Robin P M Gauff
email: gauff.robin@yahoo.de

Christophe D Lejeusne

Laure Arsenieff

Olivier Bohner

Jérôme Coudret

Florian Desbordes

Alise Jandard

Stéphane Loisel

Gaëtan Schires

Emmanuel Wafo

Dominique Davoult

variability and predation on the survival of ascidian recruits of a native and alien species

Keywords: Competitive release, facilitation, biotic resistance, invasive species, predation, ascidians

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

Introduction

With increasing globalization and interconnectivity of trade among countries, accidental and voluntary species introductions have multiplied with the use of trade ships, a trend that is predicted to further increase in coming years [START_REF] Levine | Forecasting Biological Invasions with Increasing International Trade[END_REF][START_REF] Seebens | Predicting the spread of marine species introduced by global shipping[END_REF][START_REF] Carrasco | Biodiversity conservation in a telecoupled world[END_REF]. The ecological consequences of the spread of non-indigenous species (NIS), if they become invasive, can be drastic, with some invaders completely restructuring ecosystems, potentially leading to the loss of biodiversity and ecosystem services [START_REF] Pejchar | Invasive species, ecosystem services and human well-being[END_REF]. This has led to a large body of literature focusing on the conditions of successful transport and naturalization in the new environment of these species, identifying several filters that constitute strong selective barriers that only few species successfully overcome [START_REF] Williamson | The varying success of invaders 1[END_REF][START_REF] Jarić | The tens rule in invasion biology: Measure of a true impact or our lack of knowledge and understanding?[END_REF].

Upon survival to the transport phase, the first major obstacle a NIS may encounter in a new environment is the abiotic environment. Abiotic factors have been shown to vastly influence species' evolution and the structure of ecosystems [START_REF] Je | Changes in benthic communities along a presumed pollution gradient in Vancouver Harbour[END_REF][START_REF] Benton | The Red Queen and the Court Jester: Species diversity and the role of biotic and abiotic factors through time[END_REF][START_REF] Lewis | Biotic and abiotic factors predicting the global distribution and population density of an invasive large mammal[END_REF]). NIS seem to show higher resistance to abiotic stress and disturbance than their native counterparts [START_REF] Gröner | Stress resistance in two colonial ascidians from the Irish Sea: The recent invader Didemnum vexillum is more tolerant to low salinity than the cosmopolitan Diplosoma listerianum[END_REF][START_REF] Lejeusne | Do invaders always perform better? Comparing the response of native and invasive shrimps to temperature and salinity gradients in south-west Spain[END_REF][START_REF] Marie | Transcriptomic response to thermal and salinity stress in introduced and native sympatric Palaemon caridean shrimps[END_REF]Kenworthy et al. 2018a), which is attributed to two different processes. First, in some habitats, pre-adaptation of the considered NIS may attenuate the abiotic filter of its new environment [START_REF] Schlaepfer | A multi-species experiment in their native range indicates pre-adaptation of invasive alien plant species[END_REF][START_REF] Macdougall | The Neolithic Plant Invasion Hypothesis: the role of preadaptation and disturbance in grassland invasion[END_REF]. Second, some NIS can undergo rapid evolution within several generations due to strong selective forces acting on them [START_REF] Huey | Rapid evolution of a geographic cline in size in an introduced fly[END_REF][START_REF] Hejda | Invasion success of alien plants: do habitat affinities in the native distribution range matter?[END_REF][START_REF] Jarić | The tens rule in invasion biology: Measure of a true impact or our lack of knowledge and understanding?[END_REF][START_REF] Elst | Pre-adaptation or genetic shift after introduction in the invasive species Impatiens glandulifera?[END_REF].

Biotic interactions, through a complex interplay between NIS and native species, may also condition successful introductions and invasions, and are the focus of numerous theories on the apparent success (or failure) of NIS in various ecosystems. Being a major regulator of ecosystem functioning through regulation of prey populations and a driver of community structure, top-down regulation often occupies a central role in many of these theories [START_REF] Hunter | Playing Chutes and Ladders: Heterogeneity and the Relative Roles of Bottom-Up and Top-Down Forces in Natural Communities[END_REF][START_REF] Shurin | A cross-ecosystem comparison of the strength of trophic cascades[END_REF][START_REF] Heck | The primacy of top-down effects in shallow benthic ecosystems[END_REF][START_REF] Riginos | Savanna tree density, herbivores, and the herbaceous community: Bottom-up vs. top-down effects[END_REF]. In the Biotic Resistance Hypothesis [START_REF] Elton | The Ecology of Invasions by Animals and Plants[END_REF], the combined antagonistic interactions among species, including competition and predation, may allow species-rich communities to resist the establishment of invasive species through their more effective use of resources than species-poor environments. However, despite its popularity in the scientific community, the Biotic Resistance Hypothesis remains controversial [START_REF] Jeschke | Support for major hypotheses in invasion biology is uneven and declining[END_REF]). Another explanation for the success of NIS is provided by the Enemy Release Hypothesis (ERH; [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF], which states that NIS may be released at least partially from their biotic regulators (predators, parasites and pathogens). As such, NIS may perform better in introduced areas and gain competitive advantages over native species [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF][START_REF] Joshi | The enemy release and EICA hypothesis revisited: Incorporating the fundamental difference between specialist and generalist herbivores[END_REF].

Although the presence of generalist predators or host-switching specialists may partially undermine this assumption, the food selectivity of generalist predators, potentially favoring native species, may benefit NIS due to competitive release [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF]. NIS success can also be explained by the Novel Weapon Hypothesis (NWH; [START_REF] Callaway | Novel weapons: Invasive success and the evolution of increased competitive ability[END_REF], implying specific adaptations of a NIS to its native habitat. In the absence of coevolution with NIS, native species may lack an evolutionary response to an NIS, leading to higher competitive performance of the invader [START_REF] Ni | Exploring the Novel Weapons Hypothesis with invasive plant species in China[END_REF]. This NWH can involve direct competition and allelopathic interactions between NIS and natives, as extensively described for plant species [START_REF] Bretz | Invasive plants versus their new and old neighbors: A mechanism for exotic invasion[END_REF][START_REF] Ni | Exploring the Novel Weapons Hypothesis with invasive plant species in China[END_REF]. However, such compounds may also be part of predator avoidance and thus indirectly result in competitive superiority [START_REF] Hay | Synergisms in Plant Defenses against Herbivores: Interactions of Chemistry, Calcification, and Plant Quality[END_REF][START_REF] Cappuccino | Invasive exotic plants suffer less herbivory than non-invasive exotic plants[END_REF].

While still debated, these hypotheses provide a complex interplay of ideas in the context of predation and acknowledge its importance. Predation may be a key component in determining the success or failure of NIS [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF][START_REF] Cappuccino | Invasive exotic plants suffer less herbivory than non-invasive exotic plants[END_REF][START_REF] Joshi | The enemy release and EICA hypothesis revisited: Incorporating the fundamental difference between specialist and generalist herbivores[END_REF][START_REF] Skein | Characteristics of native predators are more important than those of alien prey in determining the success of biotic resistance in marine systems[END_REF]. Being predominantly tested in terrestrial habitats, few studies have been conducted on marine invasions often with inconsistent results with regard to these hypotheses [START_REF] Chan | An overview of recent research in marine biological invasions[END_REF]. Nonetheless, fouling communities and especially artificial harbor or marina communities are highly exposed to NIS due to the process of primary introduction, via fouling on boat hulls and transport in ballast waters [START_REF] Sylvester | Hull fouling as an invasion vector: Can simple models explain a complex problem?[END_REF][START_REF] Murray | Adapted for invasion? Comparing attachment, drag and dislodgment of native and nonindigenous hull fouling species[END_REF]. This extensive exposure to NIS can lead to high economic and ecological costs, making it all the more important to better understand the factors behind NIS success in these ecosystems [START_REF] Ojaveer | Classification of Non-Indigenous Species Based on Their Impacts: Considerations for Application in Marine Management[END_REF]. Despite apparent resemblances between plant and fouling communities, there is no consensus on whether their functioning is actually similar, limiting the conclusions that can be transposed from terrestrial studies to marine communities [START_REF] Sutherland | Development and Stability of the Fouling Community at Beaufort, North Carolina[END_REF]. Most notably, fouling communities in marinas experience strong anthropogenic disturbances, which have been the focus of numerous studies showing their effect on surrounding species. Artificial substrata are physically and chemically distinct from their natural counterparts and high concentrations of heavy metals and persistent organic pollutants (POPs) are present [START_REF] Saloni | Impacts of multiple stressors during the establishment of fouling assemblages[END_REF][START_REF] Chase | The influence of substrate material on ascidian larval settlement[END_REF][START_REF] Kinsella | Separate and combined effects of copper and freshwater on the biodiversity and functioning of fouling assemblages[END_REF].

The selection exerted by all these disturbances may even result in differential selection and local adaptation at a very small spatial scale (ca. 50 m) with distinct communities adapted to local conditions [START_REF] Kawecki | Conceptual issues in local adaptation[END_REF][START_REF] Colautti | Contemporary evolution during invasion : evidence for differentiation , natural selection , and local adaptation[END_REF]Kenworthy et al. 2018b). Despite an apparent focus on abiotic factors, studies have also assessed the applicability of the above-mentioned ecological hypotheses on NIS success in their range of introduction. Many studies have investigated biotic resistance to invasions [START_REF] Kimbro | Biotic resistance in marine environments[END_REF]. Biotic resistance due to interspecific competition seems to have a crucial importance on the success or failure of NIS [START_REF] Fletcher | Effect of substrate deployment timing and reproductive strategy on patterns in invasiveness of the colonial ascidian Didemnum vexillum[END_REF][START_REF] Gestoso | Biotic effects during the settlement process of non-indigenous species in marine benthic communities[END_REF]), but interspecific competition may be modulated by predation (Oricchio et al. 2016a). Many studies have shown that predation strongly influences several fouling species and contributes to the biotic resistance against NIS [START_REF] Rogers | Native predators limit invasion of benthic invertebrate communities in Bodega Harbor, California, USA[END_REF][START_REF] Rodemann | Consumption pressure in coastal marine environments decreases with latitude and in artificial vs. natural habitats[END_REF][START_REF] Yorisue | Mechanisms underlying predator-driven biotic resistance against introduced barnacles on the Pacific coast of Hokkaido, Japan[END_REF]. There is however no clear consensus on the importance of predation, with some studies indicating only minor effects masked by abiotic factors, and some others even indicating a facilitation of NIS due to predation on native competitors, probably due to generalist predators' preference for native prey [START_REF] Astudillo | Seasonal heterogeneity provides a niche opportunity for ascidian invasion in subtropical marine communities[END_REF][START_REF] Gestoso | Biotic effects during the settlement process of non-indigenous species in marine benthic communities[END_REF][START_REF] Kincaid | Predators Associated with Marinas Consume Indigenous over Non-indigenous Ascidians[END_REF].

Studies however rarely include the complex interplay of biotic and abiotic factors, revealing the necessity for further research on how the combination of disturbance and generalist predation affects the successful establishment of NIS. In the present study, we aimed to assess these respective influences on the survival of young recruits of two ascidian species, the sea vase tunicate Ciona intestinalis (Linnaeus, 1767) and the Asian clubbed tunicate Styela clava Herdman, 1881, respectively native and non-indigenous in the North East Atlantic. Here we mainly focused on predatory fish, which were considered as a generalist predator model in this study. The recruit stage of tunicates is particularly vulnerable to predation due to its small size. The size-spectrum theory and the tendency of predatory fish to include small prey in their diet predicts higher predation pressure on small preys, i.e. recruits [START_REF] Osman | The control of the development of a marine benthic community by predation on recruits[END_REF][START_REF] Law | Size-Spectra Dynamics from Stochastic Predation and Growth of Individuals[END_REF]. Recruits may also be more sensitive to environmental stressors [START_REF] Saloni | Impacts of multiple stressors during the establishment of fouling assemblages[END_REF]. We assumed that environmental conditions vary in space, within the same marina, with levels of disturbance most likely organized in a gradient [START_REF] Je | Changes in benthic communities along a presumed pollution gradient in Vancouver Harbour[END_REF]Kenworthy et al. 2018b). We hypothesized that this organization results in spatial variation in recruit survival, being lower in the inner part of the marina, where pollution levels are likely maximal.

We hypothesized that this resulting gradient of survival is less visible for the studied NIS species, because NIS tend to better resist disturbances due to their selection during introduction [START_REF] Gröner | Stress resistance in two colonial ascidians from the Irish Sea: The recent invader Didemnum vexillum is more tolerant to low salinity than the cosmopolitan Diplosoma listerianum[END_REF]. Predation intensity was predicted to vary in space and to negatively affect both species.

However, we predicted that generalist predators prefer native prey, thus having a higher negative impact on the native species.

Materials and Methods

Study site

The Marina du Château in Brest, France, was chosen as our study site (48°22'43.4"N; 4°29'22.1"W). This recreational marina is part of a larger marine urban zone with a commercial and military harbor leading to a highly anthropized environment. As a typical marina, it is made of many artificial substrata that can be colonized by fouling communities, it is contaminated by elemental trace metals (such as copper and lead) and persistent organic pollutants (POPs), and it hosts many NIS (Kenworthy et al. 2018b). We focused on floating pontoons in our experimental setups because they are the most abundant substratum in the marina. This marina has significant variations in environmental conditions at small spatial scales (< 100 m), driving different fouling communities at the entrance, the middle and inner parts of the marina (Kenworthy et al. 2018b). In accordance with this previous study, the same three locations (inner, middle and entrance) were selected which were spaced 80-90 m apart. These locations show higher disturbance (pollution) at the inner part of the marina and a lower disturbance at the entrance due to higher water exchanges with the outer environment.

Study species

To assess the survival of ascidian recruits, we compared a native species and a NIS, being among the most abundant ascidian species in the whole marina. The native Ciona intestinalis (Linnaeus, 1767) is the dominant species in most marinas in Brittany (NW France), including the present one [START_REF] Bouchemousse | Contrasting global genetic patterns in two biologically similar, widespread and invasive Ciona species (Tunicata, Ascidiacea)[END_REF]). This species is characterized by rapid growth, a high reproduction rate and a short life cycle, proprieties that otherwise make it highly invasive in many parts of the world [START_REF] Jackson | A sea squirt (Ciona intestinalis)[END_REF]. The clubbed tunicate Styela clava (Herdman, 1881) was introduced from the NE Pacific into European waters, and was first recorded in Plymouth, UK in 1953. Since then, it has spread, constituting a potential pest on oyster and mussel farms [START_REF] Carlisle | Styela mammiculata n.sp., a new species of ascidian from the plymouth area[END_REF][START_REF] Davis | The impact of the ascidian Styela clava Herdman on shellfish farming in the Bassin de Thau, France[END_REF]. Both tunicate species have convergent characteristics, being highly invasive in their respective areas of introduction, especially in disturbed ecosystems such as harbors and marinas [START_REF] Jackson | A sea squirt (Ciona intestinalis)[END_REF][START_REF] Therriault | A qualitative biological risk assessment for vase tunicate Ciona intestinalis in Canadian waters: using expert knowledge[END_REF][START_REF] Davis | The impact of the ascidian Styela clava Herdman on shellfish farming in the Bassin de Thau, France[END_REF].

In situ assessment of recruit survival Large adult individuals (>15cm) of both species were sampled in spring from the middle of the marina.

Due to slower growth of S. clava, this species was sampled one month before C. intestinalis. Both species were transferred into aquaria facilities with circulating sea water. After stabilization and fattening for two weeks to improve gamete production and quality, ca. 30 individuals of each species were randomly selected for reproduction. Individuals of S. clava were separated into two pools to cross-fertilize and dissected by cutting the stipe at the base and opening the branchial sac, revealing the male and female reproductive apparatus. To avoid self-fertilization, oocytes and sperm were separately extracted with a thin pipette. Oocytes were rinsed and isolated from tissue debris on an 80 µm mesh filter with filtered (1.3 µm) seawater. On a Petri dish, oocytes from two individuals (to maintain some variability between recruits) were distributed. Sperm was assigned to a sperm pool so as to avoid fertilizing oocytes from the same individual and mixed with filtered seawater. One pool of sperm cells was introduced into Petri dishes containing the oocytes from different individuals.

Similarly, C. intestinalis individuals were dissected to harvest gametes. Oocytes were collected by concentrating them in the narrow part of the oviduct and puncturing them with a glass pipette.

Oocytes from different individuals were separated as described above for S. clava. Sperm was sampled similarly to the oocytes, by concentrating the mass in one side of the sacs and puncturing them with a pipette. As described for S. clava, C. intestinalis individuals were separated into different genitor pools and mixed accordingly. For each species, 40 petri dishes with fertilized gametes were created. Cell divisions started at 1 h post fertilization, and larvae appeared after 24 h (seawater at constant 18°C).

Recruits of both species were fed with a mix of Isochrysis affinis galbana Tahiti (T-iso RCC 1349) and Chaetoceros calcitrans ('Argenton') strains cultured at the Roscoff Culture Collection (RCC, http://roscoffculture-collection.org/). They were maintained for 15 days in the dark in a temperatureregulated environment (18°C). After this period, the Petri dishes were transferred into temperatureregulated aquaria that were brought to match the varying outside water temperature of approx. 19°C progressively over one week.

The number of C. intestinalis or S. clava individuals successfully attached on each Petri dish was counted and the position of the recruits was marked on the underside of each dish. We selected the 30 dishes with the highest abundance of recruits for each species. If the number of individuals exceeded 50 on a given Petri dish, we reduced it to this threshold to avoid recruits overcrowding. Later, all dishes were individually fixed on 20 cm × 20 cm black PET panels to provide shade and were photographed (Olympus Tough TG5). For each location in the marina, 10 such panels were randomly selected for each species. Half of them were caged with plastic coated iron wire mesh with a 10 mm mesh size to exclude macro-predators, mostly fish [START_REF] Dumont | Predation contributes to invasion resistance of benthic communities against the non-indigenous tunicate Ciona intestinalis[END_REF][START_REF] Giachetti | Macropredators as shapers of invaded fouling communities in a cold temperate port[END_REF]. Micropredators and meso-predators such as small crustacean and gastropods would still have access to caged treatments. The 20 randomly chosen panels (5 caged and 5 uncaged for each species) were suspended under the pontoon at each of the three locations (~1 m depth; 25 June 2019). After 20 and 28 days, cages and panels (but not the dishes) were cleaned to limit the effects of fouling on the recruits. Dishes were photographed to count the remaining individuals. Although fouling did occur on dishes, individuals stayed visible and could be identified on the dish and by their significantly larger size compared to natural recruits of the same species. The experiment ended 50 days after deployment. Dishes were recovered and individuals were counted. At this stage, heavy fouling on the dishes, especially due to marina-recruited C. intestinalis, made it impossible to analyze photos for both in vitro recruited species. Thus, all S. clava individuals were manually counted and collected for drying (1 week at 60°C) and weighing. All S. clava individuals present at the end of the experiment were labgrown, as shown by their position on the black dots. While at 28 days after deployment of the recruits it was easy to discriminate lab-grown recruits of C. intestinalis from marina-recruited ones due the size difference, it was not possible anymore at 50 days after deployment, especially considering that labgrown recruits' mortality was high -and likely even 100%. We thus chose to consider the survival observations for this species after 28 days rather than at 50 days to avoid a potential underestimation of survival. Statistical analyses were conducted taking this into account. Natural C. intestinalis recruits were nevertheless used to compare their biomass between treatments. Given that the biomass of natural recruits is linked to their survival, this provides additional information on C. intestinalis survival between treatments. The mass of potentially remaining lab-grown recruits can be neglected here due to their very low contribution to the total mass. For each dish, all recruits were collected and dried (1 week at 60°C) for weighing.

Feeding assay

In addition to the exclusion cages, predation intensity was directly measured with a feeding assay. It was developed to measure fish predation intensity in different environments and consists of 25 dried squid baits tied on fishing line, attached to fiberglass stakes on a 25 m long transect [START_REF] Duffy | Squidpops: A Simple Tool to Crowdsource a Global Map of Marine Predation Intensity[END_REF].

Squid, due to its firm consistence is particularly adapted to long feeding experiments since it does not detach without a strong attack from a larger predator and can only be detached as one piece. This assay has been used in various environments, including artificial habitats [START_REF] Duffy | Squidpops: A Simple Tool to Crowdsource a Global Map of Marine Predation Intensity[END_REF][START_REF] Rodemann | Consumption pressure in coastal marine environments decreases with latitude and in artificial vs. natural habitats[END_REF]. Here, a modified version of this assay was used and 25 x 3 locations x 3 dates = 225 baits were deployed. A snorkeler counted the remaining baits 1 h, 3 h, 6 h and 24 h after deployment to use survival analysis rather than comparisons of means, thus providing more detail on the bait consumption [START_REF] Gauff | Influence of predation risk on the sheltering behaviour of the coral-dwelling damselfish, Pomacentrus moluccensis[END_REF]). The bait was not deployed on the seafloor, because the height of the water column varied under each pontoon, but was suspended 1 m below the pontoons, close to the ascidian recruits. The assay was performed 17, 24 and 31 days after the deployment of ascidian recruits at the inner, middle and entrance locations respectively, with one transect per location and date.

Surveillance cameras (2 per transect, Gopro HD4) were installed. During the 60 h of footage, no fish feeding on the bait were recorded.

Contaminant assessment

Three sediment samples (~ 0.4kg) were taken by divers at each of the locations for testing the concentration of Persistent Organic Pollutants (POP) including Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and the most frequent pesticides. Additionally, for each location, five sediment samples (~ 0.4kg) were taken to quantify Metallic Trace Elements (MTE).

The analytical method for PCBs and pesticides is further detailed in our supplementary material and is fully described by [START_REF] Wafo | Polychlorinated biphenyls and DDT residues distribution in sediments of Cortiou (Marseille, France)[END_REF]. PCB determination focused on 33 individual congeners (Sup. 1) including target congeners proposed by the International Council for the Exploration of the Sea (ICES) as indicators of PCB contamination, complemented by congeners with high environmental prevalence [START_REF] Webster | Determination of polychlorinated biphenyls (PCBs) in sediment and biota[END_REF]. The list of the 16 quantified pesticides can be seen in Tab. 1. PAHs were determined following a previously described method [START_REF] Sarrazin | Determination of polycyclic aromatic hydrocarbons (PAHs) in marine, brackish, and river sediments by HPLC, following ultrasonic extraction[END_REF][START_REF] Ratier | Characterization of atmospheric emission sources in lichen from metal and organic contaminant patterns[END_REF][START_REF] Dron | Contaminant signatures and stable isotope values qualify European conger (Conger conger) as a pertinent bioindicator to identify marine contaminant sources and pathways[END_REF]) that is also further detailed in the supplementary material. We focused on 16 PAH congeners defined by the US Environmental Protection Agency (USEPA) priority list (Tab. 1, US EPA 2014). Each targeted PAH was identified based on the retention time and the mass spectrum from the chromatogram of standard solutions acquired in full scan mode. Quantification was then performed in the SIM mode for better selectivity.

For the quantification of Metallic Trace Elements (MTE) the sediment samples were dissolved in a three-acid solution (HCL, HN03, HF) and were then analyzed using High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS; [START_REF] Jacquet | Characterization of the submarine disposal of a Bayer effluent (Gardanne alumina plant, southern France): I. Size distribution, chemical composition and settling rate of particles forming at the outfall[END_REF]. The spectrometer was calibrated via an external calibration method adding In as an internal standard.

Statistical methods

Mean value and standard deviation were calculated for each contaminant. Differences between locations were identified via a Kruskal-Wallis test (R, CRAN, version 3.6.1). The survival data resulting from the feeding assay and collected from the ascidian recruits was analyzed using the 'survival' package (version 2.41-3, Therneau and Lumley 2017). The Kaplan-Meier curves for each treatment were established and compared using a log-rank pairwise comparisons with Bonferroni correction to avoid false positives due to multiple tests [START_REF] Pyke | Statistical Analysis of Survival and Removal Rate Experiments[END_REF][START_REF] Bretz | Invasive plants versus their new and old neighbors: A mechanism for exotic invasion[END_REF]). The dry mass per S. clava individual as well as the log of the total dry mass of C. intestinalis were compared between caged and uncaged treatments with a Wilcoxon test, and individual differences of dry mass between locations were identified using a Kruskal-Wallis multiple comparison test with the 'pgirmess' package (version 1.6.2, Giraudoux et al., 2018). Since sediment samples (contaminants), feeding experiments (predation rate) and the survival rates of ascidian recruits could not be paired unambiguously, we chose to use R to randomly attribute samples according to the locations they are linked to. The generated table was submitted to a nested cox regression from the 'nested cohort' package in R (version 1.3, Katki and Mark 2013). The two models, one for each species of ascidians, tested for the effect of all contaminants for which significative differences between locations were previously identified, the effect of caging and predation intensity (squid bait consumption after 24h) as well as their interaction, on the survival of ascidian recruits. The interaction term here is required since predation cannot apply to caged individuals. Both models were nested within the petri dishes which contained several ascidian recruits and location, as well as their interaction term. This process of random pairing and modulization was repeated 10⁵ times and p values and coefficients were saved in a separate table. The percentage of times p < 0.05 as well as the mean coefficient and mean hazard ratio were calculated for each model factor. We considered factors with above 25% of significative pvalues as factors of interest and factors with above 50% as factors with a clear link to survival. To further investigate the effect of the interaction term between predation risk and caging on the recruits, linear models were established testing for an effect of predation intensity on the percentage of surviving recruits at the end of the experiment in both, caged and uncaged treatments.

Results

Recruit survival

The Kaplan-Meier survival curve of the recruits showed mortality for both species (Fig. 1). Mortality for S. clava ranged from 15 to 30% within 50 days and from 60 to 100% for C. intestinalis within 28 days. A log-rank multiple comparison showed that survival for C. intestinalis (blue lines, Fig. 1) was consistently lower than for S. clava (red/orange lines, Fig. 1; p < 0.001). Location had a significant effect on the survival of caged C. intestinalis. Survival was the highest at the inner part, intermediate at the middle and the lowest at the entrance of the marina (p < 0.001). Conversely, location in the marina had no significant influence on survival of caged S. clava. Caging had a significant influence on C. intestinalis, increasing the survival at the inner and the middle part of the marina compared with the uncaged treatment (p < 0.01). At the marina entrance, mortality was 100% for the caged and uncaged treatments for this species. No significant caging effect was observed in the log-rank test for S. clava at any location.

Dry mass

After 50 days, the total dry mass of natural C. intestinalis recruits and the dry mass per lab-grown S. clava individual varied between treatments (Fig. 2). The Wilcoxon tests revealed a significant caging effect for both species, but with opposite significant effects on species' biomass: higher biomass for C. intestinalis in caged treatments than in uncaged treatments and lower biomass for S. clava in caged treatments than in uncaged treatments (p < 0.05 and p < 0.01, respectively; Fig 2). Differences in biomass according to site were only identified for uncaged C. intestinalis between the inner and middle locations (Kruskal-Wallis multiple comparison; adj. p < 0.05).

Contaminants

All tested PAHs concentrated in the sediments of the studied marina exceeded Canadian sediment quality guidelines and as well as concentrations at which 20% of sediments tested by the US Environmental Protection Agency would become toxic to model amphipods (Tab. 1;[START_REF] Ccme | Protocol for the derivation of canadian sediment quality guidelines for the protection of aquatic life[END_REF]US EPA 2005). Chrysene, phenanthrene, fluoranthene and fluorene largely exceeded values at which adverse effect on fauna are highly likely (Tab. 1). Total PCBs as well as two pesticides, lindane and pp'-DDD, also exceeded most guidelines used for comparison. Two PAHs, tPCB as well as seven pesticides had significant differences in concentration between locations (Kruskal-Wallis, p < 0.05; Tab. 1). Almost all MTE were distributed as a gradient from the inner location (max.) to the entrance location (min.). Cu, Pb and Zn showed significant differences between locations (Kruskal-Wallis, p < 0.05). Cu and Zn concentrations in sediment were significantly higher at the inner location compared to the entrance location (Kruskal-Wallis multiple comparisons, adj. p < 0.05). Most of the MTE concentrations were barely above the Canadian sediment quality guideline and above the concentration of 20% probability of toxicity, falling within a sediment quality category of good or moderate for Cu and Pb (Tab. 2; CCME 1999; US EPA 2005; Guerra-García et al. 2021).

Feeding assay

A total of 42 out of 225 baits (18.67%) were consumed in the experiment. Predation intensity was relatively low at all locations, but varied in space, ranging from 10% and 16% at inner and entrance location to 30% (middle location) consumption after 24 h. Using survival analysis on the baits, a logrank comparison showed a significant difference (p < 0.01) between the middle and the inner location.

The entrance location had an intermediate predation intensity not significatively different than either of the two other sites.

Statistical models

The results of the 10⁵ iterations of Cox models after random pairing of samples from the same location showed that while none of the tested contaminants had a clear link with survival, caging and predation intensity had an influence on C. intestinalis. Caging reduced the mortality risk by 22% for C. intestinalis while it increased it by 33% for S. clava. While predation intensity explained only a small fraction of the survival of C. intestinalis, the interaction between predation intensity and caging also seemed to better explain its survival. Neither in the caged nor in the uncaged treatment, linear models showed a significant effect of predation intensity on the percentage of surviving recruits.

Discussion

Our experiments yielded insights on the survival dynamics of the two species during their juvenile phase. They showed that the native C. intestinalis was strongly affected by environmental heterogeneity and predation (significant differences according to location variability and caged/uncaged treatments, respectively), but that the alien S. clava was less affected by either factor.

The survival of C. intestinalis varied significantly among locations within the marina, following an increasing gradient from the entrance to the inner-most part. Interestingly, the survival gradient was organized in the opposite direction of our prediction, based on a gradient of anthropogenic disturbance which should be maximal at the inner location of the marina. Metallic Trace Elements in sediments were distributed along this supposed gradient with maximal values in the innermost part of the marina, which is particularly visible for Cu, Pb and Zn. Our reasoning behind the hypothetic anthropogenic disturbance gradient was based on a sampling of those three MTE in 2016 (confirmed in the present), where copper, commonly used in anti-fouling painting on boats, has been shown to have significant effects on the community structure in this marina (Kenworthy et al. 2018b). In the present study, Benzo(g,h,i)perylene, Fluorene and total PCBs showed significant differences in concentrations between locations, but not along the supposed disturbance gradient. They had minimal values in the middle part of the marina. Additionally, seven pesticides had significant differences between locations with varying distribution profiles. It is notable that the studied marina sediment largely exceeds north American standards of sediment quality for Chrysene, Fluorene, Fluoranthene and Phenanthrene (PAH) as well as for total PCBs, with a 50% probability of toxicity on model amphipods if they were exposed to the sediment [START_REF] Ccme | Protocol for the derivation of canadian sediment quality guidelines for the protection of aquatic life[END_REF]US EPA 2005). On the contrary MTE pollution, even if above some of these sediment quality guidelines, falls within quality categories described as good or moderate for Cu and Zn as proposed in [START_REF] Guerra-García | Assessing environmental pollution levels in marinas[END_REF]. Despite the significant effect of location variability on C. intestinalis as shown by the Log-Rank test, none of the contaminants varying significantly between locations could explain the survival profile of this species in the Cox models. This might indicate that another, not measured factor, varied between locations and influenced the survival of this species. Mesopredators or micropredators like nudibranchs and caprellids, which have been shown to have strong influence on benthic communities, would still have access to the inside of the cages and could thus affect recruit survival independently of caging [START_REF] Osman | Predation on early ontogenetic life stages and its effect on recruitment into a marine epifaunal community[END_REF][START_REF] Lavender | Meso-predators: A confounding variable in consumer exclusion studies[END_REF][START_REF] Leclerc | Experimental and survey-based evidences for effective biotic resistance by predators in ports[END_REF]. If these mesopredators are impacted by anthropogenic disturbance, their abundance could vary in space and be maximal at the entrance of the marina, which would explain the observed survival pattern of C. intestinalis recruits. Unmeasured abiotic factors might also be at play. Factors like temperature, salinity and hydrodynamics, modulated by the artificial structures could impact the survival of the recruits [START_REF] Walters | Settlement and early post-settlement survival of sessile marine invertebrates on topographically complex surfaces: The importance of refuge dimensions and adult morphology[END_REF]Kenworthy et al. 2018a). To decide between these variables, future experiments should integrate a way of identifying the effect of smaller predators on survival and integrate more environmental factors [START_REF] Lavender | Meso-predators: A confounding variable in consumer exclusion studies[END_REF]. The systematically higher survival of S. clava suggests that this species is more adapted to the general marina environment and the factors impacting the survival of C. intestinalis.

However, its abundance in marinas is much lower than that of C. intestinalis, indicating this survival advantage may only occur at the recruit stage or indicate that C. intestinalis dominates due to its high fecundity and growth rate and not survival [START_REF] Jackson | A sea squirt (Ciona intestinalis)[END_REF]). The higher survival of S. clava in its recruit stage supports the idea that NIS are more resistant to abiotic stress and is consistent with studies indicating that NIS in marina environments show higher abundance in anti-fouling polluted environments [START_REF] Dafforn | Differential effects of tributyltin and copper antifoulants on recruitment of non-indigenous species[END_REF][START_REF] Piola | Pollution reduces native diversity and increases invader dominance in marine hard-substrate communities[END_REF][START_REF] Piola | The influence of antifouling practices on marine invasions[END_REF]).

In addition to the location, caging also seemed to strongly affect the survival of C. intestinalis recruits, their survival increased when they were caged. This result is shown in the Log-Rank as well as in the Cox models with an associated 22% mortality risk decrease. At the entrance location, this effect was however not observable in the Log-Rank test due to the high mortality in both caged and uncaged treatments. This probably also leads to an underestimation of the risk decrease in the Cox model. Predation intensity estimated by the feeding experiment varied in space with maximal predation pressure in the middle of the marina and a minimal pressure at the inner part of the marina. This predation intensity might be linked to the survival or C. intestinalis recruits in the Cox models since it slightly reduces their survival in 25% of iterations. The influence of the interaction of caging and predation intensity is due to the fact that predation could not apply on caged individuals. While not being able to demonstrate it, we propose that predation intensity acts negatively on the survival of C. intestinalis in uncaged treatments, while it is neutral for caged individuals. This interaction, however, further demonstrates that the increased survival in caged treatments may at least partly due to a protection against the predators partaking in the feeding experiment. As previously highlighted, the squid-bait feeding experiment targets almost exclusively fish [START_REF] Duffy | Squidpops: A Simple Tool to Crowdsource a Global Map of Marine Predation Intensity[END_REF]. However, we cannot exclude the possibility that native velvet swimming crabs Necora puber (Linnaeus, 1767) also partook in the feeding assay and the predation on C. intestinalis recruits. Everything considered, we suggest that a significant part of predation on ascidians may thus be exerted by highly mobile generalist predators. The surveillance cameras installed around the feeding assay did not record any direct attacks on baits, but camera surveillance observed the black seabream Spondyliosoma cantharus (Linnaeus, 1758) feeding on fouling communities close to the baits. This species is a plausible predator on ascidian recruits, as Sparidae can significantly influence marina communities, and also feed on squid bait (Oricchio et al. 2016b;[START_REF] Rodemann | Consumption pressure in coastal marine environments decreases with latitude and in artificial vs. natural habitats[END_REF]. Studies frequently demonstrated that C. intestinalis and its congener Ciona robusta (Hoshino & Tokioka, 1967) are vulnerable to predation in their introduced ranges, highlighting that predators may participate in biotic resistance against them in the areas that they attempt to invade [START_REF] Dumont | Predation contributes to invasion resistance of benthic communities against the non-indigenous tunicate Ciona intestinalis[END_REF][START_REF] Astudillo | Seasonal heterogeneity provides a niche opportunity for ascidian invasion in subtropical marine communities[END_REF][START_REF] Leclerc | Experimental and survey-based evidences for effective biotic resistance by predators in ports[END_REF][START_REF] Giachetti | Invasive ascidians: How predators reduce their dominance in artificial structures in cold temperate areas[END_REF]. Our results are consistent with observations of the predation on Ciona spp., although in the present case, C. intestinalis was studied in its native range. The most abundant solitary non-indigenous ascidian in the Brest marina, S. clava, was not affected by predation intensity and had a higher mortality risk if caged. Considering the invasive status of S. clava, biotic resistance -as exerted by predators -does not seem to occur for the S. clava/C. intestinalis pair, thus making the Biotic Resistance Hypothesis unlikely in this study. Styela clava, especially adults are an unappealing food item for predators due to their tough tunic [START_REF] Clarke | Biological Synopsis of the Invasive Tunicate Styela clava ( Herdman 1881 ) Canadian Manuscript Report of Fisheries and Aquatic Sciences[END_REF]). Moreover, its congener Styela plicata (Lesueur, 1823) has been shown to be unpalatable to fish due the accumulation of chemically deterrent secondary metabolites in their gonads [START_REF] Pisut | Anti-predatory chemical defenses of ascidians: Secondary metabolites or inorganic acids?[END_REF][START_REF] Koplovitz | An evaluation of chemical and physical defenses against fish predation in a suite of seagrass-associated ascidians[END_REF]. We suggest that this may also be the case for S. clava as adults or recruits. On the other hand, C. intestinalis has no chemical deterrents with regard to palatability and is readily consumed by crabs and fishes [START_REF] Teo | Toxicity and palatability of some British ascidians[END_REF][START_REF] Koplovitz | An evaluation of chemical and physical defenses against fish predation in a suite of seagrass-associated ascidians[END_REF]. Our results thus may provide support for the Novel Weapons Hypothesis (NWH; [START_REF] Hay | Synergisms in Plant Defenses against Herbivores: Interactions of Chemistry, Calcification, and Plant Quality[END_REF][START_REF] Cappuccino | Invasive exotic plants suffer less herbivory than non-invasive exotic plants[END_REF]. The non-indigenous S. clava may possess a strong predator avoidance system, whereas the native C. intestinalis may be comparatively heavily affected by predation.

The total dry mass of naturally recruited C. intestinalis was significantly lower in uncaged treatments than in caged ones. This difference in dry mass was either due to lower abundance or smaller individual size since predation would cause mortality before recruits could grow in uncaged treatments. In both cases, this difference can be attributed to higher mortality due to predation. Interestingly, no natural S. clava recruits were observed in this study. Having strong differences in reproductive season according to locality, this species seems to reproduce and recruit in late summer/autumn, explaining the absence of natural recruits [START_REF] Clarke | Biological Synopsis of the Invasive Tunicate Styela clava ( Herdman 1881 ) Canadian Manuscript Report of Fisheries and Aquatic Sciences[END_REF]. For S. clava, the individual dry mass was significantly higher in uncaged treatments than in caged treatments, suggesting a negative effect of caging on S. clava. This result is further supported by the Cox model indicating a 33% increased risk of mortality when caged. This may appear intriguing because predators cannot directly have a positive influence on S. clava. One explanation might be that the cage acted as a refuge for meso-and micropredators, which could feed on ascidians, here especially S. clava, as devoid of predation by macropredators [START_REF] Lavender | Meso-predators: A confounding variable in consumer exclusion studies[END_REF]. However, such an effect of mesopredation would uniquely affect survival and not the mean individual mass as it has been affected in our experiment. Furthermore, a previous study conducted in the same marina has shown no effect of caging (protection from large predators) on small mobile fauna (mesopredators) assemblage and abundance [START_REF] Leclerc | Habitat formation prevails over predation in influencing fouling communities[END_REF]. It thus seems unlikely that the cages acted as refuge for mesopredators and that they may be responsible for the negative effect of caging on S. clava. Another mechanism seems more likely to explain the observed results. When observing photos of the S. clava dishes, it is striking that uncaged S. clava were prominent, while they were smothered by naturally recruited C. intestinalis in caged treatments. We thus think that an indirect positive interaction might be induced by the predation on natural C. intestinalis recruits in uncaged S. clava panels, leading to a decrease in the spatial and trophic competition exerted on S. clava by C. intestinalis (Fig. 3). Considering this competition it seems to indicate that, in our study case, generalist predators facilitate the NIS S. clava through competitive release, a specific mechanism of the Enemy Release Hypothesis (ERH; [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF]. This hypothesis could be confirmed by conducting a similar experiment to ours, but mixing both species' recruits on the dishes from the beginning.

In the present study, we showed that the survival of the native C. intestinalis was highly influenced by both location and predation, itself variable in space. To date, the very few studies that have focused on the very small-scale spatial variability of communities in marina environments have not considered the possibility of spatially varying predation. For the studied ascidians, we observed a preference of predators for the native prey similar to [START_REF] Cuthbert | Resistance is futile: Lack of predator switching and a preference for native prey predict the success of an invasive prey species[END_REF] and [START_REF] Kincaid | Predators Associated with Marinas Consume Indigenous over Non-indigenous Ascidians[END_REF]. The present results support the idea that generalist predation may play a crucial role in the success of NIS due to facilitation through competitive release [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF][START_REF] Kincaid | Predators Associated with Marinas Consume Indigenous over Non-indigenous Ascidians[END_REF].

The results also provide a new rationale for how generalist predators may contribute to biotic resistance. In contrast to the many involving Ciona spp. as a non-indigenous model concluding that various predators exert biotic resistance [START_REF] Dumont | Predation contributes to invasion resistance of benthic communities against the non-indigenous tunicate Ciona intestinalis[END_REF][START_REF] Leclerc | Experimental and survey-based evidences for effective biotic resistance by predators in ports[END_REF][START_REF] Giachetti | Invasive ascidians: How predators reduce their dominance in artificial structures in cold temperate areas[END_REF], we showed that in the native range of C. intestinalis, predation exerted by mobile generalist predators potentially leads to NIS facilitation. Thus, the issue of biotic resistance may depend more on the identity and characteristics of the considered native species, NIS and predator, rather than on the studied species status (NIS vs native; [START_REF] Skein | Characteristics of native predators are more important than those of alien prey in determining the success of biotic resistance in marine systems[END_REF]. Predictions on whether a NIS encounters resistance or facilitation by local predators in its introduced range could thus be formulated by looking at how various types of predation affect it in its native environment. 14,11 ± 0,83 2,66 ± 0,47 13,18 ± 2,09 heptachlor 1,24 ± 0,2 0,72 ± 0,14 0,88 ± 0,3 0,6 2,74 heptachlor_epoxide_a 40,21 ± 4,65 21,44 ± 5,66 18,96 ± 1,66 heptachlor_epoxide_b 3,48 ± 0,29 2,76 ± 0,27 2,81 ± 0,05 isodrin 0,6 ± 0,16 0,21 ± 0,03 0,28 ± 0,07 lindane 2,03 ± 0,24 1,81 ± 0,32 1,94 ± 0,23 0,32 0,99 methoxychlor 7,43 ± 1,28 5,19 ± 0,21 2,52 ± 0,22 * (in %)

US EPA

Polychlorinated

Ciona intestinalis Styela clava

Tab. 3: Mean Coefficient, mean risk increase (Mean Hazard Ratio -1) and percentage of significant pvalues after 10⁵ iterations of random sample linking followed by a Cox model (nested within petri dish, site and their interaction) for each species, integrating all factors indicated in the first column. Black font: factor of interest; Gray background: link between survival and factor.

Tab. 1 :

 1 POP contaminants in the sediments of the three study locations. Mean values (µg/kg = ppb) with standard deviation. Differences between locations were tested via a Kruskal-Wallis test followed by a Kruskal-Wallis multiple comparison post-hoc. Reference values given by the Canadian Council of Ministers of the Environment (CCME 1999; ISQG: Interim Sediment Quality Guideline; PEL: Probable Effect Level); and the United Stated Environmental Protection Agency (US EPA 2005, Tab. 11; T20: probability of 20% of toxicity among samples; T50: probability of 50% of toxicity among samples). Gray text for concentrations: NS differences between locations (not integrated into COX models). Gray text for reference values: threshold not exceeded. Black text and gray background for reference values: threshold exceeded.

Fig. 1 :

 1 Fig. 1: Kaplan-Meier survival curves for Ciona intestinalis (A) and Styela clava (B) recruits. Inner = Inner marina location; Mid. = middle location; Entr. = entrance location of the marina. Dashed lines indicate uncaged (U) treatments; solid lines indicate caged (C) treatments. Ciona Entrance C and Ciona Entrance U overlap. Styela Inner U and Styela Mid U overlap.

Fig. 2 :

 2 Fig. 2: Log of total dry mass of naturally recruited Ciona intestinalis and dry mass per lab grown Styela clava individual. Significant differences between caged and uncaged treatments: * p < 0.05 and ** p < 0.01 (Wilcoxon test). Significant differences within the uncaged C. intestinalis (a-b) p < 0.05 (Kruskal-Wallis multiple comparison).

Fig. 3 :

 3 Fig. 3: Caged and uncaged Petri dish of Styela clava recruits after 50 days in the field. Panels originating from the inner location of the marina. Natural recruits of C. intestinalis are more visible (larger, more abundant) in the caged treatment. Individual number refers to final S. clava.

Acknowledgements

This work benefited from access to the Station Biologique de Roscoff, an EMBRC-France and EMBRC-ERIC Site. We warmly thank the Centre de Ressources Biologiques Marines (CRBM) at the Station Biologique de Roscoff for its technical support and the reproduction of ascidian recruits. We also want to thank Bastien Taormina and Elyne Dugény for their support during the feeding experiment and Noémie Coulon and Brian Sevin for helping to count surviving recruits. We thank Carolyn Engel-Gautier for English proofreading and Stéphanie Jacquet for analysing MTE contaminants in sediment samples.

We also want to thank the reviewers of this article for their valuable contributions.

Declarations

Funding Funding for this project has been provided through a PhD grant from the Sorbonne Université -Museum National d'Histoire Naturelle (Ecole Doctorale 227) and a field mission grant from the Société

Data accessibility

All data tables generated or analyzed during this study are included in this published article and its supplementary material files. Photos analyzed shall be shared upon request (corresponding author).

Code availability

Code for the program 'R' can be requested upon the corresponding author.

Compliance with Ethical Standards

We followed national and international ethical guidelines whenever applicable.

Conflicts of interest

All authors declare that they have no potential conflicts of interest with any organization or entity with financial or non-financial interest in the subject discussed in this manuscript.