-Africa: biodiversity, exploitation and policy perspectives[END_REF]. During the twentieth century, wetland losses reached 40-90% in a number of northwestern European countries [START_REF] Hull | The pond life project: a model for conservation and sustainability[END_REF]. As a result of large-scale habitat loss, many species are also endangered, thus motivating studies aiming at understanding fundamental patterns of pond communities from local to regional scales [START_REF] Oertli | Pond Conservation in Europe[END_REF].

Both scientific studies and regional surveys of ponds have provided large volumes of site-specifie data (e.g. Pond [START_REF] Pond Action | The Oxfordshire Pond Survey. A Report to the World Wide Fund for Nature (WWF-UK)[END_REF][START_REF] Boix | The faunal composition of Espolla pond (NE lberian peninsula): the neglected biodiversity of temporary waters[END_REF][START_REF] Sahuquillo | Macroinvertebrate communities in sediment and plants in coastal Mediterranean water bodies (Central lberian Peninsula)[END_REF][START_REF] Oertli | Macroinvertebrate assemblages in 25 high alpine ponds of the Swiss National Park (Cirque of Macun) and relation to environmental variables[END_REF] from which spatial patterns of biological communities can be explored. However, at a regional-continental scale, use of biological traits ( e.g. body size, feeding habits, and life-history) is likely to be more useful than species lists in patterning community organization where different geographie regions are covered, though biologists are traditionally wedded to the use of such lists [START_REF] Cayrou | Species associations in lotie macroinvertebrates and their use for river typology. Example of the Adour-Garonne drainage basin (France)[END_REF]. Traits reflect environmental conditions and may be shared among many species [START_REF] Southwood | Tactics, strategies and templets[END_REF][START_REF] Statzner | Species traits and environmental constraints: entomological research and the history of ecological theory[END_REF][START_REF] Santoul | Spatial patterns of the biological traits of freshwater fish communities in S.W. France[END_REF]; species occurrence may have a strong stochastic element. Traits may give greater insight into habitat change [START_REF] Dolédec | Species traits for future biomonitoring across ecoregions: patterns along a human-impacted river[END_REF][START_REF] Statzner | Biological trait composition of European stream invertebrate communities: assessing the effects of various trait filter types[END_REF] and their determination requires much less taxonomie expertise [START_REF] Dolédec | Accurate description of functional community structure: identifying stream invertebrates to species-level?[END_REF][START_REF] Dolédec | Accurate description of the abundance of taxa and their biological traits in stream invertebrate communities: effects of taxonomie and spatial resolution[END_REF], so that it can be utilized where limited information is available, and/or for animal groups where taxonomie knowledge is limited.

The aim of this study was to compare the biological traits of macroinvertebrate communities among four biogeographie regions of Europe (Mediterranean, Atlantic, Alpine, and Continental) defined by characteristic vegetation, climate, and geology by the European Environment Agency (EEA, 2002). To date, most of our current understanding of invertebrate traits in ponds has come from local-scale studies, where traits were examined in relation to environmental variables such as hydroperiod [START_REF] Gascon | Relation between macroinvertebrate life strategies and habitat traits in Mediterranean salt marsh ponds (Emporda wetlands, NE lberian Peninsula)[END_REF], vegetation cover (Céréghino et al., 2008b, c ), or pond successional stage [START_REF] Ruhi | Spatial and temporal patterns of pioneer macrofauna in recently created ponds: taxonomie and functional approaches[END_REF]. Although these studies showed that changes in species composition in relation to physical-chemical conditions can affect community functions in ponds, we still do not know whether large-scale changes in taxonomie compositions result in changes in community functions. Here, we examined whether spatial turnover in invertebrate taxa from Northwestern to Southwestern Europe result in distinct compositions of biological traits for pond macroinvertebrates. To this end, data on the macroinvertebrate communities of 120 ponds were collected from the published literature (occurrence matrix for 204 taxa), then biological traits were described using a fuzzy-coding method (species-traits matrix). A simultaneous analysis of two matrices performed through a co-inertia analysis (CoA) was used to investigate the spatial distribution of species-trait combinations [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF]. The results were analyzed with reference to geographie differences in environmental conditions, but also highlighted current methodological gaps and future needs.

Methods

Data selection

Previous studies provided the database on invertebrate communities (see Fig. 1). We assembled data to generate a presence/absence matrix for 204 taxa in 120 ponds (see Appendix 1 for list of taxa-Supplementary material). Since our aim was to compare traits among regions, ponds were not expected to be evenly distributed within the various regions. Instead, earlier studies provided lists of taxa originating from four biogeographie regions (Mediterranean, Atlantic, Alpine, and Continental), and three main gradients: latitude (North-South, from UK to southern Spain), elevation (0 ---+ 2,600 rn a.s.l), and hydroperiod (permanent or temporary). Biogeographie regions were defined and characterized for climate, geology, and vegetation in EEA (2002). Criteria for selection were the rating of the data based on the description of the sampling protocols (intensive sweeping of a band net through the various habitat types, replicate samples from the various habitats), and at least two sampling periods at distinct seasons, to take into account invertebrate seasonality. The distribution of selected sites is shown in Fig. 1. Species lists per site were compiled as the sum of recorded species among the two periods. If a data source provided a longer time series of invertebrate occurrences, we selected periods that bad data being representative for one annual cycle. Finally, taxa identifications bad to allow aggregation at the genus (most insect taxa, Mollusca) or family (Oligochaeta) level, which corresponded to the lowest systematic units in the trait database. Given that we wished to analyse occurrence (i.e. qualitative) data, our criteria for data selection (mostly based on sampling efforts in space and time) suggest that we could be confident that the authors of selected studies did well at describing their pond invertebrate communities, and, subsequently, that the data assembled for the present study provided a reliable presence-absence matrix.

Species traits were obtained from [START_REF] Tachet | Invertébrés d'eau douce. Systématique, biologie, écologie[END_REF] (Appendix 2-Supplementary material). These traits were adult body size, life-cycle duration, number of cycles per year, aquatic stage, reproduction mode, dispersal mode, resistance forms, respiration mode, locomotion, food, feeding habits, substrate, trophic status, salinity, temperature tolerance, saprobity, and pH tolerance. Each trait was divided into categories, and categories for each biological trait were ordinal (e.g. <2.5 mm, 2.5-5 mm, 5-10 mm, etc., for adult body size) or nominal ( e.g. egg, larva, nymph, adult, for aquatic stage) (see Appendix 2-Supplementary material). Species-trait information was then structured using a fuzzy-coding technique [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF]: scores ranged from '0', indicating 'no affinity' to '5', indicating 'high affinity' for a given species-trait category. This technique allowed us to take into account ontogenetic niche shifts and within-taxon variability in traits. For instance, a genus that mostly contains predators and a few scapers would be given an affinity of 3 for the feeding category predator, and a 1 for the category scraper. A predator that feeds on benthic algae during its earl y instars would be assigned the same coding. This procedure allowed us to build the 'species-traits matrix'.

Data analysis

First, correspondence analysis (CA) was used to ordinate the ponds according to species presence/ absence data, thus summarizing the variability of invertebrate communities, and providing insights for the discussion of the subsequent species-traits analysis. The significance of the axes was determined atP < 0.05 bytes ting the eigenvalues of the inertia matrix. Second, the species-traits matrix was studied by a 'Fuzzy Correspondence Analysis' (FCA) [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF]. Finally, a simultaneous analysis of the two matrices (species distributions and species-traits (Dolédec & Chessel, 1994). This analysis studies co-structure by maximizing covariance between faunistic and biological traits ordination scores in the first two analyses (Dray et al., 2003). The aim. of the CoA is to visua1ize spatial variations in the combinati.ons of biological traits of pond invertebrates. A Permutation oest (Dol.édec & Chessel, 1994) was used to check the significance of the resulting correlation between the two sets of data resulti.ng from the two kind of analysis (CA and FCA). We carried out 500 co-inertia analyses of the taxonomie and biological traits data sets aCter random permutation of their rows. We measured the correlation between the two tables using the RV-coefficient, a multidimensional equivalent of the ordinary correlation coefficient between two variables (Robert & Escoufier, 1976, Dolédec et al., 2006). the 500 possible. The objective is to determine the common structure between the two sets of data, and then to interpret differences in pond ecosystems in terms of combinations of biological traits of their aquatic communities. Mann-Whitney tests were employed to test significant differences in pond distribution in the CA and in the CoA according to biogeographie regions and tohydroperiod, byusing the coordinates of samples on the most relevant axis. These analyses were performed with R software.

Results

Taxa distribution

The Atlantic and Mediterraneanregions bad the highest taxon richness (145 and 160 taxa, respectively). Thirty and 53 taxa were found to occur in the Alpine and Continental regions. When a CA was carried out on the invertebrate matrix (presence/absence) (Fig. 1 ), the first two axes contributed 19.6 and 13.4% to the overall variance, respectively. Axis 2 showed a geographical gradient of ponds, which roughly corresponded to the North (top) to South (bottom) distribution of the ponds, i.e. to a latitudinal gradient of underlying variables probably related to climatic and geologie conditions. The scatterplot of the CA could be divided into three subsets along axis 2 when sampling sites were more specifically grouped according to their biogeographie origin (Fig. 2a), i.e. Mediterranean, Atlantic and Alpine, and Continental. There was also a gradient of hydroperiod (from permanent to temporary ponds) from Northern (top area of the scatterplot) to Southem regions (bottom) (Fig. 2a) (Mann-Whitney test on samples' coordinates along axis 2, P < 0.01 for ponds grouped by region and by hydroperiod). Whilst only two taxa were exclusively found in the Alpine (the coleopteran Limnius) and Continental (the odonate Epitheca bimaculata) regions, 28 and 34 taxa were exclusively recorded in the Atlantic and Mediterranean regions respectively (Fig. 2b ). Among these invertebrates, the commonest 'Atlantic' taxa were mostly trichopterans (Phryganea, Anabolia, Glyphotaelius pellucidus, and Trianodes), but also the amphipod Crangonyx, the heteropteran Callicorixa, the gastropod Armiger crista, or the turbellarian Dendrocoelum lacteum. Mediterranean ponds hosted the more distinct fauna. The most common taxa were Anostraca fairy shrimps, the tadpole shrimps Triops cancriformis and Lepidurus apus, the coleopteran Cybister lateralimarginalis, the backswimmer Anisops sardea, or the gastropod Physella. W e assume that axis 1 displayed a gradient of local conditions, because ponds from Central and Southem Europe were distributed along the ordinate. Therefore, the gradient analysis performed through CA basically portrayed the graduai biogeographie changes in the compositional structure of invertebrate communities across W estero Europe, with respect to variables acting at broad scales (axis 2) and local scales (axis 1).

Spatial patterns of invertebrate traits

A permutation test indicated that the co-inertia between the species distribution and species-trait matrices was highly significant (RV = 0.54; P < 0.001). Axes 1 and 2 expressed 24 and 22% of the overall variance, respectively. Thus, the CoA (Fig. 3a,b) of species' distributions and their biological traits made it possible to derive sorne spatial patterns of species-trait combinations across Europe. Axis 2 showed a biogeographie gradient. As in the CA, the scatterplot of the CoA could be divided into three main subsets along axis 2 when sampling sites were grouped according to their biogeographie origin (Fig. 3a), i.e. Mediterranean, Atlantic and Alpine, and Continental (Mann-Whitney tests on pond coordinates along axis 2, P < 0.01). We assume that axis 1 displayed a gradient of local conditions, because ponds from each of the three above mentioned subsets displayed taxonomie variations along the abscissa. Specifically, the grouping of ponds according to their permanent or temporary nature suggested a gradient of hydroperiod along axis 1, from temporary (left) to permanent (right) (Mann-Whitney tests on pond coordinates along axis 1, P < 0.01). Mediterranean ponds (top area of the scatterplot), either permanent or temporary, had a higher proportion of interstitial or attached organisms, feeding by sediment absorption, consuming bacteria or fine organic detritus ( e.g. Oligochaeta) (Fig. 3b ). Most species used asexual reproduction, and had cocoons as resistance form. Mediterranean pond macroinvertebrates were mostly small-sized ( <2.5 mm), but the largest species were also found to occur in these communities (>80 mm, e.g. Hirudo medicinalis, the threatened medicinal leech, a species listed in Anne x II of the Habitats Directive and in the IUCN Red List). Finally, it is worth noting that there was a higher proportion of stenothermic (narrow thermal range) invertebrates in the Mediterranean region. At the opposite of the scatterplot (right bottom corner), Continental ponds were characterized by invertebrates with short life cycles ( <1 year), and a higher proportion of rather large (20--80 mm) predatory species (e.g. the odonate Coenagrionidae) that prey on other macroinvertebrates, or even on small vertebrates. Finally, there was no clear distinction between Atlantic and Alpine ponds (all permanent). Compared with the above mentioned fauna, Atlantic and Alpine pond invertebrates were mostly medium-sized detritivores or herbivores (2.5-20 mm), with a higher proportion of grazers and filterers.

Discussion

Whilst the biological trait profiles of river invertebrates in Europe [START_REF] Statzner | Biological trait composition of European stream invertebrate communities: assessing the effects of various trait filter types[END_REF] show rather unimodal responses, pond invertebrates exhibit a higher diversity of ecological strategies, and biological traits combinations from Northem to Southem areas are more different than in rivers. Presumably, this is owing to the fact that ponds display a broader range of physical and chemical characteristics (i.e. a broader range of niche opportunities) than streams, rivers or lakes, whereas the larger water bodies tend to have similar more physical and chemical properties across regions [START_REF] Davies | Comparative biodiversity of aquatic habitats in the European agricultural landscape[END_REF]. This idea is strengthened by observations made in very small aquatic habitats with a simple structure like phytotelmata (plant-held waters such as tree-holes). In these containers, niche opportunities are rather consistent, and invertebrate species traits remain similar despite geographical changes in taxonomie composition (see e.g. [START_REF] Kitching | Food webs and container habitats: the natural history and ecology of phytotelmata[END_REF]. In ponds, it thus appeared that changes in community composition across Europe have a stronger impact on community functions than in rivers. This observation also suggests that the macroinvertebrates found in the varions regions are phylogenetically more distant in ponds than in rivers (e.g. high proportions of crustaceans in the Mediterranean, high proportions of trichopterans in the Atlantic).

Most of our study ponds in the Mediterranean Region are extreme sites, characterized by an altered salinity due to their vicinity with the sea, and/or high evaporation rate related to arid climate and fluctuating hydrology. If life-cycle characteristics and organism size are associated with latitude and climatic conditions [START_REF] Johansson | Latitudinal shifts in body size of Enallagma cyathigerum (Odonata)[END_REF], then a dominance of organisms with short life-span and/or asexual reproduction and small size was expected in Mediterranean climate areas, as these traits promote survivorship in unstable conditions [START_REF] Bonada | Macroinvertebrate community structure and biological traits related to flow permanence in a mediterranean river network[END_REF]. The biological traits of invertebrates in the Mediterranean Region suggested that species allocated much energy to reproduction and resistance (asexual reproduction was dominant, formation of cocoons). These characteristics, and others such as feeding habits based on fine particulates and microorganisms, suggest that populations are selected by unstable habitats or by habitats fluctuating in an unpredictable way (Begon et al., 1996). The higher proportion of stenothermic species (species with narrow thermal ranges) in the Mediterranean region is probably the major highlight of this study, as this probably makes sorne Mediterranean pond invertebrates more susceptible to extinction in a context of climate change (i.e. species that cannat migrate northward if habitat becomes unsuitable). Throughout Europe and elsewhere, environmental policies aiming at preserving the biological diversity of terrestrial and/or aquatic ecosystems heavily rely on action plans for the delineation of zones of ecological interest (which concentrate rare and/or threatened species, or which have patrimonial values), and environmental managers ask for explicit schemes such as distribution patterns that allow them to identify areas at greater ecological risk. It is worth noting that the Mediterranean region has the highest taxonomie richness for pond invertebrates (160 out of 204 European taxa), and that 20% of these taxa were exclusive to this region. With high proportions of stenothermic invertebrates, threats associated with climate change clearly align with zones of ecological interest, and this should add new impetus to calls for freshwater conservation policies in the area. The traits of pond invertebrates in Continental areas suggested that lesser energy was allocated to reproduction and dispersal, whereas habitat occupancy and resource use was favoured by larger body size, short life cycles (thus enabling many temporally segregated populations to utilize small ecosystems), and a higher diversity of feeding groups. These characteristics suggest higher resilience, and the occurrence of strong competitors. Those populations are believed to be selected by more stable and structured habitats allowing interspecific competition and/or resource partitioning through the spatial and temporal segregations of species (Begon et al., 1996), a description which fits better with Continental ponds. Finally, despite similarities in their biological traits, invertebrate communities of the Alpine and Atlantic regions showed large differences in terms of number of taxa (30 and 145 taxa, respectively). Alpine ponds may represent harsh environments that are physically stressful (rapid and wide daily temperature fluctuations, intense UV, strong wind, abundant precipitation, and low nutrient concentrations), so that a smaller proportion of potential colonizers could become established.

Within-region variability in traits composition was chiefly apparent along axis 1, and was probably related to the peculiarities of the sub-areas from which the data were collected. In the southemmost areas, the annual hydrologie cycle is an important variable differentiating pond communities [START_REF] Della Bella | Macroinvertebrate diversity and conservation status of Mediterranean ponds in Italy: water permanence and mesohabitat influence[END_REF]. From ephemeral pools to semipermanent and permanent ponds, periodic drying is a major constraint to invertebrates [START_REF] Jeffries | Invertebrate communities and turnover in wetlands ponds affected by drought[END_REF][START_REF] Schneider | Habitat duration and community structure in temporary ponds[END_REF][START_REF] Williams | Environmental constraints in temporary freshwaters and their consequences for the insect fauna[END_REF]. Hydrologie cycles govem the biological traits combinations of communities by increasing habitat complexity through macrophyte cover, water chemistry, and substrate size. For instance, greater habitat complexity increases the number of available microhabitats for those taxa which utilize floating and submersed macrophytes as food, substrate, or refuge from predators. Such a causal relationship, already reported by [START_REF] Bazzanti | Functional characteristics of macroinvertebrate communities in Mediterranean ponds (Central Italy): influence of water permanence and mesohabitat type[END_REF], could explain the increase in shredder, scrapers, and leaf piercer at the right side of the CoA scatterplot. However, these observations cali for finer analyses that should consider each region separately, to decipher the role of microclimatic, landscape, and pond habitat variables in selecting sets of species from the regional pool. Ideally, these analyses should be based on quantitative macroinvertebrate data from a large number of evenly distributed ponds, and on environmental variables recorded in a standardized way. Therefore, a posteriori analyses of pre-existing datasets (this study) are not recommended at the regional scale, where fieldwork can be more easily designed to ensure homogeneity and replication in the data. Finally, although our data covered 120 ponds, these were derived (rather unevenly) from relatively small sampling areas. As a result, taxa lists were quite similar within each sampling area, which implies limited representation for each region. For instance, it is likely that differences in traits decrease from the core of each region to its interface with other regions.

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A key objective of the current research on small water bodies is to develop the knowledge base and tools to enable integrated monitoring and assessment for the conservation of freshwater biodiversity [START_REF] Epcn | European Pond Conservation Network)[END_REF]. Exploration of fundamental ecological patterns is needed, to cover the range of habitats found along broad geographical, altitudinal, and environmental gradients. These analyses, in addition to giving a vital understanding of large-scale patterns, are also intended to reveal gaps in existing knowledge. Obviously, our analysis of invertebrate traits did not cover the full range of pond conditions across Europe. The very first and probably more problematic gap is the lack of accessible and curated data. W e must unfortunately acknowledge that only a very small fraction of the existing information on pond biodiversity is made available to researchers and practitioners. Although we could gather information on about one hundred ponds supposed to represent various biogeographie regions, we are still unable to meet our attempts to obtain information on a much larger set of ponds that would be evenly distributed across Europe. These data certainly exist, but the heterogeneity of the existing formats (e.g. data per sample units, aggregated data) and low accessibility ( e.g. unpublished data not found with literature databases, not disclosed by authors) remain a problematic issues. There is still, therefore, a pressing need for the incorporation of high quality national data sets into a standardized database so that they can be further analyzed in an integrated European-wide manner.

Fig

  Fig. 2 Correspondance

Fig. 3

 3 Fig.3Co-inertia analysis results: a left panel: ordination of the 120 ponds on the first two axes of the CoA (Axes 1 and 2 contributed to 24 and 22% of the overall variance, respectively); left panel: grouping of samples according to biogeographie regions; right panel: grouping of samples according to hydroperiod (P =permanent, T = temporary). Ellipses contain 90% of the ponds for each region, thus summarizing the distribution of the data. b Distribution of species-trait categories on the fust two axes (see Appendix for coding of categories-Supplementary material). Categories are positioned at the weighted average of their species

Acknowledgments This work was funded by the MA V A Foundation as part of the Pro-Pond Project. Three anonymous reviewers provided valuable comments on an earlier version of this article.


Dorothée Koppa

Jordi Figuerola

Arthur Campina

Frédéric Santouf

Régis Céréghinoa

Aecolab

Local extinction and colonisation in native and exotic fish in relation to changes in land use

Keywords: agriculture, freshwater fishes, introductions, thermal preferences, urbanisation

Distribution patterns of many native and exotic fish species are weil documented, yet little is known about the temporal dynamics of native and exotic diversity in relation to changes in land use. We hypothesised that colonisation rates would be higher for exotic fish species and that extinction rates would be higher for native species in large stream systems. We also predicted that cold-water species would be more impacted than thermaily tolerant species. To test these hypotheses, we used generalised linear mixed models to compare changes in native and exotic fish species richness over 10 years in a French drainage basin subjected to landscape alterations. Exotic fish were more susceptible to local extinction than the native ones. Extinction was greater among cold-tolerant species and at higher elevations. Colonisation by exotic species was higher at lower elevations. Although a decade of expanding urbanisation affected fish colonisation, agricultural lands experienced higher extinction rates. In the context of global changes in land use and population pressure, our study suggests that the temporal dynamics of fish diversity are driven by landscape alterations as weil as by the thermal tolerance of species.

Introduction

W orldwide, many countries have established national Biodiversity Action Plans (BAPs, e.g. USA, Australia, New Zealand, Tanzania). These policy changes were implemented in response to a growing concem that development over previous decades was having a significant impact upon biodiversity, despite considerable benefits to the citizens of nations. Under this framework, assessing the effects of landscape alterations ( e.g. urbanisation, agriculture) on community dynamics is nowa priority for biological conservation (McK.inney 2002). Much of the current research has reported declines in the diversity of target organisms ( e.g. various invertebrates, fish, mammals or plants) in relation to landscape alterations, from local to global scales. Apart from their direct effects upon diversity and ecological processes, changes in land use or habitat destruction can also promote biological invasions by exotic species. These biological invasions constitute a significant component of global environmental changes and may affect human health and wealth, ecosystem functions and native biological diversity [START_REF] Vitousek | Biological invasions as global environmental change[END_REF]. Freshwaters are particularly subjected to habitat degradation, hydrologie alterations, pollution and the spread of invasive species [START_REF] Dudgeon | Freshwater biodiversity: importance, threats, status and conservation challenges[END_REF]. For example, recent estimates indicate that 25% of the world' s freshwater fish are threatened with extinction [START_REF] Vié | Wildlife in a Changing World-an Analysis of the 2008 IUCN Red List ofThreatened Species[END_REF].

Many species present a metapopulation structure, with colonisation/extinction occurring at local scales [START_REF] Case | Spatial patterns of macroinvertebrate functional feeding groups in streams in relation to physical variables and land-cover in southwestem France[END_REF]. The equilibrium between bath processes determines trends in population ranges. However, research has focussed on characterising colonisation and waves of establishment (e.g. [START_REF] Leprieur | Fish invasions in the world's river systems: when natural processes are blurred by human activities[END_REF][START_REF] Gozlan | Current knowledge on non-native freshwater fish introductions[END_REF] and little is known about the temporal stability of bath exotic and native populations in invaded areas [START_REF] Strayer | Understanding the long-term effects of species invasions[END_REF]. In this context of species loss and biological invasions, the response oflocal communities to environmental changes can be perceived in terms of colonisation by sorne sets of species and extinction of other species over time. Local colonisation is defined as a species being detected in a location from which it was previously absent, whereas local extinction is defined as the absence of a species in a location where it was previously present [START_REF] Nichols | Estimating rates of local species extinction, colonization, and turnover in animal communities[END_REF]. Assuming that exotic species colonise ecosystems as agricultural or urban land caver increases [START_REF] Pool | Environmental drivers of fish functional diversity and composition in the Lower Colorado River Basin[END_REF], one may predict that exotic and native species differ in their colonisation/extinction responses to environmental changes [START_REF] Kopp | Environmental effects related to the local absence of exotic fish[END_REF]. Spatial patterns of native and exotic species richness have been extensively studied along gradients of disturbance (e.g. [START_REF] Kopp | Environmental effects related to the local absence of exotic fish[END_REF]. However, little is known about the temporal dynamics of native and non-native diversity in relation to changing landscapes. Although habitat degradation, climate change or biological invasions can cause immediate extinctions, there is often a considerable lag between environmental changes and the subsequent extinctions [START_REF] Olden | Conservation biogeography of freshwater fishes: recent progress and future challenges[END_REF]. Most studies of the distributions of exotic species have been based on shortterm surveys and have, thus, yielded snapshots that lack a temporal context [START_REF] Strayer | Understanding the long-term effects of species invasions[END_REF]. In light of recent urban and agricultural development, short-term assessments are likely to be inadequate to describe colonisation/extinction patterns and the roles that exotic species play over time in the areas they invade. Using long-term data on species occurrence or comparing past vs present habitat characteristics may be used to address these aims [START_REF] Olden | Conservation biogeography of freshwater fishes: recent progress and future challenges[END_REF].

In this study, we focussed on the Adour-Garonne stream system (France). The drainage basin of this system is subjected to urbanisation and extensive agriculture. River fish are the most frequently introduced freshwater organisms worldwide [START_REF] Leprieur | Fish invasions in the world's river systems: when natural processes are blurred by human activities[END_REF]; therefore, they are relevant model organisms to study the contribution of native and exotic species to biodiversity patterns in a spatial and temporal context. Our specifie aim was to assess the extent of changes in native and exotic fish species richness over the past decade. Assuming that increasing anthropogenic impact enhances the chances of successful invasion by exotic fishes and that exotic and native species differ in their responses to environmental changes ( water temperature, urbanisation, agriculture), we hypothesised higher colonisation rates for exotic species and higher extinction rates for native ones. In the context of global warming, we also predicted that cold-water species would be more prone to extinction than thermally tolerant species. To test these hypotheses, we analysed the patterns of colonisation and extinction by native and exotic fish at 100 sites from 1994--97 to 2004--07, in relation to changes in land use, the location of sites within the stream system and the thermal preferences of species (from cold to warm waters).

Materials and methods

Study area and data collection

The Adour-Garonne stream system (south-western France) has a 116 000 km 2 drainage basin. W e selected 100 sampling sites ranging from 6 to 1800m above sea level (a.s.l., see Fig. S1, available as Supplementary Material to this paper), representing rivers from high mountain, plain and coastal areas. Samples were collected by electrofishing during low-flow periods. To avoid pseudo-absence data, each site was sampled three times between 1994 and 1997 and then three times between 2004 and 2007. The sampling reaches ( -100 rn long) were similar between the periods. A species with three absences during a given period was considered as absent. The biological variables assigned to each site were the colonisation by new fish species or the extinction of species from one period to the other. As fish temperature preferences have a strong influence upon species' potential range within the river continuum [START_REF] Buisson | Modelling stream fish species distribution in a river network: the relative effects of temperature versus physical factors[END_REF], fish were divided into warm-, cool-and cold-water types with regard to their preferred thermal conditions [START_REF] Magnuson | Temperature as an ecological resource[END_REF]. We then included the thermal tolerances offish: cold-, cool-or warm-tolerant (according to Kottelat and Freyhof2007) as explanatory variables in the analysis.

For each site, a geographie information system (GIS, Mapinfo Professional 7.8, Troy, NY) was used to delineate a geographical buffer zone representing a 1 000-m radius centred on the site. This size falls within that of the 'reach buffer' defined by [START_REF] Allan | Landscapes and riverscapes: the influence ofland use on stream ecosystems[END_REF] as a buffer of 100 to several hundred metres in width on each bank and sorne hundreds of metres to a kilometre in length). In practice, the width of the buffer zone is often adapted to the landscape characteristics of the studied areas and ranges from 30 to 100 rn on each si de of the river (discussed by Compin and Céréghino 2007). Sampling sites were then characterised using elevation above sea level (rn) and three land-cover variables intended to account for anthropogenic pressure. The three land-cover variables, described for the two periods 1994--97 and 2004--07, were percentage area within a buffer zone covered by forest (areas occupied by forest and woodlands with native or exotic coniferous or deciduous trees; scrub and herbaceous vegetation associations), urban areas (industrial, commercial and transport units; artificial and nonagricultural vegetated areas) and agricultural areas (arable lands, permanent crops and pasture). Digital land-cover information was obtained from the CORINE land-cover database for Europe (European Environment Agency, http://www. eea.europa.eu!, accessed June 2010). This database was generated from orthorectified satellite images and provides thematic GIS map layers including up to 44 land-cover classes with a mapping scale of 1 : 100 000. These four variables were chosen because they characterise the location of sampling sites within the stream system and within the regionallandscape mosaic and they are easy to describe using a GIS.

Data analyses

W e analysed the relationship of different ecological factors and the patterns of colonisation and extinction in 100 different sites in four different river basins. W e fitted a generalised linear mixed model (GLMM) to the presence-absence data collected for each species in each different site in 1994--97 and 2004--07. Both basin and site within a basin were included as random factors to control for local non-independence of the data. We modelled the non-independence of data coming from the same species by assuming a common positive correlation between data from the same species coming from different localities and a zero correlation with the data of other fish species (see Blackburn and Duncan 2001 for a similar approach). We constructed two different models: one for local colonisations and another for local extinctions. For the model of local colonisations, we considered only the data of negative census for each species x locality in 1994--97 and used the presence/absence data for 2004--07 as the response variable modelling the probability of obtaining a positive count in 2004--07. For the extinctions model, we used the data of positive census for each species x locality in 1994--97 and again used the presence/ absence data as the response variable, modelling the probability of a negative census in 2004--07.

All calculations were done with the GLIMMlX procedure in SAS 9.2. (SAS Institute, Cary, NC, USA) using a binomial distributed error and a logit link function. W e followed a backwards selection procedure. The least significant variable was excluded from the model which was then recalculated until only explanatory variables increasing fit with a P < 0.05 were retained. Environmental variables were log-transformed to fit a normal distribution (elevation) or ranked when not normalised by usual transformations (% urban, % agricultural area, changes in covers ofboth land uses). As land use was classified in three exclusive categories (i.e. %urban, agricultural and forest areas ), a strong colinearity existed between the three variables. For this reason, only two of the categories (% urban and % agricultural areas) were included in further analyses, but statistically significant relationships of the same sign for both variables must also be interpreted as a negative relationship for the third one. The category excluded from the analyses was chosen at random but qualitatively identical results emerged when excluding another of the habitat change categories. Changes in land cover were calculated as the difference in the proportion of habitat between 2004-07 and 1994-97.

Results

Thirty-seven fish species were found in the Adour-Garonne basin, among which 16 were exotics (according to Keith and Allardi 2001; for complete list see

Fish extinction

The extinction model revealed that local extinction was significantly higher among cold-tolerant species {F 1 , 32 = 4.08; P=0.05; Table 1), so that the extinction rate was higher at higher elevation {F 1 , 932 = 6.79; P = 0.009). Exotic species had a higher tendency to local extinction compared with the native ones {F 1 , 32 = 18.51; P = 0.0001; Table 1 ). The intensification of agriculture (up to 46% at sorne ofthe studied sites) fostered local extinction of fish species {F 1 , 932 = 7.53; P = 0.006; Table 1).

Fish colonisation

The colonisation model revealed that colonisation has been greater at low elevations {F 1 , 2792 = 76.95; P < 0.0001; Table 2).

Colonisation appeared to be lower for both cold-and thermallytolerant species, eithernative or exotic {F 1 , 34 = 1 0.07; P = 0.003 and F 1 , 34 = 5.02; P = 0.02 respectively). Finally, the expansion of urban lands between 1994-97 and 2004-07 (up to 17%) positively affected colonisation {F 1 , 2792 = 21.8; P < 0.0001; Table 2).

Discussion

In the Adour-Garonne stream system, fish colonisation was higher at low elevations. According to the 'biotic resistance hypothesis', the higher fish richness at downstream sites [START_REF] Kopp | Environmental effects related to the local absence of exotic fish[END_REF]) should prevent the establishment of non-native species through competitive exclusion (Leprieur et al. 2008; Olden et al. 201 0). However, because colonisation was higher in these areas, we can infer that higher species richness did not limit the capacity of exotic species to establish. Instead, the hypothesis of 'biotic acceptance' can be applicable in our system. This hypothesis predicts that the factors that support greater native richness in a region (e.g. abundant resources and habitat heterogeneity) also promote the establishment of non-native species. Therefore, biological interactions may have played a negligible role in the observed patterns, contrary to environmental factors.

Cold-tolerant species, which experienced the greatest extinction rates, are typically confined to mountain streams in the Adour-Garonne basin. According to Bal combe et al. (20 11), these cold-water tolerant species are particularly sensitive to increased stream temperature, especially when they are located at their temperature limits [START_REF] Morrongiello | Climate change and its implications for Australia's freshwater fish[END_REF]) because temperature has a direct physiological effect on these fish. As fish are poikilotherms, water temperature strongly controls population dynamics through metabolism, growth and fecundity (Lobon-Cervia et al. 1996), thus, forming a key physicochemical habitat filter [START_REF] Poff | Landscape filters and species traits: towards mechanistic understanding and prediction in stream ecology[END_REF]) that determines species' potential refuge sites along the river continuum [START_REF] Murawski | Climate change and marine fish distributions: forecasting historical analogy[END_REF]. Mountain streams may also represent harsh environments which are physically stressful (higher river competence and erosive forces generated through the combination of slope with other variables such as water depth and current velocity and snowmelt floods). These natural environmental factors, combined with low connectivity among suitable sites in cold headwater streams, may be responsible for low levels of colonisation at high altitude sites [START_REF] Gido | Invasion ofNorth American drainages by alien fish species[END_REF].

At the same time, the intensification of agriculture (including pasture lands in mountainous areas) may have fostered local extinction of fish species. The agriculturallandscape of southwestern France and western Europe in general, has radically changed over the past decades (Meeus et al. 1990) as a result of land-use conversion, intensification of production systems and abandonment of traditional practices. The principal pressures causing biodiversity loss in the countryside are habitat fragmentation, degradation and destruction due to land-use change. Important sources of agriculture-derived pollution include the inflow of nutrients, pesticides and heavy metals from diffuse and point sources [START_REF] Allan | Landscapes and riverscapes: the influence ofland use on stream ecosystems[END_REF]. Fragmentation impacts the connectivity between suitable habitats [START_REF] Fahrig | Effects of habitat fragmentation on biodiversity[END_REF] and ultimately affects community dynarnics [START_REF] Denoël | Conservation ofnewt guilds in an agricultural landscape of Belgium: the importance of aquatic and terrestrial habitats[END_REF].

Expansion of urban lands positively affected colonisation. Although our model does not allow us to distinguish whether expansion of urban lands favours native or exotic fish colonisation, close scrutiny of the dataset revealed that colonizers were mainly exotic species such as Pseudorasbora parva (topmouth gudgeon) and Si/urus glanis (European catfish). A recent study in the United States revealed that non-native species dominate watersheds supporting high densities of dams, roads and urban and agricultural landscapes, whereas watersheds characterised by upstream land protection support fish communities with a strong complement of native species (Pool et al. 201 0). According to [START_REF] Leprieur | Fish invasions in the world's river systems: when natural processes are blurred by human activities[END_REF], the 'human activity hypothesis' (i.e. by disturbing naturallandscapes and increasing the importation of non-native species, human activities facilitate greater levels of establishment) best explains the patterns of non-native fish richness in river basins. In our study area, landscapes characterised by increasing proportions of urban land use were successfully colonised by exotic species, thus, supporting the idea that association with humans increase the chances of successful dispersal and establishment in non-native species.

In conclusion, the colonisation and extinction patterns highlighted in this study only partially matched our a priori expectations. Over a decade, exotic fish were more susceptible to local extinction than the native one s. The 'three tens' rule (one imported species in 10 appears in the wild, one in 1 0 of the se become established and one in 10 of established non-indigenous species becomes a pest) which was initially formulated for exotic plants [START_REF] Williamson | The varying success of invaders[END_REF] probably does not apply to exotic fish, because these animais are intentionally introduced by humans in potentially suitable habitats. Therefore, it is likely that extinction in exotic fish was not related to unsuccessful settlement, but rather to external factors such as water temperature or flow variability [START_REF] Costelloe | Are alien fish disadvantaged by extremely variable follow regimes in arid-zone rivers?[END_REF].

Regardless of their biogeographie status ( either native or exotic ), cold-tolerant fish were more prone to extinction than the cool-or thermally tolerant ones. This may be due to the rapidity of climate changes which are predicted to exceed the ability of many species to adapt to new environmental conditions (Morrongiello et al. 2011; Olden et al. 2011). Therefore, our results support the hypotheses that: (i) ali fish communities are susceptible to invasion regardless of native species richness [START_REF] Moyle | Biological invasions of fresh water: empirical rules and assembly theory[END_REF][START_REF] Gido | Invasion ofNorth American drainages by alien fish species[END_REF]; and (ii) water temperature, or its changes, is a major driver offish extinction in human-impacted landscapes. In light of future climate scenarios, our study further suggests that mountain ranges are the areas at greatest extinction risk for poikilotherms and our ability to detect responses of native and exotic species to landscape alterations using a combination of simple environmental variables exemplifies a cost-effective technique for assessing areas at greater invasion risk in large stream systems.

Table 1 .

 1 Models analysing the patterns of fish extinction for cold-tolerant species ( cold), cool-tolerant species (cool), thermally-tolerant species (warm) and for native vs. exotic species Only variables with P < 0.05 are interpreted as statistically significant. Estimates correspond to slope values associated to each factor and are only reported for significant effects retained in the final model

	Extinction	F-value	d.f.	P-value	Estimate ± s.e.
	Elevation	6.79	1,932	0.009	0.5448 ± 0.2090
	Cold	4.08	1,32	0.05	Non-cold, -0.7309 ± 0.6320; cohl, 0
	Cool	O.o?	1,31	0.79
	Warm	0.13	1,31	0.72
	Native vs exotic	18.51	1,32	0.0001	Exotics, 1.2880 ± 0.2994; native, 0
	Urbanisation	0.28	1,931	0.60
	Agriculture	7.53	1,932	0.006	0.0103 ± 0.0037
	Change urban land cover	0.54	1,931	0.46
	Change agriculturalland cover	0.14	1,931	0.70

Table 2 .

 2 Models analysing the patterns of fish colonisation (for legend see Table1) Only variables with P < 0.05 are interpreted as statistically significant. Estimates correspond to slope values associated to each factor and are only reported for significant effects retained in the final model

	Colonisation	F-value	d.f.
	Elevation	76.95	1,2692
	Cold	10.07	1,34
	Cool	0.78	1,33
	Warm	5.02	1,34
	Native vs exotic	0.67	1,33
	Urbanisation	0.69	1,2691
	Agriculture	1.03	1,2691
	Change urban land cover	21.80	1,2692
	Change agriculturalland cover	1.11	1,2692

Table 81

 81

	P-value	Estimate ± s.e.
	<0.0001	-1.2457 ± 0.1420
	0.003	Non-cohl, 1.2534 ± 0.3894; cold, 0
	0.38
	0.02	Non-warm, 0.6912 ± 0.3085; warm, 0
	0.42
	0.41
	0.31
	<0.0001	0.0796 ± 0.0170
	0.29

, available as Supplementary Material to this paper). Our models indicate that elevation and land-use variables could explain local extinction and colonisation patterns in river fish in this basin.

Acknowledgements

We wish to thank the French Office National de l'Eau et des Milieux Aquatiques (ONEMA) and more specifically Dr N. Poulet, for providing us with the fish data. Three anonymous reviewers and Dr A. Boulton made useful comments on an earlier version of this paper.