Gwendaline Le Hen

Paride Balzani

Peter Haase

Antonín Kouba

Chunlong Liu

J Nagelkerke

Nikola Theissen

David Renault

Ismael Soto

Phillip J Haubrock

Leopold A J Nagelkerke

Alien species and climate change drive shifts in a riverine fish community and trait compositions over 35 years

Keywords: Rhine river, freshwater fish, long-term study, alien species, functional ecology, traits

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

Introduction

Extinction and speciation processes constitute the natural renewal of biodiversity [START_REF] Johnson | Biodiversity losses and conservation responses in the Anthropocene[END_REF]. At global scales, the growing extent of human impacts on ecosystems worldwide, including changes in climatic conditions and land-use, overexploitation of resources, or introduction of alien species [START_REF] Sala | Global biodiversity scenarios for the year 2100[END_REF][START_REF] Cowx | Freshwater fish conservation: options for the future[END_REF], increases the frequency of occurrence of these processes. At regional scales, the man-made alterations of habitat quality greatly favour the increase of extinction rates, and cause a staggering decline in biodiversity [START_REF] Dirzo | Defaunation in the Anthropocene[END_REF][START_REF] Johnson | Biodiversity losses and conservation responses in the Anthropocene[END_REF]IPBES, 2019).

Among the above-mentioned drivers of biodiversity decline, biological invasions play a central role, as growing numbers of alien species are introduced (deliberately or accidentally) beyond their native range [START_REF] Blackburn | A unified classification of alien species based on the magnitude of their environmental impacts[END_REF][START_REF] Turbelin | Mapping the global state of invasive alien species: patterns of invasion and policy responses[END_REF][START_REF] Seebens | Global rise in emerging alien species results from increased accessibility of new source pools[END_REF].

Once successfully established, some alien species can proliferate locally before spreading geographically, and colonising new habitats, causing ecological and socio-economic impacts on recipient ecosystems [START_REF] Arim | Spread dynamics of invasive species[END_REF]. Invasive alien species can threaten biodiversity at all organisational scales, in all regions, ecosystems, or habitats [START_REF] Bellard | Alien species as a driver of recent extinctions[END_REF][START_REF] Mollot | The effects of invasive species on the decline in species richness: a global meta-analysis[END_REF][START_REF] Blackburn | Alien versus native species as drivers of recent extinctions[END_REF][START_REF] Pilotto | Meta-analysis of multidecadal biodiversity trends in Europe[END_REF][START_REF] Pyšek | Scientists' warning on invasive alien species[END_REF]. Biodiversity from freshwater ecosystems is no exception: in addition to being critically affected by anthropogenic activities (e.g. change in land use, climatic changes), environmental stressors can magnify the deleterious impacts of alien species, potentially leading to species extinction and to the loss of ecosystem functions [START_REF] Sala | Global biodiversity scenarios for the year 2100[END_REF][START_REF] Gherardi | Invasive crayfish in Europe: the impact of Procambarus clarkii on the littoral community of a Mediterranean lake[END_REF][START_REF] Villéger | Homogenization patterns of the world's freshwater fish faunas[END_REF][START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF].

Invasive alien fishes have been known to modify aquatic ecosystems (e.g. by altering community structure and the trophic web) and displace native species through competition and/or predation [START_REF] Cambray | Impact on indigenous species biodiversity caused by the globalisation of alien recreational freshwater fisheries[END_REF][START_REF] Gherardi | A review of allodiversity in Lake Naivasha, Kenya: Developing conservation actions to protect East African lakes from the negative impacts of alien species[END_REF][START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF][START_REF] Haubrock | Knowledge gaps in economic costs of invasive alien fish worldwide[END_REF]. These impacts have been recently monetised, and economic losses incurred by alien fishes were ACCEPTED MANUSCRIPT / CLEAN COPY reported as being mainly related to damages, resource losses, and impaired social welfare [START_REF] Haubrock | Knowledge gaps in economic costs of invasive alien fish worldwide[END_REF]. Alien fish species can also negatively affect taxonomic diversity [START_REF] Cambray | Impact on indigenous species biodiversity caused by the globalisation of alien recreational freshwater fisheries[END_REF][START_REF] Gallardo | Global ecological impacts of invasive species in aquatic ecosystems[END_REF], with potential consequences for the functional structure of the community. In these circumstances, the loss of native species can result in the concomitant losses of certain traits, whereas new species are prone to introduce "novel" traits or even replace those lost [START_REF] Nock | Functional traits[END_REF][START_REF] Renault | Advancing biological invasion hypothesis testing using functional diversity indices[END_REF].

Information on ecological preferences and biological (i.e. functional) traits (later on referred to as simply ecological and biological traits) are characteristic to each individual or species, determining its biology and functioning within a community, as well as shaping the ecological space it occupies. Traits are often considered plastic, in several cases slowly changing either by developmental acclimation or by on-time adjustments, allowing their constant optimisation or the maintenance of biological performance, even when environmental factors vary qualitatively or quantitatively [START_REF] Hooper | Effects of biodiversity on ecosystem functioning: a consensus of current knowledge[END_REF]Mason et al., 2005;[START_REF] Nock | Functional traits[END_REF]. In invaded habitats, the loss of individuals and species can be associated with the loss of traits, and this can have profound impacts on community structure and functioning when unique and/or specialised traits are lost [START_REF] Clavel | Worldwide decline of specialist species: toward a global functional homogenization?[END_REF][START_REF] Dias | Functional erosion and trait loss in fish assemblages from Neotropical reservoirs: The man beyond the environment[END_REF][START_REF] Pilotto | Decline in niche specialization and trait β-diversity in benthic invertebrate communities of Central European low-mountain streams over 25 years[END_REF]. In several instances, taxonomic and functional changes induced by alien species, and the consecutive loss of native species, have resulted in the homogenisation of freshwater communities. Indeed, ~40% of European fish assemblages that experienced taxonomic differentiation were also functionally homogenised [START_REF] Olden | On defining and quantifying biotic homogenization[END_REF][START_REF] Villéger | Homogenization patterns of the world's freshwater fish faunas[END_REF][START_REF] Villéger | Functional homogenization exceeds taxonomic homogenization among European fish assemblages[END_REF][START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF]. Functional homogenisation of all local communities within a region (e.g. metacommunities) can increase vulnerability to large-scale environmental events by synchronising local biological responses across individual communities [START_REF] Olden | Ecological and evolutionary consequences of biotic homogenization[END_REF][START_REF] Clavel | Worldwide decline of specialist species: toward a global functional homogenization?[END_REF][START_REF] Villéger | Functional homogenization exceeds taxonomic homogenization among European fish assemblages[END_REF]. Although some taxonomic and functional changescommonly described with a series of trait metrics (see Baker et al., 2021) induced by alien ACCEPTED MANUSCRIPT / CLEAN COPY fish are known to occur in European lakes [START_REF] Zhao | Accounting for intraspecific diversity when examining relationships between non-native species and functional diversity[END_REF], not much is known about riverine ecosystems. Furthermore, changing climatic or environmental conditions can induce shifts in the establishment rates and concomitantly abundance of alien fish [START_REF] Strayer | Boom-bust dynamics in biological invasions: towards an improved application of the concept[END_REF]. This also induces functional changes-an aspect pertaining to the compositional and functional dimension of biological invasions that has not been studied in depth (but see [START_REF] Crozier | Plastic and evolutionary responses to climate change in fish[END_REF].

The Rhine river, one of the most heavily anthropogenically impacted freshwater rivers in Europe with a total length of 1,250 km [START_REF] Tittizer | Ausbreitung aquatischer Neozoen (Makrozoobenthos) in den europäischen Wasserstrassen, erläutert am Beispiel des Main-Donau-Kanals. In Güteentwicklung der Donau, Rückblick und Perspektiven[END_REF][START_REF] Van Der Velde | Ecological impact of crustacean invaders: general considerations and examples from the Rhine River[END_REF][START_REF] Uehlinger | The Rhine river basin[END_REF], provides numerous services such as transportation, power generation and drinking water [START_REF] Cioc | The Rhine: an eco-biography, 1815-2000[END_REF][START_REF] Uehlinger | The Rhine river basin[END_REF]. The opening of the Rhine-Main-Danube canal in 1992 links the Rhine catchment via the Main tributary with the Danube river, and serves as a major pathway facilitating the spread of alien species [START_REF] Tittizer | Ausbreitung aquatischer Neozoen (Makrozoobenthos) in den europäischen Wasserstrassen, erläutert am Beispiel des Main-Donau-Kanals. In Güteentwicklung der Donau, Rückblick und Perspektiven[END_REF][START_REF] Bij De Vaate | Geographical patterns in range extension of Ponto-Caspian macroinvertebrate species in Europe[END_REF][START_REF] Balzani | Knowledge needs in economic costs of invasive species facilitated by canalization. 19[END_REF]. However, while information on the taxonomic identity of alien species present in highly anthropogenically altered ecosystems like the Rhine river is available, not much is known about the changes in the community and trait composition that have occurred as a response to these introductions [START_REF] Nehring | Aquatic alien species in Germany-listing system and options for action[END_REF][START_REF] Rabitsch | The times are changing: temporal shifts in patterns of fish invasions in central European fresh waters[END_REF], nor how environmental changes over time drove these.

In the current era of globalisation, ongoing climatic changes, and an increased interest in the protection of natural resources, long-term studies have become increasingly necessary to provide a better understanding of introduction processes, reconstruct introduction events, and community dynamics [START_REF] Franklin | Importance and justification of long-term studies in ecology[END_REF][START_REF] Hooper | Effects of biodiversity on ecosystem functioning: a consensus of current knowledge[END_REF][START_REF] Strayer | Understanding the long-term effects of species invasions[END_REF]). Yet, longterm data has rarely been used to disentangle the effects of alien species on the taxonomic and trait diversity of fish communities, nor to investigate effects of climatic or environmental changes (but see [START_REF] Guareschi | Invasive species influence macroinvertebrate biomonitoring tools and functional diversity in British rivers[END_REF]. Using long-term biomonitoring data from the Rhine river in western Germany , we investigated changes in the taxonomic and trait ACCEPTED MANUSCRIPT / CLEAN COPY composition in invaded fish communities over time. To this end, we specifically investigated if changes in the communities' taxonomic and trait composition were driven by disappearances of native fishes or by replacements by alien fishes as well as climatic changes over time and consequently, if identifiable changes were indicative of taxonomic and functional (i.e. trait) homogenisation and subsequently, responding to environmental changes. We tested the following hypotheses: (i) the increase in alien fish abundance will be at the expense of native species, resulting in a decrease in the overall native communities' abundance, (ii) the arrival and establishment of alien fishes will introduce novel functional traits, reshaping the ecological space occupied by the community, (iii) changes in environmental parameters, and more particularly climatic factors, will drive compositional changes.

Methods

Data collection

To investigate the changes in composition of freshwater fish communities over spatiotemporal scales, we used a time series of fish abundance from the Rhine river in western Germany. Fish samplings were conducted in the Rhine river between 1984-2018, although not continuously (Table S1), over different seasons (i.e. spring and summer) and from 31 sites. In total, the samplings covered ~170 km of the Rhine river from the border to the Netherlands upstream (6°12'55''N-7°11'55''N; 50°39'49''E-51°49'46''E; Figure 1). Fish were collected at each site following a standardised protocol by bank fishing from the right river bank through continuous electrofishing. Over the study period, 403 samplings were conducted, capturing a total of 43,888 individuals identified at the species level. For each fish species, abundance (i.e. number of individuals per sampling) data were averaged across sites for each year.

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Fish biological and ecological traits

The sampled fish species were characterised with traits extracted from three resources: www.freshwaterecology.info (Schmidt-Kloiber & Hering, 2015), www.fishbase.org [START_REF] Froese | FishBase. World Wide Web electronic publication[END_REF] and scholar.google.com (Google Scholar sources). When trait data were missing at the species level, information was retrieved from the genus. We retained a total of 25 traits ACCEPTED MANUSCRIPT / CLEAN COPY split into: (i) 13 ecological traits, sub-divided into 43 'modalities' (e.g. if the considered ecological trait is 'habitat', the different modalities correspond to the diversity of habitats which the species can occupy; Table S2) describing the environmental tolerance of species, and (ii) 12 biological traits with 37 modalities reflecting characteristics of species (Table S2; [START_REF] Devin | Biological and ecological characteristics of invasive species: a gammarid study[END_REF].

Community metrics

To investigate changes in the composition of the fish community over the investigated period , five commonly used taxonomic metrics were computed for each site and year:

(i) total abundance, (ii) species richness, (iii) Shannon diversity, (iv) Pielou evenness and (v) temporal species turnover (Table S3; [START_REF] Baranov | Complex and nonlinear climate-driven changes in freshwater insect communities over 42 years[END_REF]. Taxonomic metrics were computed using the R package 'vegan' [START_REF] Oksanen | {vegan}: Community Ecology Package. R package version 2[END_REF]. The turnover of overall communities, native communities and alien communities were calculated using the turnover function implemented in the R package 'codyn' [START_REF] Hallett | {codyn}: Community Dynamics Metrics[END_REF]. In addition, we calculated the beta-diversity (i.e. as a measure of the ratio between regional and local species diversity) across all sites sampled within each year as a proxy for taxonomic homogenisation using beta.div.comp function implemented in the R package 'adespatial' [START_REF] Dray | {adespatial}: Multivariate Multiscale Spatial Analysis[END_REF].

Trait metrics

To investigate trait changes in fish communities over time, all trait modalities (i.e. ecological and biological) were 'fuzzy coded' with the prep.fuzzy.var.function following [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF] using the R package 'ade4' [START_REF] Dray | {ade4}: implementing the duality diagram for ecologists[END_REF]. The fuzzy coding procedure indicates to which extent a taxon exhibits each trait category by proportionally scaling them between 0 and 1 [START_REF] Schmera | A proposed unified terminology of species traits in stream ecology[END_REF]. This is essential as it simplifies the synthesis of trait information and enables the synthesis of diverse kinds of biological information (Baker et al., ACCEPTED MANUSCRIPT / CLEAN COPY 2021). Then, four trait metrics: trait richness (TRic), trait divergence (TDiv), trait dispersion (TDis) and trait evenness (TEve; Table S3; Baker et al., 2021;[START_REF] Renault | Advancing biological invasion hypothesis testing using functional diversity indices[END_REF] were calculated using the alpha.fd.multidim function from the R package 'mFD' [START_REF] Magneville | {mFD}: an R package to compute and illustrate the multiple facets of functional diversity[END_REF]. Trait metrics were computed for ecological and biological traits separately.

Abiotic and biotic predictors

Site-specific characteristics were used to investigate the spatiotemporal changes in fish communities. To assess the effects of climatic variables, we extracted the average daily temperature and precipitation of the 12 months before the sampling event from a gridded European-scale observation-based dataset for each year and site (spatial resolution: 0.1 °C; www.ecad.eu/E-OBS/; [START_REF] Cornes | An ensemble version of the E-OBS temperature and precipitation data sets[END_REF]. Lastly, we estimated the richness and abundance of alien species in each year of each time series as biotic predictors to investigate their effects on the community.

Statistical analyses

To investigate changes in taxonomic and trait composition of communities over time, canonical analysis of principal coordinates (CAPs) were performed based on the Bray-Curtis dissimilarity index [START_REF] Bray | An ordination of the upland forest communities of southern Wisconsin[END_REF]. The CAPs were based on (i) species abundance and the community weighted means (CWM, i.e. the mean value for each trait for the whole community weighted by the abundance of the species carrying the trait) of (ii) ecological and (iii) biological traits for each year. Further, permutational multivariate analysis of variance (PERMANOVA, permutations = 9,999) based on the Bray-Curtis dissimilarity index were performed using adonis2 function of the R package 'vegan' [START_REF] Oksanen | {vegan}: Community Ecology Package. R package version 2[END_REF] on the species abundance matrix to identify the significance of abiotic (i.e. latitude, temperature, precipitation) and biotic predictors (i.e. alien species abundance and richness) and then the significance of ecological ACCEPTED MANUSCRIPT / CLEAN COPY and biological trait modalities. Then, two PERMANOVAs (Bray-Curtis dissimilarity index, permutations = 9,999) were separately performed on the matrices of the ecological and biological traits CWM to identify the significance of abiotic and biotic predictors.

Finally, taxonomic and trait metrics as well as climatic variables were modelled as the response variables over time. In addition, we identified periods in which community and traitbased metrics significantly changed over time by fitting an autoregressive AR (1) process [START_REF] Nathan | On the application of generalised additive models to the detection of trends in hydrologic time series data[END_REF] for the residuals enabled the computation of derivatives of fitted splines using the method of finite differences to estimate the rate of change (slope) in the fitted smoother. This produced diagnostic plots of the time series with which periods of significant changes were identified and superimposed within the respective time series upon the respective trends over time [START_REF] Simpson | Modelling palaeoecological time series using generalised additive models[END_REF]. Based on the results from the autoregressive models we identified significant changes to have occurred in ~2010. Accordingly, we splitted the study period into two (1984-2010: 25 years; 2010-2018: 8 years; Figure S1) to identify the effects of alien fishes in the community. Taxonomic and trait metrics were modelled using the gam function from the R package 'mgcv' [START_REF] Wood | {mgcv}: mixed GAM computation vehicle with automatic smoothness estimation[END_REF]. All metrics were modelled using a negative binomial distribution except for species abundance and richness, modelled using a poisson and gaussian distribution respectively. Hence, each model was composed of the specific response variable (i.e. taxonomic, biological and ecological trait metrics) and the explanatory variables: 'year', 'latitude', 'temperature' and 'precipitation'. Considering non-linear trends over time, our models included a cubic regression smooth spline for the predictor "year" [START_REF] Wood | Generalized additive models: an introduction with R. Chapman and hall/CRC[END_REF] and a smoothed spline for abiotic predictors.

All analyses were conducted in the R environment, version 4.1.1 (R Core Team, 2021).

The level of significance was set at α < 0.05.

Results

Over the 35 years of this study, 43 fish species (32 native and 11 alien species) were caught in the lower part of the Rhine river (Table S4). The Cyprinidae was the most diverse family in terms of species (20 out of the 43 sampled species), followed by Salmonidae (4 species), Gobiidae (4 species) and Percidae (3 species). The 12 remaining families were represented by a single species (Table S4; Figure S2). Over the studied period, the average annual temperature and precipitation increased by ~0.94 °C (~ 0.03 °C year -1 ) and ~0.26 mm (~0.008 mm year -1 ), respectively. While temperature fluctuated throughout the period, peaking in 2006, precipitation steeply inclined after 2010 (Figure S3).

Compositional changes in communities over time

Between 1984 and 2018, total community richness increased by 49%, albeit declining between 2004 and 2010 before increasing again until 2018 (Figure 2a). Starting from 198 individuals/m² in 1984, the overall community abundance largely declined to 23 individuals/m² in 2010 (-88%), and then consequently increased by 678% up to 180 individuals/m² until 2018 (Figure 2b). Despite the slow increase at the beginning of the studied period, the richness and abundance of alien species considerably increased after 2010 (Figure 2) and peaked in 2013 with a maximum of 8 alien species and again in 2018 with 7 alien species. The common carp Cyprinus carpio (first recorded in 1993), the rainbow trout Oncorhynchus mykiss (first recorded in 1998) and the European catfish Silurus glanis (first recorded in 2004) were the first alien species that were detected and successfully established in the studied stretch of the Rhine river (Table S4). While C. carpio, O. mykiss, and S. glanis represented 5.0-8.3% of the total species richness before 2006 (Figure 3a), their abundance remained negligible (0.1-0.4%; Figure 3b). In 2010, the abundance of five identified alien fish species increased to 14.9%. In the period 2010-2013, six novel alien fish species established in the river: the pumpkinseed ACCEPTED MANUSCRIPT / CLEAN COPY the round goby N. melanostomus, the topmouth gudgeon Pseudorasbora parva, and the European bitterling Rhodeus amarus (Table S4), constituting a total of 29.6% of the total species richness and 52.1% of the total communities abundance in 2013 (Figure 3). The establishment of the six novel species increased alien species richness, which peaked in 2018 (30.4%; Figure 3a), alongside with their abundance, totalling 69.9% in 2015 (Figure 3b). Considering solely the alien community, species from the genus Neogobius sp.

were first detected in 2010. In this genus, N. melanostomus dominated the alien fish community ACCEPTED MANUSCRIPT / CLEAN COPY in 2018 (98.25% of the total alien communities abundance, Figure 4b). For the native fish communities, the roach Rutilus rutilus was the most abundant species at the beginning of the study period (75.14%, Figure 4a) and declined sharply in 2010 (2.30%). The bleak Alburnus alburnus, which represented 1.76% of the total native community abundance in 1984, and the ide Leuciscus idus, 1.52% in 1993, became more abundant in 2018, respectively occupying 24.47% and 22.00% (Figure 4a).

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The communities' beta-diversity increased until 2010 before decreasing towards 2018 (Figure S4). The total community turnover remained constant over time (Figure 5a), with the turnover of the native fish communities slightly increasing in recent years from 0.48 to 0.60, albeit being non-significant (R² = 0.135; p = 0.238; Figure 5b). The turnover of the alien fish community decreased significantly after 2006, from 0.89 to 0.34 (R² = 0.210; p = <0.01; Figure 5c). The applied ordination suggested a clear segregation in the community's taxonomic composition, with years after 2010 characterised by a significant increase in alien species richness and abundance, as well as changes in temperature and precipitation, as confirmed by the applied PERMANOVA (Figure 6a; Table S5). Moreover, the applied PERMANOVA identified all community weighted means CWM (both ecological and biological) to ACCEPTED MANUSCRIPT / CLEAN COPY significantly vary over time (Table S6), as within each trait, modalities of both ecological (Figure S5) and biological traits (Figure S6) changed in their relative prevalence, showing notable differences in their respective proportion before and after 2010. After 2010, the community was dominated by species characterised by smaller adult fishes and larvae, shorter life span, moderate relative fecundity, bigger eggs, and provision of parental care (Figure S6; Table S6). Regarding ecological traits, the post-2010 community was characterised by fishes preferring rheophilic conditions in a demersal habitat, and feeding in benthic habitats (Figure S5; Table S6). A comparable pattern was identified for the CWMs of ecological and biological trait compositions (Figure 6b,c), as confirmed by the PERMANOVA (Table S7). Changes after 2010 were significantly related to an increase in alien species' richness and abundance as well as increases in temperature and precipitation (Figure S3).

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Temporal patterns in the community taxonomic and trait metrics

Without separating the time series into two distinct periods, we identified a fluctuation in richness (i.e. a low around the year 2010 encompassed by two periods of high species richness), a decline in abundance, and an incline in Shannon diversity as well as inclines in both ecological and biological trait dispersion (p < 0.05; Figure S7). Between 1984 and 2010, species richness did not change significantly (Figure 7a; Table S8), whereas species abundance significantly decreased from on average 198 (± 82) to 23 (± 28) individuals/m² (R² = 0.624; p = <0.01; Figure 7b). Species richness and abundance were significantly positively affected by latitude, temperature and precipitation (Table S8). No significant changes in Shannon diversity and Pielou's evenness were detected (Figure 7c,d). With regard to trait metrics, only an increase in trait dispersion (TDis) of ecological traits was detected (R² = 0.292; p = 0.01; Figure 7g).

After 2010, species richness and abundance increased significantly from approximately six to ten species, and from on average 23 (± 28) to 180 (± 176) individuals/m² (richness: R² = 0.425; p = <0.01; abundance: R² = 0.731; p = <0.01; Figure 7a,b). No further significant change was detected (Figure 7; Table S8). While species richness after 2010 was positively related with latitude, the identified increase in abundance was primarily driven by the increasing richness (and abundance) of alien species as well as an increase in temperature, precipitation, and latitude. Changes in Shannon diversity and Pielou's evenness were only significantly negatively related to the abundance of alien species (Table S8).

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Discussion

The direct and indirect impacts of alien fishes and meteorological conditions are major drivers of biodiversity loss in freshwater ecosystems worldwide [START_REF] Villéger | Functional homogenization exceeds taxonomic homogenization among European fish assemblages[END_REF][START_REF] Gallardo | Global ecological impacts of invasive species in aquatic ecosystems[END_REF][START_REF] Bellard | Insights from modeling studies on how climate change affects invasive alien species geography[END_REF][START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF], often leading to taxonomic homogenisation [START_REF] Olden | Ecological and evolutionary consequences of biotic homogenization[END_REF][START_REF] Pilotto | Meta-analysis of multidecadal biodiversity trends in Europe[END_REF][START_REF] Haubrock | Knowledge gaps in economic costs of invasive alien fish worldwide[END_REF]. Our data, collected with a standardised protocol over the period 1984-2018, revealed significant changes in taxonomic and trait composition following the introduction of alien fishes after 2010 and concomitant changes in climatic conditions. We further identified changes in community composition following declines in the richness and to some extent also the abundance of native communities until 2010, before indicating taxonomic homogenisation, while in the case of the abundanceafter alien fish species were detected to dominate in 2015-reached levels comparable to those ACCEPTED MANUSCRIPT / CLEAN COPY measured in the first year, while richness reached a considerably higher level. While climatic changes (i.e. in temperature and precipitation) likely benefited some alien species reflecting characteristic traits, these however did not determine the transition to a community dominated by aliens as both variables expressed no period of significant change during the study period (Figure S1m,n).

Patterns in community composition before 2010

Trends in the abundance and richness of alien species can shift following external changes [START_REF] Rahel | Assessing the effects of climate change on aquatic invasive species[END_REF][START_REF] Früh | Physicochemical and morphological degradation of stream and river habitats increases invasion risk[END_REF], and have destabilising effects on invaded communities [START_REF] Cardoso | Incorporating invasive alien species into ecological assessment in the context of the Water Framework Directive[END_REF][START_REF] Strayer | Long-term changes in a population of an invasive bivalve and its effects[END_REF]. The three alien fish species already present before 2010 in the Rhine river (C. carpio, O. mykiss, and S. glanis) are well-known for their substantial impacts on ecosystems and native species (e.g. through habitat degradation, competition, predation and the spread of diseases; [START_REF] Cambray | Impact on indigenous species biodiversity caused by the globalisation of alien recreational freshwater fisheries[END_REF][START_REF] Copp | Voracious invader or benign feline? A review of the environmental biology of European catfish Silurus glanis in its native and introduced ranges[END_REF][START_REF] Gherardi | A review of allodiversity in Lake Naivasha, Kenya: Developing conservation actions to protect East African lakes from the negative impacts of alien species[END_REF][START_REF] Guillerault | Does the non-native European catfish Silurus glanis threaten French river fish populations?[END_REF]. Yet, their abundances remained at relatively low levels (<0.5% of the overall community abundance), whereas these species can reach considerably high biomasses with detectable impacts [START_REF] Driver | The effects of size and density of carp (Cyprinus carpio L.) on water quality in an experimental pond[END_REF]. The overall and significant decline in native species abundances by -90% is however unlikely to have occurred due to their presence [START_REF] Copp | Voracious invader or benign feline? A review of the environmental biology of European catfish Silurus glanis in its native and introduced ranges[END_REF]. Consistent with this assumption, several studies have reported that the impacts of poorly abundant alien species on communities are often undetectable or absent [START_REF] Parker | Impact: toward a framework for understanding the ecological effects of invaders[END_REF][START_REF] Ricciardi | Predicting the impacts of an introduced species from its invasion history: an empirical approach applied to zebra mussel invasions[END_REF][START_REF] Thiele | Impact assessment revisited: improving the theoretical basis for management of invasive alien species[END_REF]. Rather, there could be additional stressors not encompassed by our data, e.g. anthropogenic alterations in the water quality of the Rhine river [START_REF] Tittizer | Ausbreitung aquatischer Neozoen (Makrozoobenthos) in den europäischen Wasserstrassen, erläutert am Beispiel des Main-Donau-Kanals. In Güteentwicklung der Donau, Rückblick und Perspektiven[END_REF][START_REF] Van Der Velde | Ecological impact of crustacean invaders: general considerations and examples from the Rhine River[END_REF], leading to a decline in native species and increasing diversity across sites. Changes in climatic conditions or the nutrient load of the Rhine following improvements in the sewage treatment plants in the early 1990s [START_REF] Malle | Cleaning up the river Rhine[END_REF] have limited the availability of prey (e.g. zoo-and phytoplankton), and this may partially ACCEPTED MANUSCRIPT / CLEAN COPY explain the community composition pattern found (pers. comm. Nikola Theissen). The opening of the Rhine-Main-Danube canal in 1992 likely contributed further to the observed decline in native fish communities, as their composition and numbers considerably changed from 1993 onwards. While the direct presence of Ponto-Caspian species is unlikely, increased shipping and perturbation have likely contributed to the identified declines.

Patterns in community composition after 2010

In the Rhine river, the arrival of new alien species was likely linked to the opening of the Rhine-Main-Danube canal in 1992 [START_REF] Tittizer | Ausbreitung aquatischer Neozoen (Makrozoobenthos) in den europäischen Wasserstrassen, erläutert am Beispiel des Main-Donau-Kanals. In Güteentwicklung der Donau, Rückblick und Perspektiven[END_REF][START_REF] Bij De Vaate | Geographical patterns in range extension of Ponto-Caspian macroinvertebrate species in Europe[END_REF]. Indeed, three out of the six alien fish species (N. fluviatilis, P. kessleri and N. melanostomus) are native to the Ponto-Caspian region, and it took 18 years before they arrived and established [START_REF] Kottelat | Handbook of European freshwater fishes[END_REF][START_REF] Froese | FishBase. World Wide Web electronic publication[END_REF]. The introduction of other alien fishes (L. gibbosus, P. parva and R. amarus) is most likely linked to intensified aquaculture and angling [START_REF] Kottelat | Fishes of Mongolia. A check-list of the fishes known to occur in Mongolia with comments on systematics and nomenclature[END_REF][START_REF] Kottelat | Handbook of European freshwater fishes[END_REF][START_REF] Froese | FishBase. World Wide Web electronic publication[END_REF]. It is, therefore, not surprising that our results suggest compositional changes (driven by the spatiotemporal spread of highly abundant Ponto-Caspian fish species) to have progressed along a latitudinal gradient. This finding however highlights the effectiveness of riverine ecosystems to facilitate the spread of alien species through the increase of propagule pressure [START_REF] Lockwood | The role of propagule pressure in explaining species invasions[END_REF][START_REF] Cassey | Dissecting the null model for biological invasions: A meta-analysis of the propagule pressure effect[END_REF]. Furthermore, the introduction of these alien species after 2010 led to profound changes in the taxonomic composition and homogenisation of invaded communities. While this is in line with previous studies (Van der Veer & Nentwig, 2015; [START_REF] Olden | On defining and quantifying biotic homogenization[END_REF], the overall communities' turnover remained stable due to a slight increase in the native species' turnover, and a decrease in alien species' turnover after 2010, depicting the process of alien species reshaping invaded communities.

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After 2010, the richness of alien fish increased to ~30% of the overall community richness, constituting up to 70% of the overall abundance (an increase in the overall communities abundance from 2010 by +1,031% in 2014 or +678% in 2018) before declining by ~33% and possibly stabilising at lower abundance levels in 2018, resembling typical invader boom-bust dynamics [START_REF] Strayer | Boom-bust dynamics in biological invasions: towards an improved application of the concept[END_REF]Soto et al., in press). Compositional changes have often been observed in the invasion of freshwater ecosystems [START_REF] Clavero | Homogenization dynamics and introduction routes of invasive freshwater fish in the Iberian Peninsula[END_REF][START_REF] Gallardo | Global ecological impacts of invasive species in aquatic ecosystems[END_REF][START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF]. In our study, the observed changes can be ascribed to the establishment of six additional alien species between 2010 and 2013, with L. gibbosus, N. fluviatilis, P. kessleri, N. melanostomus, P. parva, and R. amarus contributing 69.9% of the total community abundance in 2015. Although all of these are comparably small species (≤ 20 cm), they can reach high densities and thus, biomass, accompanied by notable impacts to invaded ecosystems (see e.g. [START_REF] Britton | Trophic interactions and consequent impacts of the invasive fish Pseudorasbora parva in a native aquatic foodweb: a field investigation in the UK[END_REF][START_REF] Števove | Ontogenetic variations in the diet of two invasive gobies, Neogobius melanostomus (Pallas, 1814) and Ponticola kessleri (Günther, 1861), from the middle Danube (Slovakia) with notice on their potential impact on benthic invertebrate communities[END_REF]. These alien species probably benefited from previous declines of native species and environmental changes [START_REF] Marvier | Habitat destruction, fragmentation, and disturbance promote invasion by habitat generalists in a multispecies metapopulation[END_REF][START_REF] Früh | Physicochemical and morphological degradation of stream and river habitats increases invasion risk[END_REF][START_REF] Heringer | Acacia invasion is facilitated by landscape permeability: The role of habitat degradation and road networks[END_REF], while their added abundance has resulted in the overall community reaching comparable abundances to those measured in 1984underlining the turnover from native to alien species communities [START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF] suggesting that total community abundances are somewhat limited due to the productivity of the ecosystem [START_REF] Storch | The more-individuals hypothesis revisited: the role of community abundance in species richness regulation and the productivity-diversity relationship[END_REF]. Indeed, as ectothermic animals, fish species are sensitive to environmental changes [START_REF] Magnuson | Temperature as an ecological resource[END_REF]. Several studies have highlighted the negative impacts of temperature increases and precipitation shifts on native fish communities [START_REF] Magnuson | Temperature as an ecological resource[END_REF][START_REF] Marchetti | Effects of flow regime on fish assemblages in a regulated California stream[END_REF][START_REF] Lamouroux | Fish assemblages and stream hydraulics: consistent relations across spatial scales and regions[END_REF] but also on aquatic communities in general [START_REF] Durance | Climate change effects on upland stream macroinvertebrates over a 25-year period[END_REF][START_REF] Chessman | Climatic changes and 13-year trends in stream macroinvertebrate assemblages in New South Wales, Australia[END_REF][START_REF] Heino | Climate change and freshwater biodiversity: detected patterns, future trends and adaptations in northern regions[END_REF]). Yet, their effects remain difficult to predict [START_REF] Haase | Moderate warming over the past 25 years has already reorganized stream invertebrate communities[END_REF][START_REF] Baranov | Complex and nonlinear climate-driven changes in freshwater insect communities over 42 years[END_REF]. Concomitantly, we found temperature and precipitation to incline over the studied period, consequently driving compositional changes in the investigated ACCEPTED MANUSCRIPT / CLEAN COPY community. Although observed increases in temperature (~ 0.94 °C) and precipitation (~ 0.26 mm) seem low, even minor changesas well as fluctuationscan lead to substantial (i.e. seasonal) changes in native communities [START_REF] Magnuson | Temperature as an ecological resource[END_REF][START_REF] Lamouroux | Fish assemblages and stream hydraulics: consistent relations across spatial scales and regions[END_REF] and benefit alien species [START_REF] Rahel | Assessing the effects of climate change on aquatic invasive species[END_REF][START_REF] Radinger | The role of connectivity in the interplay between climate change and the spread of alien fish in a large Mediterranean river[END_REF].

As such, the increase in water temperature has likely permitted invasive fish species to cross physiological thresholds that were formerly limiting or slowing down their biology.

The introduction of alien species after 2010 induced further changes in the community's traits composition. These were mainly driven by the alien species N. melanostomus, which dominated the communities' abundance since the first year it was observed by contributing up to ~98% of the total abundance of alien fish in 2018. The dominance of N. melanostomus which can severely impact invaded ecosystems [START_REF] Kipp | Impacts of the Eurasian round goby (Neogobius melanostomus) on benthic communities in the upper St. Lawrence River[END_REF], may be linked with the demonstrated presence of Asian clam Corbicula fluminea in Rhine river [START_REF] Merschel | Rare earth elements in the aragonitic shell of freshwater mussel Corbicula fluminea and the bioavailability of anthropogenic lanthanum, samarium and gadolinium in river water[END_REF], used as its main food resource [START_REF] Nagelkerke | Functional feeding traits as predictors of invasive success of alien freshwater fish species using a food-fish model[END_REF][START_REF] Coughlan | When worlds collide: Invader-driven benthic habitat complexity alters predatory impacts of invasive and native predatory fishes[END_REF]. Other alien fish species not belonging to the Neogobius genus were observed with less than 100 individuals throughout the entire period. This dominance of N. melanostomus suggests that comparably less abundant species occupied a less dominant role [START_REF] Grabowska | The role of intraspecific competition in the dispersal of an invasive fish[END_REF]. However, it is possible that the sampling method applied here (i.e. electrofishing) was not adequate to capture the total abundance, e.g. C. carpio or S. glanis, which tend to occupy deep benthic zones. For this reason, it is possible that the abundances of these species were underestimated (compared to the abundances observed by [START_REF] Singh | Invasion of an exotic fish-common carp, Cyprinus carpio L.(Actinopterygii: Cypriniformes: Cyprinidae) in the Ganga River, India and its impacts[END_REF]Singh et al., or Šmejkal et al., 2015)).

Patterns in trait composition

Taxonomic changes in communities over time are often accompanied by trait changes (Baker et al., 2021;[START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF][START_REF] Liu | Biological invasions reveal how niche change affects the transferability of species distribution models[END_REF]. Here, we found trait changes to follow a ACCEPTED MANUSCRIPT / CLEAN COPY similar pattern to the shift in taxonomic composition, with the communities' trait structuring driven by alien species richness and abundance, as well as changes in temperature and precipitation. On the other hand, no significant changes in trait metrics were observed in parallel to compositional changes. This result is peculiar, as it contrasts with other studies showing alien species simultaneously leading to both taxonomic changes and functional homogenisation [START_REF] Pool | Taxonomic and functional homogenization of an endemic desert fish fauna[END_REF][START_REF] Villéger | Functional homogenization exceeds taxonomic homogenization among European fish assemblages[END_REF][START_REF] Pilotto | Decline in niche specialization and trait β-diversity in benthic invertebrate communities of Central European low-mountain streams over 25 years[END_REF]. However, functional homogenisation may be hard to detect in the early stages of an invasion, in particular during the establishment and possible lag phase, which may explain the discrepancy of our results with the available literature. This additionally stresses the importance of computing different diversity indices to properly prove impacts of alien invasions on recipient communities.

In the present work, the only trait metric changing over time was trait dispersion (TDis) of ecological traits before 2010. Often, TDis is expected to increase at the onset of the invasion process, in particular when alien species introduce novel traits which extend trait space [START_REF] Renault | Advancing biological invasion hypothesis testing using functional diversity indices[END_REF]). Yet, this metric rose before alien species started to invade the investigated communities, and rather accompanied the increase in beta-diversity. Betadiversity is widely known to increase when the studied habitats become more heterogeneous, augmenting dissimilarity, and likely increasing the dispersion of species traits that cope with a large diversity of environmental conditions. The demise (at least in abundance, also evidenced by the temporal species turnover analysis) of ecologically similar species in the native community is consistent with this assumption. This has increased trait divergence (TDiv) among species from the different habitats, in addition to removing the less adapted individuals (species). After 2010, TDis remained stable, even if beta-diversity declined, and confirmed the maintenance of species' TDiv among habitats.

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Other ecological and biological trait metrics were stable over time, providing unexpected, yet interesting insights. The non-significant changes in trait richness indicate that the demise of species carrying a certain set of traits is accompanied by acquiring new (alien) species carrying new sets of trait modalities not expressed by the native community [START_REF] Mcgeoch | Measure and reduce the harm caused by biological invasions[END_REF]. Furthermore, it should be considered that due to its zoogeographic history, the natural fish communities of western Europe are relatively species-poor [START_REF] Kottelat | Handbook of European freshwater fishes[END_REF]. As such, it is possible that introductions of alien species, which are often functionally and especially morphologically different (e.g. the asp Leuciscus aspius) as compared with other fish species, lead to an initial increase in trait diversity and richness. This does not mean that the demised species were functionally replaced, but rather that the losses in traits were counterbalanced by the invader carrying the same or similar traits, as well as bringing new traits into the community, resulting in a similarly occupied trait space. In other words, invaders may have a high degree of functional identity as compared with native species, suggesting, here again, that environmental filtering may have been strong and contributed to the selection of particular traits.

The shifts in ecological and biological traits observed after 2010 were mainly driven by one abundant alien species, namely N. melanostomus. This can explain trait metrics not indicating significant changes, albeit the high abundance (and thus also biomass and density) of this species likely impacting the studied sites. This lack of an identifiable trait change, however, could also be caused by a high redundancy between the traits of native and alien species [START_REF] Loreau | Does functional redundancy exist?[END_REF][START_REF] Baiser | The relationship between functional and taxonomic homogenization[END_REF][START_REF] Villéger | Homogenization patterns of the world's freshwater fish faunas[END_REF] or by alien species compensating for declining alien species by invading empty niches [START_REF] Herbold | Introduced species and vacant niches[END_REF][START_REF] Liew | Dammed rivers: impoundments facilitate fish invasions[END_REF][START_REF] Nagelkerke | Functional feeding traits as predictors of invasive success of alien freshwater fish species using a food-fish model[END_REF]. High functional redundancy has been shown to increase both community resilience and ecological stability [START_REF] Pillar | Functional redundancy and stability in plant communities[END_REF][START_REF] Biggs | Does functional redundancy affect ecological stability and resilience? A review and meta-analysis[END_REF] ACCEPTED MANUSCRIPT / CLEAN COPY 2020), and likely arises when environmental conditions are restricting (reduced phenotypic variation in trait expression and characteristics). The possibility of a high functional redundancy between alien and native species in the Rhine river could indicate that ecosystem functions [START_REF] Vilà | How well do we understand the impacts of alien species on ecosystem services? A pan-European, cross-taxa assessment[END_REF][START_REF] Vilà | Ecological impacts of invasive alien plants: a meta-analysis of their effects on species, communities and ecosystems[END_REF] were maintained despite the detected taxonomic turnover and alterations in the ratio of several expressed biological and ecological traits. This further suggests that alien species can, while simultaneously altering invaded habitats and recipient biodiversity when becoming abundant, maintain functional roles or fill previously arisen voids.

By contributing to stabilise community resilience in perturbed habitats, alien species may seemingly help to preserve an ecosystem's functioning in the short-term, although the longterm effects remain unknown raising the controversy of the effects of alien species [START_REF] Schlaepfer | The potential conservation value of non-native species[END_REF][START_REF] Russell | The rise of invasive species denialism[END_REF]. Finally, our study advocates the need for the inclusion of additional or new traits introduced with the establishment of alien fish species which are not considered in www.freshwaterecology.info (Schmidt-Kloiber & Hering, 2015), further increasing the resolution of functional changes [START_REF] D'andrea | Challenges in linking trait patterns to niche differentiation[END_REF].

Conclusion

Our results underline the importance of analysing long-term trends in invasion dynamics and facilitating drivers, which has often been limited by a lack of adequate long-term data or the non-continuity of annual sampling over time [START_REF] Daufresne | Long-term changes within the invertebrate and fish communities of the Upper Rhône River: Effects of climatic factors[END_REF][START_REF] Durance | Climate change effects on upland stream macroinvertebrates over a 25-year period[END_REF][START_REF] Comte | Do stream fish track climate change? Assessing distribution shifts in recent decades[END_REF][START_REF] Jourdan | Effects of changing climate on European stream invertebrate communities: A long-term data analysis[END_REF][START_REF] Haubrock | Two centuries for an almost complete community turnover from native to non-native species in a riverine ecosystem[END_REF][START_REF] Haubrock | Knowledge gaps in economic costs of invasive alien fish worldwide[END_REF]. This has concomitantly limited insights into changing community functioning or ecological changes due to changes in alien species' trait composition. This was also the case in this work, as highlighted by the lack of information on the fish community between 1984 and 1993. Yet, having identified a disconnect between visible responses in taxonomic composition, and no detectable changes in functional metrics due to the invasion of alien species, suggests that both aspects ACCEPTED MANUSCRIPT / CLEAN COPY pertaining to fish invasions must be considered in conjunction and separated at the same time.

More specifically, taxonomic changes directly reflect biodiversity alterations, often at the expense of native species, which possibly cause changes in the occurrence of trait modalities and hence, potentially also functional changes, which can, but may not necessarily impact ecosystem functioning.

Our results further lead to the question, why native species declined pre-2010 and communities became more diverse, before alien species became abundant and increased taxonomic homogenisation. This finding highlights the importance of considering further variables to investigate the temporal effects of species introductions. These variables could reflect habitat fragmentation [START_REF] Cowx | Freshwater fish conservation: options for the future[END_REF], the regulation of water flow [START_REF] Poff | The natural flow regime[END_REF][START_REF] Albert | Scientists' warning to humanity on the freshwater biodiversity crisis[END_REF], changes in the land use [START_REF] Albert | Scientists' warning to humanity on the freshwater biodiversity crisis[END_REF], pollution [START_REF] Matthews | Similarities in fish distribution and water quality patterns in streams of Arkansas: congruence of multivariate analyses[END_REF][START_REF] Taylor | Fish species-environment and abundance relationships in a Great Plains river system[END_REF][START_REF] Lappalainen | Latitudinal gradients in niche breadth and position-regional patterns in freshwater fish[END_REF], the pressure of fishing [START_REF] Cowx | Freshwater fish conservation: options for the future[END_REF][START_REF] Butchart | Global biodiversity: indicators of recent declines[END_REF])predictors which were not adequately available over the entire period to be considered in our models.

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Figure 1 .

 1 Figure 1. Representation of 31 fish sampling sites along the Rhine river (Germany).

Figure 2 .

 2 Figure 2. (a) Total species richness and (b) total community abundance of the Rhine river fish community from 1984 to 2018 indicating the average values ± the respective standard deviation for each sampled year and the respective share of native (blue) and alien species (red). Numbers on the right indicate the (a) total species richness and the (b) total community abundance of native and alien fish in 2018.

Figure 3 .

 3 Figure 3. (a) Relative species richness and (b) relative community abundance of native (in blue) and alien (in red) fish species of the Rhine river from 1984 to 2018.

Figure 4 .

 4 Figure 4. Relative abundance of the six most abundant (a) native and (b) alien fishes during the study period. Number on top of each bar indicates the total abundance of all (a) native and (b) alien species collected in that respective year.

Figure 5 .

 5 Figure 5. Temporal turnover rate displaying appearances and disappearances of fish species in the Rhine river from 1984 to 2018. (a) Overall turnover in the whole community. (b) Turnover for native fishes. (c) Turnover for alien fishes. Please note that there was no detectable turnover in the alien community before 2006 despite the presence of several, continuously present alien fish species.

Figure 6 .

 6 Figure 6. Canonical analysis of principal coordinates (CAP) on (a) the taxonomic community composition (based on species abundances), the community weighted means (CWMs) of (b) ecological and (c) biological traits across years. Blue arrows indicate significant abiotic and biotic predictors. Values along the arrows indicate the correlation with the dominant axis.

Figure 7 .

 7 Figure 7. Generalised additive models (GAMs) exploring the relationship between (a-d) taxonomic and (e-h) trait metrics of the studied communities. Trait metrics are divided into ecological (black lines) and biological (blue lines) traits. Solid lines indicate significant change over time, whereas dashed lines represent non-significant trends. See table S8 for other significant predictors.

Figure S2 .

 S2 Figure S2. Distribution of species in trait space based on both biological and ecological traits.

Figure S3 .

 S3 Figure S3. Generalised additive models (GAMs) exploring the relationship between (a) temperature and (b) precipitation during the study period. Solid lines indicate significant change over time.

Figure S4 .

 S4 Figure S4.Beta-diversity over time using the Jaccard similarity Index (solid black lines) and the response of GAM model (blue line) using "year" as a unique predictor. Dashed line split the period into before the arrival of alien species (< 2010) and after (> 2010).

Figure S5 .

 S5 Figure S5. Relative abundance of each ecological trait modalities over time.

Figure S6 .

 S6 Figure S6. Relative abundance of each biological trait modalities over time.

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 2 Figure 2

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 3 Figure 3

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 4 Figure 4

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

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 S1 Figure S1

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 S2 Figure S2

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Table S2 .

 S2 Definitions of all ecological and biological traits used in this study; extracted from www.freshwaterecology.info(Schmidt-Kloiber & Hering, 2015).

	Time series	Sampling years Df SumOfSqs	Total sampled years R2 F	Pr(>F)	Period
	Biological Species	Family	Status	Ecological Period
	Abramis brama Latitude	Cyprinidae 1 5.670	Native 0.063 1984-2018 35.044	0.0001
	Body length Alburnoides bipunctatus Alien species abundance	Body length in cm Cyprinidae 1 10.454	Habitat 2017 0.115 Native 64.617	0.0001	Where species living and
	Alburnus alburnus	Cyprinidae	Native	1984-2018		feeding
	Shape factor Anguilla anguilla Alien species richness	Ratio between the length and Anguillidae Native 1 7.222 0.080 Habitat (fiBS) 1984-2018 44.640	0.0001	Flow conditions of the
	Barbatula barbatula Precipitation	the width Nemacheilidae 1 1.283	Native 0.014 2004-2017 7.929	0.0001	habitat
	Barbus barbus	Cyprinidae	Native	1984-2018
	Swimming factor Blicca bjoerkna Temperature	Ratio of the minimum depth Cyprinidae Native 1 1.854 0.020 Feeding habitat 1984-2004 11.460	0.0001	Feeding in the bottom or
	Carassius gibelio Carassius carassius Residual	of the caudal peduncle to the Cyprinidae Native maximum caudal fin depth Cyprinidae Native 397 64.229 0.708 2013-2015 1984		in the water column
	Life span Chondrostoma nasus Total	Life span in years Cyprinidae 402 90.711	Reproduction (fiBS) 1984-2018 1.000 Native	Habitat reproduction
	Cobitis taenia	Cobitidae	Native	2013
	Female maturity Cottus gobio	Age of females mature for Cottidae	Native	Spawning habitat 2004		Flow conditions of the
	Cyprinus carpio	the first time Cyprinidae	Alien	1993-2018		spawning habitat
	Absolute fecundity Esox lucius	Number of oocytes produces Esocidae Native	Habitat degradation 1984-2017	Tolerance or not to
	Gasterosteus aculeatus	per female Gasterosteidae	Native	tolerance 2015-2017		habitat degradation
	Gobio gobio	Cyprinidae	Native	1984-2010
	Relative fecundity Gymnocephalus cernua	Number of oocytes produces Percidae Native	Water quality tolerance 1993-2015	Tolerance to usual water
	Lampetra fluviatilis	per gram Petromyzontidae	Native	2004		quality parameters
	Egg diameter Lepomis gibbosus	Egg diameter in mm Centrarchidae	Alien	Temperature tolerance 2013-2017	Eury-or stenothermal
	Leuciscus aspius	Cyprinidae	Native	1993-2018
	Spawning time	Season of fish spawning		Oxygen tolerance		Tolerance to low oxygen
	Leuciscus idus	Cyprinidae	Native	1993-2018
						concentration
	Leuciscus leuciscus	Cyprinidae	Native	1984-2018
	Reproductive behaviour Lota lota	One or several spawning Lotidae	Native	Toxicity tolerance 2000-2018	Tolerance to toxic
	Neogobius fluviatilis	events in the potential season Gobiidae Alien	2013-2018		contamination
	Ponticola kessleri	Gobiidae	Alien	2010-2018
	Parental care	Protection of eggs and/or		Acid tolerance		Tolerance to acidification
	Neogobius melanostomus	Gobiidae	Alien	2010-2018
		larvae or not
	Oncorhynchus mykiss	Salmonidae	Alien	1998-2010
	Larval length Perca fluviatilis	Length of larval fish in cm Percidae	Native	Salinity 1984-2018		Salinity habitat
	Platichthys flesus	Pleuronectidae	Native	1993-2018
				Feeding diet (adult)	Type of feeding diet
	Proterorhinus marmoratus	Gobiidae	Alien	2006
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Table S6 .

 S6 Results of the PERMANOVA on community species composition using abundance-weighed ecological and

	894	temp_tol_eury life_span_ls1		1 1	0.949 3.136		0.010 0.035	21.831 70.445	0.0001 0.0001
	895	biological traits as predictors.
		oxy_tol_im life_span_ls2		1 1	5.006 2.527		0.055 0.028	115.140 56.758	0.0001 0.0001
		Ecological traits Ecological traits
		oxy_tol_intol maturity_ma1		1 1	3.665 0.712		0.040 0.008	84.299 15.988	0.0001 0.0001
			Df	Df SumOfSqs SumOfSqs R2	R2 F	F	Pr(>F)	Pr(>F)
		tox_tol_im maturity_ma2		1 1	0.917 1.195		0.010 0.013	21.100 26.843	0.0001 0.0001
		habitat_ben Latitude	1	1 0.622	16.739	0.044	0.185 25.237	384.976 0.0001	0.0001
		tox_tol_intol maturity_ma3		1 1	0.841 2.069		0.009 0.023	19.352 46.474	0.0001 0.0001
		habitat_dem acid_tol_im maturity_ma4 Alien species abundance	1	1 1 1 2.282	2.324 0.281 1.310	0.162	0.026 0.003 0.014 92.612	53.450 6.453 29.420 0.0001	0.0001 0.0001 0.0001
		habitat_fibs_indifferent acid_tol_intol abs_fecundity_fe1 Alien species richness	1	1 1 1 0.813	11.853 0.457 6.164	0.058	0.131 0.005 0.068 33.006	272.589 10.507 138.471 0.0001	0.0001 0.0001 0.0001
		habitat_fibs_rheophil salinity_fbm abs_fecundity_fe2 Precipitation	1	1 1 1 0.080	0.213 0.367 0.942	0.006	0.002 0.004 0.010 3.234	4.908 8.446 21.168 0.0193	0.0001 0.0001 0.0001
		feeding_habitat_ben salinity_fbr rel_fecundity_fr1 Temperature	1	1 1 1 0.490	7.496 0.461 0.925	0.035	0.083 0.005 0.010 19.869	172.392 10.595 20.771 0.0001	0.0001 0.0001 0.0001
		reproduction_lithopelagophilic diet_invert rel_fecundity_fr2 Residual	397	1 1 1 9.782	0.196 0.165 0.451	0.695	0.002 0.002 0.005	4.508 3.790 10.127	0.0001 0.0001 0.0001
		reproduction_lithophilic diet_omni egg_ed1 Total	402	1 1 1 14.068	6.982 0.368 1.082	1.000	0.077 0.004 0.012	160.575 8.470 24.304	0.0001 0.0001 0.0001
		reproduction_marine diet_parasit egg_ed2 Biological traits		1 1 1	5.686 0.091 0.681		0.063 0.001 0.008	130.772 2.099 15.296	0.0001 0.0388 0.0001
		reproduction_ostracophilic		1	0.076		0.001	1.742		0.048
		Residual spawning_time_st1 Latitude	1	372 1 0.661	16.175 0.429	0.032	0.178 0.005 22.641	9.638	0.0001	0.0001
		reproduction_phytolithophilic		1	1.149		0.013	26.431	0.0001
		Total repr_behav_fra Alien species abundance	1	402 1 5.882	90.711 0.379	0.286	1.000 0.004 201.484	8.508	0.0001	0.0001
		reproduction_phytophilic		1	0.632		0.007	14.534	0.0001
		Biological traits repr_behav_pro Alien species richness	1	1 2.164	0.563	0.105	0.006 74.129	12.653 0.0001	0.0001
		reproduction_psammophilic		1	0.154		0.002	3.535		0.0001
		body_length_bl1 parental_care_no Precipitation	1	1 1 0.091	16.563 0.266	0.004	0.183 0.003 3.128	372.089 5.971 0.0341	0.0001 0.0001
		spawning_habitat_eury		1	2.360		0.026	54.278	0.0001
		body_length_bl2 larval_length_ll1 Temperature	1	1 1 0.209	8.241 0.271	0.010	0.091 0.003 7.172	185.126 6.089 0.001	0.0001 0.0001
		spawning_habitat_limno		1	0.624		0.007	14.339	0.0001
		shape_factor_sh1 larval_length_ll2 Residual	397	1 1 11.590	6.093 0.190	0.563	0.067 0.002	136.878 4.260	0.0001 0.0001
		hab_degr_tol_im		1	1.273		0.014	29.281	0.0001
		shape_factor_sh2 Residual Total	402	1 377 20.598	7.764 16.782	1.000	0.086 0.185	174.419	0.0001
		hab_degr_tol_intol		1	1.323		0.015	30.431	0.0001
		shape_factor_sh3 Total		1 402	6.112 90.711		0.067 1.000	137.295	0.0001
		wat_qual_tol_im		1	0.366		0.004	8.409		0.0001
		swimming_factor_sw1		1	4.451		0.049	99.983	0.0001
		wat_qual_tol_intol		1	1.521		0.017	34.980	0.0001
		swimming_factor_sw2		1	1.416		0.016	31.805	0.0001

Table S8 .

 S8 Coefficients of significant predictors in generalised additive models (gam) exploring the relationship between taxonomic and trait metrics over study time. Abbreviations "NS" indicate non significative predictors and "AS" refer to alien species.

	Period	Metrics	R²	Year Latitude Temperature Precipitation AS abundance AS richness
	1984 to 2006 Richness	0.110 NS	0.81	1.54	3.60	NS	NS
		Abundance	0.624 5.92	8.56	7.86	8.33	NS	NS
		FDis ecological	0.292 0.84	NS	NS	NS	NS	NS
	2010 to 2018 Richness	0.425 4.06	1.65	NS	NS	NS	1.00
		Abundance	0.731 4.93	7.64	8.21	7.70	2.97e-03	6.83e-02
		Shannon diversity 0.353 NS	NS	NS	NS	-0.003	NS
		Pielou evenness	0.477 NS	NS	NS	NS	-0.003	NS
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Table S6 .

 S6 Results of the PERMANOVA on community species composition using abundance-weighed ecological and biological traits as predictors.

	Ecological traits
	Df		SumOfSqs	R2	F	Pr(>F)
	habitat_ben	1	16.739	0.185	384.976	0.0001
	habitat_dem	1	2.324	0.026	53.45	0.0001
	habitat_fibs_indifferent	1	11.853	0.131	272.589	0.0001
	habitat_fibs_rheophil	1	0.213	0.002	4.908	0.0001
	feeding_habitat_ben	1	7.496	0.083	172.392	0.0001
	reproduction_lithopelagophilic	1	0.196	0.002	4.508	0.0001
	reproduction_lithophilic	1	6.982	0.077	160.575	0.0001
	reproduction_marine	1	5.686	0.063	130.772	0.0001
	reproduction_ostracophilic	1	0.076	0.001	1.742	0.048
	reproduction_phytolithophilic	1	1.149	0.013	26.431	0.0001
	reproduction_phytophilic	1	0.632	0.007	14.534	0.0001
	reproduction_psammophilic	1	0.154	0.002	3.535	0.0001
	spawning_habitat_eury	1	2.36	0.026	54.278	0.0001
	spawning_habitat_limno	1	0.624	0.007	14.339	0.0001
	hab_degr_tol_im	1	1.273	0.014	29.281	0.0001
	hab_degr_tol_intol	1	1.323	0.015	30.431	0.0001
	wat_qual_tol_im	1	0.366	0.004	8.409	0.0001
	wat_qual_tol_intol	1	1.521	0.017	34.98	0.0001
	temp_tol_eury	1	0.949	0.01	21.831	0.0001
	oxy_tol_im	1	5.006	0.055	115.14	0.0001
	oxy_tol_intol	1	3.665	0.04	84.299	0.0001
	tox_tol_im	1	0.917	0.01	21.1	0.0001
	tox_tol_intol	1	0.841	0.009	19.352	0.0001
	acid_tol_im	1	0.281	0.003	6.453	0.0001
	acid_tol_intol	1	0.457	0.005	10.507	0.0001
	salinity_fbm	1	0.367	0.004	8.446	0.0001
	salinity_fbr	1	0.461	0.005	10.595	0.0001
	diet_invert	1	0.165	0.002	3.79	0.0001
	diet_omni	1	0.368	0.004	8.47	0.0001
	diet_parasit	1	0.091	0.001	2.099	0.0388
	Residual	372	16.175	0.178
	Total	402	90.711	1
	Biological traits
	body_length_bl1	1	16.563	0.183	372.089	0.0001
	body_length_bl2	1	8.241	0.091	185.126	0.0001
	shape_factor_sh1	1	6.093	0.067	136.878	0.0001
	shape_factor_sh2	1	7.764	0.086	174.419	0.0001
	shape_factor_sh3	1	6.112	0.067	137.295	0.0001
	swimming_factor_sw1	1	4.451	0.049	99.983	0.0001
	swimming_factor_sw2	1	1.416	0.016	31.805	0.0001
	life_span_ls1	1	3.136	0.035	70.445	0.0001
	life_span_ls2	1	2.527	0.028	56.758	0.0001

Table S6

 S6 Click here to access/download;Table (Editable version);Supplementary table 6.docx

	maturity_ma1	1	0.712	0.008	15.988	0.0001
	maturity_ma2	1	1.195	0.013	26.843	0.0001
	maturity_ma3	1	2.069	0.023	46.474	0.0001
	maturity_ma4	1	1.31	0.014	29.42	0.0001
	abs_fecundity_fe1	1	6.164	0.068	138.471	0.0001
	abs_fecundity_fe2	1	0.942	0.01	21.168	0.0001
	rel_fecundity_fr1	1	0.925	0.01	20.771	0.0001
	rel_fecundity_fr2	1	0.451	0.005	10.127	0.0001
	egg_ed1	1	1.082	0.012	24.304	0.0001
	egg_ed2	1	0.681	0.008	15.296	0.0001
	spawning_time_st1	1	0.429	0.005	9.638	0.0001
	repr_behav_fra	1	0.379	0.004	8.508	0.0001
	repr_behav_pro	1	0.563	0.006	12.653	0.0001
	parental_care_no	1	0.266	0.003	5.971	0.0001
	larval_length_ll1	1	0.271	0.003	6.089	0.0001
	larval_length_ll2	1	0.19	0.002	4.26	0.0001
	Residual	377	16.782	0.185
	Total	402	90.711	1
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Table S7 .

 S7 Results of the PERMANOVA on abundance-weighed ecological and biological traits using abiotic and biotic predictors.Table S7Click here to access/download;Table (Editable version);Supplementary table 7.

	Ecological traits
	Df		SumOfSqs	R2	F	Pr(>F)
	Latitude	1	0.622	0.044	25.237	0.0001
	Alien species abundance	1	2.282	0.162	92.612	0.0001
	Alien species richness	1	0.813	0.058	33.006	0.0001
	Precipitation	1	0.08	0.006	3.234	0.0193
	Temperature	1	0.49	0.035	19.869	0.0001
	Residual	397	9.782	0.695
	Total	402	14.068	1
	Biological traits
	Latitude	1	0.661	0.032	22.641	0.0001
	Alien species abundance	1	5.882	0.286	201.484	0.0001
	Alien species richness	1	2.164	0.105	74.129	0.0001
	Precipitation	1	0.091	0.004	3.128	0.0341
	Temperature	1	0.209	0.01	7.172	0.001
	Residual	397	11.59	0.563
	Total	402	20.598	1

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Table S8 .

 S8 Coefficients of significant predictors in generalised additive models (gam) exploring the relationship between taxonomic and trait metrics over study time. Abbreviations "NS" indicate non significative predictors and "AS" refer to alien species.

	Period	Metrics	R²	Year	Latitude Temperature Precipitation	AS abundance	AS richness
		Richness	0.11	NS	0.81	1.54	3.6	NS	NS
	1984 to 2006	Abundance	0.624	5.92	8.56	7.86	8.33	NS	NS
		FDis ecological	0.292	0.84	NS	NS	NS	NS	NS
		Richness	0.425	4.06	1.65	NS	NS	NS	1
		Abundance	0.731	4.93	7.64	8.21	7.7	2.97E-03	6.83E-02
	2010 to 2018	Shannon diversity	0.353	NS	NS	NS	NS	-0.003	NS
		Pielou evenness	0.477	NS	NS	NS	NS	-0.003	NS

Table S8

 S8 Click here to access/download;Table (Editable version);Supplementary table 8.

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Table S1. Description of each time series

TableS4. Species sampled in the Rhine river between 1984 and 2018, their family, status (native/alien, the latter highlighted

in red) and sampled occurrences.

TableS5. Results of the PERMANOVA on community species composition using abiotic and biotic predictors.

TableS7. Results of the PERMANOVA on abundance-weighed ecological and biological traits using abiotic and biotic

predictors.

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Acknowledgements

The authors express their sincere thanks to Michele Mugnai for helpful comments on a previous version of this manuscript and the "Commission Bourses de Mobilité à l'Étranger de l'Université de Rennes 1" and the ERASMUS + program for their financial support. The authors further acknowledge the help of Dr. Lep Omis. PH and PJH received funding from the EU Horizon 2020 project eLTER PLUS (Grand Agreement No 871128). IS is supported by the Grant Agency of the University of South Bohemia, project number 065/2022/Z. ACCEPTED MANUSCRIPT / CLEAN COPY

Supplements

ACCEPTED MANUSCRIPT / CLEAN COPY Table S3. Definitions of all metrics and measurements used in this study.

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Metric Definition

Species richness Number of species (Baker et al., 2021) Abundance

Number of individuals per species

Shannon diversity A function of both richness and evenness [START_REF] Maurer | Measurement of species diversity in Biological Diversity: Frontiers in Measurement and Assessment[END_REF] Pielou evenness Distribution of abundances across all species within a community (Baker et al., 2021) Trait richness (TRic)

The amount of niche space occupied by all species within a given community (Mason et al., 2005) Trait divergence (TDiv)

The degree to which the abundance distribution utilises differences in traits within the community (a measure of how spread or clumped species are within the niche space, weighted by the relative abundance) (Mason et al., 2005;[START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] Trait dispersion (TDis)

The average distance of individual species to the group centroids of all species [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] Trait evenness (TEve)

The regularity in the distribution of the abundance in trait space of the organisms composing an ecological unit [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF][START_REF] Schleuter | A user's guide to functional diversity indices[END_REF]

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ACCEPTED MANUSCRIPT / CLEAN COPY The amount of niche space occupied by all species within a given community (Mason et al., 2005) Trait divergence (TDiv)

The degree to which the abundance distribution utilises differences in traits within the community (a measure of how spread or clumped species are within the niche space, weighted by the relative abundance) (Mason et al. 2005;[START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] Trait dispersion (TDis) The average distance of individual species to the group centroids of all species [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] Trait evenness (TEve) The regularity in the distribution of the abundance in trait space of the organisms composing an ecological unit [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF][START_REF] Schleuter | A user's guide to functional diversity indices[END_REF] Table S3 Click here to access/download;

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: