Jean-Charles Leclerc
email: leclercjc@gmail.com

Frédérique Viard

Elizabeth González Sepúlveda

Christian Díaz

José Neira Hinojosa

Karla Pérez Araneda

Francisco Silva

Antonio Brante

Aulacomya Atra Native

Semimytilus Algosus Native

Mytilus Cf Edulis Cryptogenic

Crustacea Austromegabalanus Psittacus Native

Balanus Laevis Native

Verruca Laevigata Native

Ciona Robusta Nis

Corella Eumyota Cryptogenic

Didemnidae

Paramolgula Sp Native

Pyura Chilensis Native

Egg Masses Unassigned

Non-indigenous species contribute equally to biofouling communities in international vs local ports in the Biobío region, Chile

Keywords: Propagule pressure, non-indigenous species, cryptogenic species, maritime traffic, predators, diversity Annelida Terebellidae/Spionidae (Tubes) Unassigned Bivalvia Unassigned Crepipatella fecunda Native Asterocarpa humilis NIS

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

Introduction

Biological introductions, characterized by sudden changes of the natural distribution of species due to human-mediated transports, are among the most pervasive global changes [START_REF] Simberloff | Impacts of biological invasions: what's what and the way forward[END_REF]. Human-mediated transport is indeed breaking biogeographic boundaries and contribute to the homogenization of ecosystems at global scale [START_REF] Villéger | Homogenization patterns of the world's freshwater fish faunas[END_REF][START_REF] Capinha | The dispersal of alien species redefines biogeography in the Anthropocene[END_REF]. In the marine realm, most of the attempts to eradicate established NIS have failed [START_REF] Ojaveer | Ten recommendations for advancing the assessment and management of non-indigenous species in marine ecosystems[END_REF][START_REF] Ojaveer | Classification of non-indigenous species based on their impacts: considerations for application in marine management[END_REF]. This poor success in management and eradication is likely due to a combination of factors, including notably late detection, high dispersal abilities of many NIS and a large diversity of spreading vectors [START_REF] Bax | Marine invasive alien species: a threat to global biodiversity[END_REF].

Regarding the need for early detection, surveillance should target with a high priority the points of entry of NIS, in particular ports and aquaculture facilities in the marine realm.

Shipping and leisure boating (ballast water and sediment, hull biofouling) as well as aquaculture are indeed the most critical contributors to invasion in coastal ecosystems at a global scale [START_REF] Drake | Hull fouling is a risk factor for intercontinental species exchange in aquatic ecosystems[END_REF][START_REF] Molnar | Assessing the global threat of invasive species to marine biodiversity[END_REF][START_REF] Murray | Recreational boating: a large unregulated vector transporting marine invasive species[END_REF][START_REF] Sylvester | Hull fouling as an invasion vector: can simple models explain a complex problem?[END_REF][START_REF] Nunes | Gateways to alien invasions in the European seas[END_REF]. Current international shipping hauls almost 90 % of globally traded goods and shipping intensity is consistently increasing [START_REF] Kaluza | The complex network of global cargo ship movements[END_REF][START_REF] Tournadre | Anthropogenic pressure on the open ocean: The growth of ship traffic revealed by altimeter data analysis[END_REF]. Tthe longstanding recognition of the importance of ballast water and associated sediments in NIS (including harmful organisms and pathogens) transportation has led to the International Convention for the Control and Management of Ships' Ballast Water and Sediments (BWM Convention) which entered into force on September 2017. In contrast,, no international regulation regarding hull fouling organisms -such as Craft Risk Management Standard for Biofouling in New Zealand (http://www.mpi.govt.nz/) or Biofouling Management Plan in California (http://www.slc.ca.gov/) -has been set up yet. However, in 2011, the IMO adopted Guidelines for the Control and Management of Ships' Biofouling [START_REF] Mepc | Guidelines for the control and management of ships' biofouling to minimize the transfer of invasive aquatic species[END_REF][START_REF] Mepc | Report of the Marine Environment Protection Committee on its 66th session[END_REF] and is currently preparing a new project (GloFouling) to tackle this major issue. Submerged ship hulls can indeed be colonized by diverse sessile and encrusting organisms that may dislodge or reproduce in the destination port-of-call during the ship stopover. Besides, many of these sessile species are habitat-builders for a myriad of invertebrates [START_REF] Sellheim | Effects of a nonnative habitat-forming species on mobile and sessile epifaunal communities[END_REF][START_REF] Thomsen | Impacts of marine invaders on biodiversity depend on trophic position and functional similarity[END_REF][START_REF] Leclerc | Habitat formation prevails over predation in influencing fouling communities[END_REF]. As such, hull biofouling is a major pathway of introduction and spread of NIS, through both direct and facilitative processes [START_REF] Bax | Marine invasive alien species: a threat to global biodiversity[END_REF][START_REF] Drake | Hull fouling is a risk factor for intercontinental species exchange in aquatic ecosystems[END_REF][START_REF] Sylvester | Hull fouling as an invasion vector: can simple models explain a complex problem?[END_REF][START_REF] Moser | Quantifying the total wetted surface area of the world fleet: a first step in determining the potential extent of ships' biofouling [journal article[END_REF][START_REF] Pinochet | Presence of the tunicate Asterocarpa humilis on ship hulls and aquaculture facilities in the coast of the Biobío Region, south central Chile[END_REF].

Following their primary introduction, the rapid spread of marine NIS is favored by coastal hardening [START_REF] Mineur | Changing coasts: marine aliens and artificial structures[END_REF][START_REF] Airoldi | Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale[END_REF]. The increasing rate of coastal artificial structures, coined 'ocean sprawl' (sensu [START_REF] Duarte | Is global ocean sprawl a cause of jellyfish blooms?[END_REF], has emerged as protection against environmental perturbations (rising sea level, erosion, storms and flooding) and to support the development of human activities (shipping, urbanization, aquaculture, energy extraction and recreation) [START_REF] Firth | Ocean sprawl: challenges and opportunities for biodiversity management in a changing world[END_REF]. Beyond habitat-degradation and fragmentation, coastal artificial structures provide novel habitats for colonization by various species -among which NIS prevail as compared to neighboring natural reefs -and alter connectivity and local species pools [START_REF] Mineur | Changing coasts: marine aliens and artificial structures[END_REF][START_REF] Dafforn | Marine urbanization: an ecological framework for designing multifunctional artificial structures[END_REF][START_REF] Bishop | Effects of ocean sprawl on ecological connectivity: impacts and solutions[END_REF]. Interestingly ocean sprawls with shipping and boating are jointly enhancing colonization and propagule pressures (sensu [START_REF] Lockwood | The more you introduce the more you get: the role of colonization pressure and propagule pressure in invasion ecology[END_REF]): besides, being points of entry, ports and marinas are "invasion hubs" [START_REF] Airoldi | Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale[END_REF]. NIS establishment, spread and impacts are however context dependent, relying on a complex combination of invader traits, resource availability, abiotic conditions, and community structure of the invaded habitat [START_REF] Fridley | The invasion paradox: reconciling pattern and process in species invasions[END_REF][START_REF] Thomsen | Impacts of marine invaders on biodiversity depend on trophic position and functional similarity[END_REF]. The infrastructures and logistics associated to the maritime activities alter environmental factors such as temperature, salinity, hydrodynamics, sediment resuspension, contaminants (nutrients, organic matter and metals) and incident light -all susceptible to affect fouling communities and potentially select tolerant NIS, from species-specific settlement to biotic interactions [START_REF] Bulleri | The introduction of coastal infrastructure as a driver of change in marine environments[END_REF][START_REF] Dafforn | Marine urbanization: an ecological framework for designing multifunctional artificial structures[END_REF][START_REF] Johnson | Anthropogenic activities promoting the establishment and spread of marine non-indigenous species post-arrival[END_REF][START_REF] Lawes | Multiple stressors in sediments impact adjacent hard substrate habitats and across biological domains[END_REF]. While environmental conditions artificially created may be unique at the local scale, they tend to be similar across distant locations, hence removing the potential mismatch between port-of-calls and contributing to the biotic homogenization within and among oceans [START_REF] Streftaris | Globalisation in marine ecosystems: The story of non-indigenous marine species across European seas[END_REF][START_REF] Seebens | The risk of marine bioinvasion caused by global shipping[END_REF][START_REF] Seebens | Predicting the spread of marine species introduced by global shipping[END_REF].

In SE Pacific and more specifically along the Chilean coasts, particular attention has been paid to the numerous NIS introduced for aquaculture purposes. However, the scarcity of dedicated surveys for the presence of NIS in the marine environment is noteworthy [START_REF] Castilla | Down under the southeastern Pacific: marine non-indigenous species in Chile[END_REF][START_REF] Castilla | Chapter 26, Marine bioinvasions in the Southeastern Pacific: status, ecology, economic Impacts, conservation and management[END_REF][START_REF] Pinochet | Presence of the tunicate Asterocarpa humilis on ship hulls and aquaculture facilities in the coast of the Biobío Region, south central Chile[END_REF][START_REF] Villaseñor-Parada | Ecología de invasiones marinas en Chile continental:¿ Qué sabemos y que nos falta por saber?[END_REF]. Past and recent research have suggested that that very few species have successfully established along SE Pacific coasts as compared to other regions in the world [START_REF] Castilla | Chapter 26, Marine bioinvasions in the Southeastern Pacific: status, ecology, economic Impacts, conservation and management[END_REF][START_REF] Villaseñor-Parada | Ecología de invasiones marinas en Chile continental:¿ Qué sabemos y que nos falta por saber?[END_REF].. Among possible causes, unique oceanographic properties characterized by dissolved-oxygen deficit in coastal waters together with cold north-flowing current north of 42°S and low temperature and salinity south of 42°S, had been proposed as a mechanism enabling substantial "abiotic resistance" to regional NIS establishment and spread [START_REF] Castilla | Chapter 26, Marine bioinvasions in the Southeastern Pacific: status, ecology, economic Impacts, conservation and management[END_REF]. Recent modelling approaches regarding international trades also suggested that this environmental mismatch between source regions and SE Pacific coastline should reduce invasion risks [START_REF] Keller | Linking environmental conditions and ship movements to estimate invasive species transport across the global shipping network[END_REF][START_REF] Seebens | The risk of marine bioinvasion caused by global shipping[END_REF]. Paradoxically, those barriers are elusive and may change over time (e.g. seasonality and stochasticity of upwellings): they could be overwhelmed locally by expanding international traffic, which is increasing colonization and propagule pressures [START_REF] Melbourne | Invasion in a heterogeneous world: resistance, coexistence or hostile takeover?[END_REF][START_REF] Lockwood | The more you introduce the more you get: the role of colonization pressure and propagule pressure in invasion ecology[END_REF][START_REF] Simberloff | The role of propagule pressure in biological invasions[END_REF][START_REF] Sylvester | Hull fouling as an invasion vector: can simple models explain a complex problem?[END_REF][START_REF] Miller | Arctic shipping and marine invaders[END_REF] and by "ocean sprawl" which is increasing the NIS establishment and spread probabilities [START_REF] Streftaris | Globalisation in marine ecosystems: The story of non-indigenous marine species across European seas[END_REF][START_REF] Mineur | Changing coasts: marine aliens and artificial structures[END_REF][START_REF] Seebens | The risk of marine bioinvasion caused by global shipping[END_REF][START_REF] Airoldi | Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale[END_REF].

Over the last decade, the number and extent of artificial structures have dramatically expanded along the Chilean coastline in order to protect inhabited shores from storms and tsunamis and to promote economic development, through shipping trades, mining and aquaculture [START_REF] Aguilera | Spatial variability in community composition on a granite breakwater versus natural rocky shores: lack of microhabitats suppresses intertidal biodiversity[END_REF][START_REF] Aguilera | Artificial defences in coastal marine ecosystems in Chile: Opportunities for spatial planning to mitigate habitat loss and alteration of the marine community structure[END_REF]. Between 18° and 41°S (3000 km of linear coastline), these infrastructures account approximately to 200 km, among which 70% are concentrated in northern (18-22°S and 23°S), central (ca. 33°S) and southern (36°S and 41°S) regions [START_REF] Aguilera | Artificial defences in coastal marine ecosystems in Chile: Opportunities for spatial planning to mitigate habitat loss and alteration of the marine community structure[END_REF]. In parallel, Chilean ports receive international ships from diverse regions (mainly Asia, North America, Europe and Australasia). Shipping pathways have been assumed to be responsible for the introduction of 30-38 % of the NIS established in Chile [START_REF] Castilla | Chapter 26, Marine bioinvasions in the Southeastern Pacific: status, ecology, economic Impacts, conservation and management[END_REF]. Nonetheless, the legal regulation of marine NIS surveillance has just started to be implemented in Chile Chilean authorities have enacted a Legal Procedure DIRECTEMAR A-51/002, October, 14th, 2002, based on the Ballast Water Convention, which is still under revision for being concordant with the IMO statements (Lloyd's Register 2014).

However, there are currently no regulation, policies or national strategic plan to target biofouling in relation with international, regional or local shipping. In addition, research regarding NIS in Chile is generally focused on the ecology of some already-described invaders and there has been -to our knowledge -no attempt to implement dedicated NIS surveys neither in artificial nor in natural environments [START_REF] Villaseñor-Parada | Ecología de invasiones marinas en Chile continental:¿ Qué sabemos y que nos falta por saber?[END_REF]. In this context there are serious knowledge gaps and uncertainties regarding the extent of NIS contribution to the fouling communities established on hard substrates (natural and artificial). Such data are urgently needed for accurate reports of NIS presence, distribution and establishment of baselines in order to implement dedicated surveillance programs of the biofouling communities in Chilean ports and nearby artificial structures. The present study aimed to investigate the diversity and structure of hard bottom communities, and specifically identify the non-indigenous components of those communities in South-Central Chile. We targeted both international and local ports, the former being primary entry points for introduction mediated by international maritime traffic while the latter being potential destination of NIS primarily introduced. To this end, a series of standardized settlement panels were deployed twice (summer and winter) in 3 international and 3 local ports, before being randomly collected after 1, 3 and 7 months. In parallel, rapid assessment visual surveys were conducted to compare the established communities in the neighboring substrates of the panels. Finally, various abiotic parameters (metal and organic matter concentration, light, and temperature) were measured to check for potential confounding effects when comparing the biofouling communities in international vs. local ports. We hypothesised that NIS richness and abundance measured on 1 month-old panels -fouled by early settlers thus being proxies of the colonization and propagule pressures -would be higher in international than in local ports. After 3 and 7 months of developments, patterns in diversity and assemblage structure were expected to differ, with potential greater contributions of NIS and cryptogenic species in international as compared to local ports.

Methods

Sampling design

The study was performed along approximately 100 km of coastline in the Bíobío region (Chile) between August 2016 and October 2017. Within the region, a total of six fully marine sites (ports) were sorted according to their shipping traffic (Fig. 1): three international ports (cargo and tanker moorings) located in Coronel (37.0304°S, 73.1540°W) Biodiversity assays combined the deployment of settlement panels and rapid assessment surveys using scuba diving. In August 2016 (winter trial) and March 2017 (summer trial), a series of settlement panels (black polypropylene, 15 × 15 cm) were deployed vertically upon two experimental units (90 × 100 cm) made of a plastic fence (mesh 2.5 × 2.5 cm, Fig. 1), at two plots separated by 20-50 m within each site (local and international ports) at ca. -4 m.

Depending on the site conformation and substratum availability (e.g. jetties or mooring buoys), the experimental units were either attached to concrete/steel pilings (Coronel, San Vicente, Lirquén, Coliumo), large rocks (Chome) or to floating longline (Llico). A total of 24 panels (12 per plot) were deployed per site on each occasion. After 1, 3 and 7 months, 8 panels (4 at random per plot) were retrieved using polypropylene rubble bags (mesh < 0.5 mm) to minimize mobile fauna loss, and then stored (for up to 4 hours) within a tank filled with sea water until processing in the laboratory. Panels retrieved after 1 month of development are occupied by early-settlers (less than one-month old) and thus were hypothesized to point out mainly differences in recruitments: colonization and propagule pressures (sensu [START_REF] Lockwood | The more you introduce the more you get: the role of colonization pressure and propagule pressure in invasion ecology[END_REF]; see also [START_REF] Sylvester | Hull fouling as an invasion vector: can simple models explain a complex problem?[END_REF] were estimated by richness and abundances, respectively. Panels left for a few weeks are indeed effective to detect new arrivals [START_REF] Bishop | Repeated rapid assessment surveys reveal contrasting trends in occupancy of marinas by non-indigenous species on opposite sides of the western English Channel[END_REF]Bishop, Wood, Yunnie, et al. 2015) and to comprehend propagule pressure s.l. dynamics [START_REF] Sorte | Patterns and processes of compositional change in a California epibenthic community[END_REF][START_REF] Bouchemousse | Do settlement dynamics influence competitive interactions between an alien tunicate and its native congener?[END_REF]. Panels retrieved after 3 and 7 months were analysed to examine differences in community assembly, resulting from species interactions [START_REF] Stachowicz | Biodiversity, invasion resistance, and marine ecosystem function: reconciling pattern and process[END_REF][START_REF] Lord | Predicting competitive shifts and responses to climate change based on latitudinal distributions of species assemblages[END_REF].

In order to complement the settlement panel datasets, rapid assessment surveys were conducted by the same diver (JCL) during each trial (in November 2016 and June 2017) within established subtidal communities of hard substrates surrounding the experimental units in all locations. During 30 minutes, all species encountered were given a score of semi-abundance according to the SACFOR scale (Superabundant, Abundant, Common, Frequent, Occasional, Rare), which takes into account the individual size and growth form [START_REF] Hiscock | Marine nature conservation review: rationale and methods[END_REF]. These surveys were conducted between ca. -1.5 m and -5 m and generally covered the horizontal distance between the experimental units (20-50m) depending on the site conformation (e.g. distance between pilings). Most sessile taxa as well as mobile taxa > 10 mm were visually identified at the lowest taxonomic level possible in the field. To ensure accurate identification, prior to this study, a series of preliminary fieldworks were performed in each study site. During these dives, specimens were photographed, collected, identified under a dissecting microscope and preserved in 95% EtOH to form a local reference collection. This preliminary work was used to prepare detailed field-worksheets and train the observer eye. In addition, during the study, a few specimens were photographed and collected to confirm field identification back in the laboratory.

Data collection

Environmental parameters

Environmental conditions of each site were assessed from a series of parameters: incident light, temperature, sediment pH, sediment organic matter content and concentration of different metals. Temperature (°C) and illuminance (Lum ft -2 ) were measured in situ at 10 min intervals between March and June 2017 using data loggers (onset HOBO® data-loggers Pendant Temp-Light, Onset Computer Corporation) deployed within each site. Owing to the biofouling undergone by the loggers, light data gathered more than 4 weeks after installation were not considered. Sediment parameters were determined from superficial sediment (first cm) samples (n = 3-4) collected below experimental units in June 2017 and stored at -20°C until analyses. In the laboratory, sediment samples were lyophilized and pulverized. The pH was measured in 1:2.5 sediment to water ratio using an electrode. The organic matter content (%OM) was determined after calcination at ca. 550ºC. Metal contents were determined using total Ray X reflexion fluorescence analysis [START_REF] Towett | Quantification of total element concentrations in soils using total X-ray fluorescence spectroscopy (TXRF)[END_REF], details provided in Appendix S1).

Diversity and community structure assays

In the laboratory, panels were removed from their bags, cleared from cable tiles and left with all remaining bag contents in seawater tanks until sessile fauna returned to their natural, untense state. Sessile taxa (mostly fauna, see Results) were identified under a dissecting microscope and their abundances were assessed using cover. To avoid edge effects in their distribution, a 15 mm perimeter was excluded from analysis, giving a 120 × 120 mm working area. Species cover was estimated under 100 random intersection points out of 169 created between evenly spaced lengths of string of 150 × 150 mm quadrat. Any species identified out of these intersection points was given a cover of 0.5 %. Species layering was taken into account, therefore the total cover frequently exceeded 100%. Following sessile taxa identification, panels were washed through a 500 µm mesh sieve to separate mobile fauna, ultimately stored in EtOH 95%. All specimens were identified at the lowest taxonomic level possible (generally species) by the same observer (JCL) and occasionally verified by external experts (see acknowledgments). Throughout the survey, voucher specimens were collected, dissected when appropriate and preserved in 95% EtOH in order to fill in the local reference collection (for further morphological and/or molecular examination). When appropriate, some specimens were maintained in tanks with bubbling air stone and filled with seawater at ambient temperature until they developed diagnostic size and/or characteristics. Molecular barcoding (using COI)

was also employed whenever necessary and possible. While this approach did not always enable identification at the species level (e.g. because of the absence of reliable molecular reference data), it allowed to compare juveniles and adults, the latter being identified (by JCL) upon morphological characteristics and assign juveniles to identified adults. The identified specimens were categorized as 'native', 'non-indigenous' (NIS), 'cryptogenic' or 'unassigned' according to the literature (e.g. [START_REF] Moyano | Southern Pacific Bryozoa: a general view with emphasis on Chilean species[END_REF][START_REF] Galea | Hydroids and hydromedusae (Cnidaria: Hydrozoa) from the fjords region of southern Chile[END_REF][START_REF] Turon | Ascidian fauna (Tunicata, Ascidiacea) of subantarctic and temperate regions of Chile[END_REF]) and public databases (EASIN, WORMS/WRIMS, [START_REF] Pagad | World Register of Introduced Marine Species[END_REF]). The cryptogenic species, from unknown/uncertain origin (sensu [START_REF] Carlton | Biological invasions and cryptogenic species[END_REF], found in this study displayed a cosmopolitan distribution and were potentially non-indigenous to the study area. Cryptogenic and NIS, both candidates for further introduction and spread, were thus considered in analyses [START_REF] Dafforn | Shallow moving structures promote marine invader dominance[END_REF][START_REF] Leclerc | Habitat formation prevails over predation in influencing fouling communities[END_REF]. When appropriate, taxa were also sorted according to their main function within the food web (carnivores, suspension-deposit-feeders, herbivores).

Statistical analyses

Patterns in environmental conditions were explored using a principal component analysis (PCA), based on normalized data. Data related to sediment conditions (pH, %OM, metal concentrations) were replicated (n = 3-4 per sites) and were all included as active variables in the PCA. All samples were given the same values for light and temperature (average and range over deployment period), therefore these data were included as supplementary (i.e.

illustrative or inactive) variables [START_REF] Lê | FactoMineR: an R package for multivariate analysis[END_REF]. Environmental patterns were examined with a two-way design using a permutational multivariate analysis of variance (PERMANOVA, [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF], with 4999 permutations. Factors were 'maritime traffic' (hereafter 'traffic', fixed, 2 levels: international and local) and 'site' (random, nested within traffic). This analysis was based on a Euclidean distance matrix generated from normalized data of the active variables of the PCA.

Patterns in species richness, abundance and community structure of sessile taxa were examined with a four-way design using PERMANOVAs with 4999 permutations. Factors were 'traffic' (fixed, 2 levels: international and local ports), 'starting deployment period' ('starting date', random, 2 levels: August 2016 and March 2017), 'age of the settlement panel at the time of collection' ('age', fixed, 3 levels: 1, 3 and 7 months) and 'site' (random, nested within traffic). A few experimental units were lost over the course of the experiment, therefore the corresponding term (plot) was not included in analyses. Nonetheless, there were between 4 and 8 replicate panels available for each combination of Site (Traffic) × Starting date × Age.

Univariate analyses were based on Euclidian distance matrices whereas multivariate analyses were based on Bray-Curtis similarity matrices generated from either raw or transformed data.

In order to down-weight the importance of most abundant species (and homogenize multivariate dispersion), multivariate data were always square root-transformed. The homogeneity in univariate or multivariate dispersion was checked among the levels of the lowest interaction term Site (Traffic) × Starting date × Age using PERMDISP [START_REF] Anderson | PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods[END_REF]. No transformation allowed homoscedasticity to be achieved in univariate data, except in one case (total richness). Given the balance of the design and the large number of samples, univariate PERMANOVAs (analogous to ANOVAs) were considered robust enough to cope with this issue and were run on untransformed data [START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF]. As for multivariate data, samples were also ordinated using principal coordinate (PCO) analyses to support PERMANOVA results [START_REF] Anderson | PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods[END_REF]. When appropriate, PERMANOVAs were followed by pairwise comparisons and P-values were estimated using Monte Carlo procedure. Finally, semiquantitative data resulting from rapid assessment surveys on surrounding communities were analyzed graphically, by considering the number of reports (i.e. taxa) within each category of abundances at both sampling periods. These analyses were presented separately for nonindigenous and cryptogenic species as well as native space occupiers and potential predators.

Statistical analyses were performed either on all variables combined (including unassigned taxa as well as abiotic data in the case of community structure: bare surface, grazing scars, dead biota), native taxa, non-indigenous or cryptogenic species [START_REF] Thomsen | To include or not to include (the invader in community analyses)? That is the question[END_REF]. By analysing sub-component of communities separately, Bray-Curtis similarity could not always be computed because of the presence of empty samples (e.g. panels not colonized by NIS):

corresponding pairs of samples were thus removed from analyses (see degree of freedom in PERMANOVA results). Environmental data were analysed using R environment [START_REF] Lê | FactoMineR: an R package for multivariate analysis[END_REF]; R Development Core Team 2012) while community analyses were performed using PRIMER 7 [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF].

Results

Environmental parameters

According to the principal component analysis (PCA), all study sites were quite welldiscriminated by their abiotic conditions (Fig. 2, Appendix S1). While the PERMANOVA confirmed these differences (Site (Traffic): F4,13 = 5.01, P < 0.001), it failed to detect a multivariate effect of the traffic category (i.e. international vs. local ports, F1,13 = 2.75, P = 0.099). In spite of a large within-site variability, the first PCA axis (50.1% of the variation) however suggested differences between ports according to their category. These differences are predominantly explained by higher concentrations of a series of metals (except Hg), but lower light (inactive variable) in international as compared to local ports.. There is however some overlap among sites: most metal concentrations and light levels were similar in Coliumo (local),

Lirquén and San Vicente (international ports). This is exemplified on the second axis of the PCA (26% of variation) which discriminates Coronel samples (57% of contribution) and the local port located in Llico (5%) from the four other sites. This discrimination is mainly dueby decreasing order of importance -to higher %OM, pH and concentrations of copper, lead and zinc. Coronel also experienced greater mean values and variation in temperature.

Diversity and community structure

During the study (combining panels examination and rapid assessment survey), a total of 119 taxa were identified, including 10 algae, 41 sessile and 37 mobile animals. Complete lists and species authorities are provided in Appendix S2. Among these taxa, 10 were classified as non-indigenous and 15 as cryptogenic. Importantly, these taxa included novel records for the region: Bougainvillia muscus, Ectopleura larynx, E. crocea, Hydractinia sp. and Exochella nov.

sp. and Asterocarpa humilis (for the latter, details are provided in [START_REF] Pinochet | Presence of the tunicate Asterocarpa humilis on ship hulls and aquaculture facilities in the coast of the Biobío Region, south central Chile[END_REF]. Within established hard bottom communities examined through rapid assessment surveys at both periods, NIS and cryptogenic species contributed to 13% and 26% of the sessile taxa richness, respectively. The highest proportions of NIS (23%) and cryptogenic species (25%) were found in Lirquén (Appendices S3, S5). Nonetheless, NIS were often rare or occasional (Fig. 3) and some of them were only found in specific habitats (e.g. crevices, mooring lines and abandoned ropes). In comparison, several native species were classified as frequent, common or abundant in both categories of ports (Fig. 3). Less NIS (i.e. cumulative numbers over sites) were reported in local (3 and 7 in November 2016 and March 2017, respectively) than in international ports (13 and 18) (Appendices S3, S5). Six NIS were found only in international ports, with three of them Bougainvillia muscus (rare to frequent), Ectopleura crocea (rare to abundant), Ciona robusta (absent to rare) observed in at least two sites. For cryptogenic species, the number of reports was greater, but with less difference than for NIS, between local (12-15) and international ports (21-20), with two of them only recorded in international ports (e.g. Clytia linearis, varying from rare to abundant depending on site and sampling date, Appendix S3).

On experimental panels, over the whole experiment, a total number of 6 and 9 NIS settled on the panels in local and international ports, respectively (details in Appendix S4). In local ports, half of the NIS were observed in one site only, and none in the three study sites. In international ports, 5 were also found in one site only but 3 NIS were observed in the three study sites. The number of cryptogenic species was more balanced between the two categories with 12 and 14 taxa reported in local and international ports, respectively. Like for NIS, the number of occurrence was however higher across sites in international ports with 6 cryptogenic species observed in the three international ports and only one in the three local ports.

Significant three-way interactions (Site × Starting date × Age) were observed with regards to either richness, sessile taxa cover or community structure, whether considering all, non-indigenous, cryptogenic or native species (Table 1-3). However, despite larger number of NIS observed in international ports and with higher occurrence, no effect of the traffic category (international vs. local ports) could be detected, regardless of the deployment period or its duration (Table 1-3, Fig. 456).

After one month, an average of 4 taxa settled on the panels, with an average coverage of 34% of the panel area (22% in September 2016, 45% in October 2017). Only a few nonindigenous specimens were observed at this stage (2 in local and 6 in international ports on total, both sampling periods combined), with up to 1 taxa for a maximum cover of 2% in Lirquén (Fig. 4-5, Appendices S6-7). No clear pattern could be detected for cryptogenic species

(5 and 9 taxa in local and international ports, respectively) richness and cover, although they represented half of the richness and up to 87% of the cover in Llico (Fig. 4-5, Appendices S6-7). The PCO illustrates the three-way interaction (Site × Starting date× Age, Table 3): traffic category did not explain settlement patterns, but important site-to-site differences were observed within each category and depending on the period (Fig. 6). NIS poorly influenced these patterns, which are more likely due to some species-specific recruitments. For instance, the native barnacle Austromegabalanus psicattus showed greater settlement in Llico during the second trial (March 2017) as compared to all other sites (Fig. 6).

After 3 months, site-to-site differences -though depending on the period -increased (Fig. 4-6, Appendices 6-8). The average richness per panel varied between 3 (Chome) and 9

(Lirquén) after the first deployment and between 3 (Chome) and 10 (San Vicente) after the second one (Appendix S6). Total cover of sessile taxa varied between 40% (Chome) and 180%

(San Vicente) after the first deployment and between 20% (Lirquén) and 100% (in Llico, Coliumo and Coronel) after the second (Appendix S7). At this stage of development (both periods combined), a total of 4 and 5 NIS was observed on panels in local and international ports, respectively (5 and 14 cryptogenic species). All non-indigenous, cryptogenic and native species apparently contributed to differences among sites (Appendices S6-7, Table 1-2). The global multivariate structure allowed to discriminate all sites and to illustrate within sitesimilarity between panels deployed in winter and summer (the first two axes of the PCO explained a total of 44%, Fig. 6). Importantly, it also illustrates the absence of clear discrimination according to the traffic categories (Table 3): for instance, grazing scars (apparently due to urchins, JCL pers. obs.) -correlated with bare surface -contributed to community structure in both Chome (local) and Lirquén (international port), at least in summer, while barnacle A. psicattus was an important space occupier in both Llico (local) and San Vicente (international port). Similarly, by analysing separately non-indigenous, cryptogenic and native assemblage structures, it is clear that several species contributed to spatial patterns, regardless of traffic categories (Fig. 6). For instance, while B. muscus contributed to NIS community structure in all international ports, Exochella n. sp. was abundant in both Coliumo (local port, where the species has been observed for the first time) and San Vicente (international). After 7 months, panels were generally fouled by more species and in greater abundances (involving either NIS, cryptogenic, native or a combination of all, depending on site or sampling occasion, Appendix S5), but the observed patterns were generally consistent with those observed after 3 months (Fig. 4-6, Appendices S6-8). At this stage, a total of 5 and 8 NIS and a total of 8 and 9 cryptogenic species were observed on panels from local and international ports, respectively.

Discussion

International ports are often coined as 'introduction hotspots' or' invasion hubs' because maritime traffic occurring in these areas is expected to enhance the colonization and propagule pressures. The largest number of NIS observed in international ports in our study supports this scenario. However, a roughly similar number of NIS was also observed in local port. Thus, conversely to our working hypotheses, no significant effect of the traffic category (international vs. local ports) could be detected on the biotic component, at any stage of development of the biofouling community. NIS early settlement was low in all sites, including in international ports, as measured on experimental panels collected after 1 month at any trial period. While contrasting biofouling communities developed 3 and 7 months after panel deployment, NIS played either a minor role or an equal contribution across both categories of ports. Altogether, important site-to-site differences were observed regarding abiotic conditions, richness, sessile taxa cover and community structure, but without discriminating ports according to their traffic categories.

Overall, unique communities developed in each study site irrespective of their traffic category and in spite of some variations in environmental parameters (metal concentration, light) between them. For instance, higher metal concentrations were reported in the international ports, as shown in other studies [START_REF] Dafforn | The challenge of choosing environmental indicators of anthropogenic impacts in estuaries[END_REF]. Resuspension of sedimentbound contaminants, such as metals, may influence biofouling communities by affecting either settlement cues or the biofilm. In the more contaminated site of this study (Coronel), the average concentrations of copper, lead and zinc were of 41, 34 and 73 mg kg -1 , respectively. While such values are in the range of some impacted estuaries (e.g. in New South wales) [START_REF] Dafforn | The challenge of choosing environmental indicators of anthropogenic impacts in estuaries[END_REF], they are below the reference range of possible adverse biological effects [START_REF] Macdonald | Development and evaluation of sediment quality guidelines for Florida coastal waters[END_REF] [START_REF] Galea | Hydroids and hydromedusae (Cnidaria: Hydrozoa) from the fjords region of southern Chile[END_REF][START_REF] Galea | Additions to the hydroids (Cnidaria: Hydrozoa) from the fjords region of southern Chile[END_REF]). These campaigns resulted in novel records of various non-native (Bougainvillia muscoides, B. pyramidata) and cryptogenic hydroids (e.g. Clytia linearis, Amphisbetia operculata ), some of them widely distributed [START_REF] Galea | Hydroids and hydromedusae (Cnidaria: Hydrozoa) from the fjords region of southern Chile[END_REF][START_REF] Galea | Additions to the hydroids (Cnidaria: Hydrozoa) from the fjords region of southern Chile[END_REF]).

Interestingly, among the novel taxa herein recorded, Ectopleura crocea has been observed in all studied international ports. This common biofouling species is considered non-native in many regions worldwide, including in the NE Pacific along which -decades ago -its hydrants have been demonstrated to survive long distances and disperse from the hulls of sailing vessels [START_REF] Carlton | Biogeography and dispersal of coastal marine organisms: experimental studies on a replica of a 16th-century sailing vessel[END_REF]. First time report does not mean that the introduction is recent (e.g. [START_REF] Provan | Tracking the invasive history of the green alga Codium fragile ssp. tomentosoides[END_REF], especially in under-studied taxonomic groups. Like other non-native species reported for the first time, including in this study, this species has probably been introduced along the SE Pacific, and more specifically in Chile, a long time ago, spread and established widely, likely beyond the geographic span of this study. In order to test whether the recruitment patterns herein observed result from local populations or external sources, thorough surveys of ship tanks and hulls, as well as small craft hulls, arriving in the studied ports would have been needed. For the time being and according to worldwide literature on the topic, it seems however unlikely that arriving international ships do not transport NIS [START_REF] Bax | Marine invasive alien species: a threat to global biodiversity[END_REF][START_REF] Drake | Hull fouling is a risk factor for intercontinental species exchange in aquatic ecosystems[END_REF][START_REF] Gollasch | Critical review of the IMO international convention on the management of ships' ballast water and sediments[END_REF]. A recent study of international ship hulls in Talcahuano port, located in our study area, revealed the presence of the non-indigenous tunicate Asterocarpa humilis -putativelly native from New Zealand -within their sea chest [START_REF] Pinochet | Presence of the tunicate Asterocarpa humilis on ship hulls and aquaculture facilities in the coast of the Biobío Region, south central Chile[END_REF]. Although no effect of the traffic category could be have been detected

on recruitments over the studied period, the larger number of NIS reported in international ports suggests that international shipping have influenced their local establishment at some point.

Nonetheless, the environment of the receiving ports may mismatch with the abiotic requirements of many introduced species for reproduction and propagule survival [START_REF] Castilla | Chapter 26, Marine bioinvasions in the Southeastern Pacific: status, ecology, economic Impacts, conservation and management[END_REF][START_REF] Seebens | The risk of marine bioinvasion caused by global shipping[END_REF]). In addition, regardless of their survival, some propagules may simply not be retained: although generally constructed within bays, none of the study ports were enclosed by breakwaters or seawalls, therefore it is possible that the propagule pressure was locally limited in favour of outward dispersal [START_REF] Floerl | Boat harbour design can exacerbate hull fouling[END_REF][START_REF] Bishop | Effects of ocean sprawl on ecological connectivity: impacts and solutions[END_REF].

Dispersal and survival modelling based on local oceanographic features and species life history traits would help to test whether these hypotheses could explain the generally low propagule pressure and the current distribution of NIS among all sampling sites.

After 3 and 7 months, NIS contribution to community structure was highly variable -in terms of identity and abundances -among sites and there was no indication that international maritime traffic contributed to this pattern. While NIS richness and cover respectively increased in 4 and 3 sites between 1 and 3 months, they only increased in 1 and 3 sites between 3 and 7 months, irrespective of their associated traffic (Appendix S9). In comparison, native richness and cover respectively increased in 4 and 5 sites between 1 and 3 months and both in 4 sites between 3 and 7 months. The apparent poor contribution of NIS to community development patterns is potentially in agreement with [START_REF] Castilla | Chapter 26, Marine bioinvasions in the Southeastern Pacific: status, ecology, economic Impacts, conservation and management[END_REF] who suggested that the uniqueness of the Chilean coastline in terms of oceanographic conditions could contribute to limit the regional establishment and spread of NIS, less 'adapted' than most native species under specific conditions (e.g. [START_REF] Castilla | Invasion of a rocky intertidal shore by the tunicate Pyura praeputialis in the Bay of Antofagasta, Chile[END_REF][START_REF] Caro | Competition between a native mussel and a non-indigenous invader for primary space on intertidal rocky shores in Chile[END_REF]. In order to be validated, such a hypothesis would however require thorough test of the propagule survival and growth of the NIS herein found (as well as native species), under controlled conditions. With regards to the temporal changes in richness and cover undergone by native species (generally increasing, Appendix S9) on experiment panels, the influence of these species in limiting NIS establishment (i.e. biotic resistance hypothesis) deserves further experimental scrutiny [START_REF] Stachowicz | Biodiversity, invasion resistance, and marine ecosystem function: reconciling pattern and process[END_REF][START_REF] Clark | Propagule pressure and disturbance interact to overcome biotic resistance of marine invertebrate communities[END_REF][START_REF] Lord | Predicting competitive shifts and responses to climate change based on latitudinal distributions of species assemblages[END_REF]. In all the studied international ports, pilings were generally completely fouled (JCL, pers. obs.) and various native sessile taxa were classified as common, abundant or even super-abundant (Figure 3), i.e.

with species-specific covers varying between 10 to more than 80% depending on the growth forms (crust or massive)-. In parallel, only a few NIS fell in these categories, most of them being rare or occasional (Fig. 3). The native assemblages may have contributed to limit NIS establishment in the studied sites by limiting at least one of the most limiting resources of biofouling communities: free space [START_REF] Elton | The ecology of invasions by plants and animals[END_REF][START_REF] Stachowicz | Biodiversity, invasion resistance, and marine ecosystem function: reconciling pattern and process[END_REF]. Should the influence of native space occupiers in limiting NIS establishment be further investigated in the study area, the complexity of possible interactions (e.g. facilitation, predation) between native and nonindigenous taxa could not be overlooked [START_REF] Bulleri | Beyond competition: incorporating positive interactions between species to predict ecosystem Invasibility[END_REF][START_REF] Rogers | Native predators limit invasion of benthic invertebrate communities in Bodega Harbor, California, USA[END_REF][START_REF] Leclerc | Habitat formation prevails over predation in influencing fouling communities[END_REF]. Various NIS (e.g. Ectopleura crocea, Bugulina flabelatta, Asterocarpa humilis, Diplosoma listerianum) and cryptogenic species (e.g. Alcyonidioides mytili, Amathia cf.

gracilis, Plumularia cf. setacea) were, for instance, herein frequently observed in abundance when developing upon massive native habitat builders, such as Pyura chilensis and Austromegabalanus piscattus. In the present survey, several native predators (e.g. fish, urchins, crabs, Caprellid amphipods) were also observed and generally classified as frequent or common depending on study sites, in either local or international ports (Fig. 3, Appendix S5).

Nonetheless, multivariate analyses suggested that grazing (apparently due to sea urchins) influences the community with contrasting dynamics in at least two sites -an international (Lirquén) and a local port (Chome) -by increasing the free space (Fig. 6). As such, the specific functions of native predators in controlling some NIS populations may deserve further investigation [START_REF] Dumont | Predation contributes to invasion resistance of benthic communities against the non-indigenous tunicate Ciona intestinalis[END_REF][START_REF] Rogers | Native predators limit invasion of benthic invertebrate communities in Bodega Harbor, California, USA[END_REF], although careful attention should be paid regarding direct and indirect effects [START_REF] Connell | Predatory fish do not always affect the early development of epibiotic assemblages[END_REF].

In a more regional context, the influence of local shipping and ocean sprawl in favoring NIS establishment and spread should also be considered [START_REF] Wasson | Biological invasions of estuaries without international shipping: the importance of intraregional transport[END_REF][START_REF] Mineur | Changing coasts: marine aliens and artificial structures[END_REF][START_REF] Aguilera | Spatial variability in community composition on a granite breakwater versus natural rocky shores: lack of microhabitats suppresses intertidal biodiversity[END_REF][START_REF] Johnson | Anthropogenic activities promoting the establishment and spread of marine non-indigenous species post-arrival[END_REF]. For instance, unlike all the studied international ports, Coliumo jetty did open to regional traffic (fishing boats) only one year before the beginning of our survey and its construction dated back a couple of years following 2010 tsunami -common catastrophic events along the Chilean coast [START_REF] Aguilera | Artificial defences in coastal marine ecosystems in Chile: Opportunities for spatial planning to mitigate habitat loss and alteration of the marine community structure[END_REF]. At this site, only seldom and poorly abundant native taxa were initially observed on pilings (Appendix S5), still covered by antifouling paint. Later on (e.g. in June 2017), several NIS were recorded (e.g.

Exochella nov. sp., Bugula neritina, Bugulina flabelatta, Asterocarpa humilis, Diplosoma listerianum) and some of them (e.g. Exochella nov. sp.) showed important increase in cover (and apparent overgrowth of native taxa) on experimental panels between sampling dates (Appendix S9). These results strengthen the hypothesis that free space may be an important limiting factor for NIS establishment [START_REF] Stachowicz | Biodiversity, invasion resistance, and marine ecosystem function: reconciling pattern and process[END_REF]. In this context, it is noteworthy that NIS cover varied on average between 14 and 29% after 7 months, which are values greater than those observed within the surrounding communities (Fig. 3). Unless they get colonized by abundant native taxa, the creation of novel artificial structures may thus contribute to the local establishment of NIS, and potentially act as stepping stones for subsequent spread [START_REF] Wasson | Biological invasions of estuaries without international shipping: the importance of intraregional transport[END_REF][START_REF] Mineur | Changing coasts: marine aliens and artificial structures[END_REF][START_REF] Airoldi | Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale[END_REF], potentially facilitated by regional transport [START_REF] Murray | Recreational boating: a large unregulated vector transporting marine invasive species[END_REF][START_REF] Ferrario | Role of commercial harbours and recreational marinas in the spread of non-indigenous fouling species[END_REF]) -herein mainly due to fishing barge and crafts:

in the present survey, Exochella nov. sp. was detected for the first time underneath the local jetty of Coliumo in March 2017 and later recruited and contributed to community structure on experimental panels in both Coliumo and San Vicente (international port). If Chilean authorities and research groups were to implement NIS surveillance programs, the present study performed locally, along with others conducted abroad (e.g. [START_REF] Murray | Recreational boating: a large unregulated vector transporting marine invasive species[END_REF]Bishop, Wood, Yunnie, et al. 2015;[START_REF] Johnson | Anthropogenic activities promoting the establishment and spread of marine non-indigenous species post-arrival[END_REF], strongly supports that the diversity of putative vectors (including hull biofouling of local crafts) and stepping stones (including local mooring facilities) should be taken into account.

In conclusion, there is no clear evidence that international ports, in which intense international maritime traffic is occurring, play the most important role for NIS establishment in the study region. Without challenging the fact that international ports are invasion hubs, our surveys also pointed out the importance of local ports for NIS establishment and potential subsequent spread, including in nearby international ports from where they can be afterwards transported over longer distance. Overall, NIS recruitment appeared low during the study period and NIS contribution to community structure depended on study sites, regardless of traffic categories. Site-specific processes are most likely to explain these patterns, among which the influence of environmental conditions, regional transport, the availability of free space to be colonized and NIS interactions with the recipient communities should deserve further attention.

Acknowledgements. We are grateful to M Altamirano, R Reed and O Marin for diving assistance, J Martínez and J del Carmen for sailing assistance and to B Pedreros, A Carillo, A Gallegos, N Fernández, A Araya, V Molina, E Fernandez, G Vargas, MJ Ferro, P Valenzuela and N Cofré and the CIBAS institute for help with logistics in the study sites. We are also grateful to three anonymous reviewers for their comments that helped to improve the manuscript. We finally thank D Gordon and V Häussermann for help with Exochella and Acontiaria identification, respectively. AB was supported by a FONDECYT grant, N°1170598. cryptogenic and native species on the panels collected after 1 and 7 months in all study sites for each trial period ( w : winter and s : summer). Vector plots of variables correlated with the PCO axes are indicated within the same panels, with r > 0.6 for all species and r > 0.4 for NIS, cryptogenic and native species. Detailed data (including 3 months-panels) are presented in Appendix S8.

  Figure 1. Location of the study sites along Bíobío region: International (white symbols) and local ports (black symbols).

Figure 2 .

 2 Figure 2. Principal Component Analyses (PCA) describing environmental conditions in international and local ports (individual factor map, left panel). Active (black arrows) and inactive (dashed grey arrows) variables are indicated on the vector plot (right panel).

Figure 3 .

 3 Figure 3. Number of non-indigenous, cryptogenic and native species (space occupiers and benthic predators) encountered within communities surrounding panels for the two categories of ports, depending on their traffic (international and local ports). Data are presented as the average number of reports (species or taxa) per site and sampling occasion (± S.D.) within each category of abundance (SACFOR). Detailed data are presented in Appendix S5.

Figure 4 .

 4 Figure 4. Sessile taxa (total, NIS, cryptogenic and native) richness on the experimental panels collected after 1, 3 and 7 months within each traffic category. Data are averaged (± S.D.) for all panels deployed in winter (monochrome) and summer (dashed). Detailed data are presented in Appendix S6.

Figure 5 .

 5 Figure 5. Sessile taxa (total, NIS, cryptogenic and native) cover (%) on the experimental panels collected after 1, 3 and 7 months within each traffic category. Data are averaged (± S.D.) for all panels deployed in winter (monochrome) and summer (dashed).. Detailed data are presented in Appendix S7.

  and about 10 times lower than highly contaminated sediments (from Port Kembla, New South Wales) which have recently been used to demonstrate impacts on bacterial biofilm

interactions and macro-fouling communities

[START_REF] Lawes | Multiple stressors in sediments impact adjacent hard substrate habitats and across biological domains[END_REF]

. Under the conditions herein measured, only experimental approaches considering possible interactions with a series of other stressors

[START_REF] Johnston | Direct and indirect effects of repeated pollution events on marine hard-substrate assemblages[END_REF][START_REF] Lawes | Multiple stressors in sediments impact adjacent hard substrate habitats and across biological domains[END_REF]

), which may display more complex dynamics (such as the dissolved-oxygen in the study area,

[START_REF] Ahumada | Circulation and fertility of waters in Concepcion Bay[END_REF][START_REF] Valle-Levinson | Flow induced by upwelling winds in an equatorward facing bay: Gulf of Arauco, Chile[END_REF]

would allow to disentangle among oceanographic and anthropogenic factors which influence the studied communities.

NIS represented less than 1% of the surface colonized on settlement panels collected after 1 month at both occasions, suggesting that all study sites undergo a similar and low propagule pressure by NIS. In addition, most of these NIS apparently settled on panels with relative abundances representative of those also identified within the surrounding established communities or in older panel assemblages (e.g. Bougainvillia muscus in Lirquén, Ectopleura crocea in Coronel, Bugulina flabellata in Lirquén and Llico, Exochella nov. sp. in Coliumo and San Vicente; Fig.

6

, Appendix S2) -a pattern quite consistent with the one observed for native species (e.g. Austromegabalanus psicattus in Llico, Semimytilus algosus in Coronel, Balanus laevis in Coliumo and San Vicente). It is therefore possible that a significant part of the observed propagule pressure mainly results from local (site-specific) populations, and thus involve more complex histories of species introductions. In the present study, most of the novel records were hydroids which, as compared to well-studied groups in

Chile (e.g. Bryozoans, Viviani 1969;[START_REF] Moyano | Southern Pacific Bryozoa: a general view with emphasis on Chilean species[END_REF]

, have received less attention until recent campaigns in Patagonian fjords

Table legends Table 1 .

 legends1 Results of PERMANOVA tests for differences in sessile taxa richness among levels of the main factors (traffic, starting date, age and site) and their interactions. Tests are presented for all, non-indigenous, cryptogenic and native species, separately. Post-hoc pairwise tests are summarized in Appendix S9.

	Richness		Total			NIS			Cryptogenic		Native
	Transformation		square-root			none			none			none
	PERMDISP		F 1,35 = 1.199, P = 0.464		F 1,35 = 5.478, P < 0.001		F 1,35 = 2.429, P = 0.002		F 1,35 = 5.227, P < 0.001
	Source	df	MS Pseudo-F	P	MS	Pseudo-F	P	MS	Pseudo-F	P	MS	Pseudo-F	P
	Traffic = T	1	12.2	3.14 0.160	14.9	1.64 0.328	76.1	3.85 0.111	131.9	3.23 0.170
	Starting date = S	1	0.0	0.00 0.994	8.2	1.87 0.240	2.1	0.23 0.650	1.0	0.23 0.642
	Age = A	2	19.4	13.43 0.001	20.0	6.49 0.010	6.9	1.19 0.414	122.2	8.17 0.003
	Site(T) = Si	4	3.3	3.04 0.162	11.7	2.60 0.206	20.2	2.19 0.228	35.9	8.26 0.036
	T × S	1	1.0	0.99 0.359	0.3	0.07 0.791	2.4	0.26 0.629	7.0	1.65 0.262
	T × A	2	1.6	1.12 0.425	0.1	0.52 0.799	8.4	2.07 0.175	6.0	0.61 0.737
	S × A	2	0.3	0.68 0.518	1.7	1.58 0.254	2.7	1.25 0.358	1.5	0.46 0.658
	Si(T) × S	4	1.1	13.43 <0.001	4.5	15.40 <0.001	9.2	15.90 <0.001	4.3	6.79 <0.001
	Si(T) × A	8	1.2	2.61 0.099	1.6	1.42 0.310	5.0	2.25 0.140	14.2	4.34 0.028
	T × S × A	2	0.6	1.43 0.296	0.7	0.66 0.534	0.2	0.08 0.926	1.2	0.36 0.711
	Si(T) × S × A	8	0.5	5.74 <0.001	1.1	3.78 0.001	2.2	3.85 <0.001	3.3	5.12 <0.001
	Res.	232	0.1			0.3			0.6			0.6
	Total	267

Table 2 .

 2 Results of PERMANOVA tests for differences in sessile taxa cover among levels of the main factors (traffic, starting date, age and site) and their interactions. Tests are presented for all, non-indigenous, cryptogenic and native species, separately. Post-hoc pairwise tests are summarized in Appendix S9.

	Cover (%)		Total			NIS			Cryptogenic		Native
	Transformation		none			none			none			none
	PERMDISP		F 1,35 = 2.933, P < 0.001		F 1,35 = 11.050, P < 0.001	F 1,35 = 8.518, P < 0.001		F 1,35 = 6.804, P < 0.001
	Source	df	MS Pseudo-F	P	MS	Pseudo-F	P	MS	Pseudo-F	P	MS	Pseudo-F	P
	Traffic = T	1 30415.0	0.43 0.763	3848.3	1.70 0.327	22184.0	1.95 0.277	24686.0	6.09 0.057
	Starting date = S	1	5568.8	1.02 0.360	2148.7	3.38 0.146	7011.5	0.67 0.454	5124.7	0.49 0.513
	Age = A	2 135330.0	8.13 0.004	9494.6	5.15 0.022	2790.4	0.53 0.797	19033.0	4.18 0.034
	Site(T) = Si	4 66246.0	11.90 0.023	2524.8	3.91 0.094	16589.0	1.54 0.336	3880.6	0.36 0.839
	T × S	1 18956.0	3.47 0.148	162.0	0.25 0.640	546.8	0.05 0.834	1968.3	0.19 0.658
	T × A	2	3733.2	0.74 0.662	920.9	1.12 0.447	3071.6	0.78 0.635	5732.6	1.55 0.280
	S × A	2 11498.0	3.58 0.087	1122.3	1.44 0.287	3047.5	2.02 0.197	1715.9	0.58 0.581
	Si(T) × S	4	5569.2	12.64 <0.001	645.2	3.29 0.014	10749.0	42.26 <0.001	10663.0	119.35 <0.001
	Si(T) × A	8	5670.3	1.73 0.232	893.2	1.13 0.438	5129.3	3.34 0.053	3631.8	1.20 0.405
	T × S × A	2	3892.8	1.21 0.343	645.9	0.83 0.467	833.8	0.55 0.596	2079.5	0.70 0.522
	Si(T) × S × A	8	3278.6	7.44 <0.001	791.5	4.03 <0.001	1537.4	6.04 <0.001	3032.9	33.95 <0.001
	Res.	232	440.8			196.4			254.4			89.3
	Total	267

Table 3 .

 3 Results of PERMANOVA tests for differences in community structure among levels of the main factors (traffic, starting date, age and 794 site) and their interactions. Tests are presented for all, non-indigenous, cryptogenic and native species separately. Post-hoc pairwise tests are 795

	796	summarized in Appendix S9.
		Community	Total				NIS				Cryptogenic			Native
			square-
		Transformation	root				square-root			square-root			square-root
		PERMDISP	F 1,35 = 5.628, P < 0.001		F 1,26 = 3.049, P = 0.017		F 1,35 = 8.216, P < 0.001		F 1,27 = 7.580, P < 0.001
					Pseudo-				Pseudo-				Pseudo-				Pseudo-
		Source	df	MS	F	P	df	MS	F	P	df	MS	F	P	df	MS	F	P
		Traffic = T	82673.0	1.31 0.365	1 10978.0	1.05 0.440	1 119060.0	2.53 0.150	1 20885.0	1.47	0.146
		Starting date = S	15105.0	1.54 0.238	1 2576.5	1.44 0.340	1 12569.0	1.46 0.262	1 7040.3	1.95	0.212
		Age = A	57578.0	3.67 0.008	2 6360.8	1.38 0.293	2 13968.0	1.21 0.288	2 21733.0	1.76	0.127
		Site(T) = Si	64219.0	6.44 <0.001	4 24980.0	7.83 0.001	4 52250.0	5.29 <0.001	4 34322.0	3.64 <0.001
		T × S	7766.1	0.79 0.549	1 3304.5	1.64 0.280	1	5998.5	0.70 0.616	1 7770.6	1.99	0.201
		T × A	7594.0	0.95 0.552	2 2316.0	0.97 0.565	2	8961.3	1.08 0.403	2 15629.0	1.21	0.356
		S × A	6876.6	1.12 0.380	2 3014.7	0.81 0.605	2	6108.0	0.88 0.563	2 7671.4	1.34	0.310
		Si(T) × S	9972.9	19.27 <0.001	4 3492.9	3.49 0.001	4	9885.4	8.01 <0.001	4 9612.5	8.58 <0.001
		Si(T) × A	10725.0	1.71 0.017	6 6553.4 c	1.38 0.376	8 12917.0	1.62 0.030	5 10475 c	1.77	0.118
		T × S × A	3919.7	0.64 0.769	1 2217.8 c	0.51 0.627	2	3587.6	0.52 0.905	1 9209.2 c	1.66	0.204
		Si(T) × S × A	6289.0	12.15 <0.001	2 4595.8 c	4.60 <0.001	8	7957.3	6.45 <0.001	4 5951.4 c	5.31 <0.001
		Res.	232	517.5			121 1000.0			202	1233.6			165 1119.8
		Total	267				147				237				192

c : the corresponding term has one or more empty cells 797 Figures Figure 1.

Table S3 .

 S3 List and relative abundance of species identified during rapid assessment surveys performed around experimental units within each site at two dates (November 2016 & June 2017). Relative abundances were estimated using the SACFOR scale: ******: Superabundant, *****: Abundant, ****: Common, ***: Frequent, **: Occasional, *: Rare. Status and main trophic group (TG) are indicated: carnivores (C), suspension feeders (SF), herbivores (G), primary producers (PP)

	Anthothoe chilensis Austromegabalanus psittacus Sicyases sanguineus	Native Native Native	C SF C		***** ****	*	*	**** *	**** *	*	**	****** *****	***** **** * *
	Phymactis papillosa Balanus laevis Helcogrammoides cunninghami	Native Native Native	C SF C					*	**	* ****	***	*	*** *	*** * *	* ****	* **
	Oulactis concinnata Notobalanus flosculus Leptonotus blainvilleanus	Native Native Native	C SF C		* *						**** ***** *	***	**	*
	Traffic Site TG Date C SF C Unassigned SF Status Native Native Native Native C Llico Unassigned SF Unassigned C Native C Status Cryptogenic SF Native C Chlorophyta Bryozoa Chlorophyta Unassigned PP Cryptogenic SF Native SF Unassigned NIS SF Native C Ochrophyta Ochrophyta NIS SF Native C Unassigned * Unassigned PP Native PP Cauloramphus spiniferum Taxon Acontiaria sp. 2 Verruca laevigata Blenniidae Traffic Ischyroceridae (Tubes) Prolatilus jugularis Site Aetea sp. Caprellidae Otaria flavescens Taxon Alcyonidiodies mytilii Rhynchocinetes typus Ulva sp. Amathia cf. gracilis Petrolisthes laevigatus Ulva sp. Bugula neritina Homalaspis plana Ectocarpales Bugulina flabellata Metacarcinus edwardsii Ectocarpales Macrocystis pyrifera Cryptogenic SF Romaleon setosum Native C Macrocystis pyrifera Celleporella cf. hyalina Cryptogenic SF Cancer plebejus Native C Bacillariophyceae Unassigned *** Rhodophyta Antithamnionella ternifolia NIS PP Electra monostachys Cryptogenic SF Cancer porteri Native C Rhodophyta Crustose Corallinales Unassigned PP Exochella n. sp. NIS SF Taliepus dentalus Native C Antithamnionella ternifolia Unassigned Gigartinales Unassigned PP Lagenicella variablilis Native SF Pilumnoides perlatus Native C Crustose Corallinales Unassigned Mazaella laminiorides Native PP Schyzoporella maulina Native SF Neotrypaea uncinata Native C Gigartinales Non-clacareous crust Unassigned PP Scruparia ambigua Native SF Caridea Native C Mazzaella laminarioides Native ** Rhodymeniales Unassigned PP Membranipora isabelleana Native SF Alpheidae Native C Non-clacareous crust Unassigned * Tricellaria acuelata Native SF Echinodermata Rhodymeniales Unassigned Porifera Clathria sp. Unassigned SF Annelida Heterocucumis godeffroyi Native SF Ciliophora Haliclona sp. Sabellariidae Unassigned SF Ophiactis sp. Unassigned SF Ciliophora indet. Unassigned ** Unassigned SF Leucosolenia sp. Unassigned SF Spirorbis sp Cryptogenic SF Patiria chilensis Native C Brachiopoda Mollusca Asteriidae Unassigned C Discinisca cf. tenuis Unassigned Cnidaria Amphisbetia operculata Cryptogenic SF Choromytilus chorus Native SF Arbacia dufresnii Native C Porifera Bougainvillia sp. (B. muscus ) NIS SF Aulacomya atra Native SF Stichaster striatus Native C Leucosolenia sp. Unassigned * Clytia linearis Cryptogenic SF Semimytilus algosus Native SF Tetrapygus niger Native C Cnidaria Coryne eximia Cryptogenic SF Mytilus cf. edulis Cryptogenic SF Meyenaster gelatinosus Native C Amphisbetia operculata Cryptogenic Ectopleura crocea NIS SF Hiatella sp. Unassigned SF Chordata Bougainvillia sp. (B. muscus ) NIS * Ectopleura larynx NIS SF Chiton cumingsii Native H Aplidium peruvianum Native SF Clytia linearis Cryptogenic ** Eudendrium sp. Unassigned SF Acanthina monodon Native C Asterocarpa humilis NIS SF Coryne eximia Cryptogenic * Halecium sp. Unassigned SF Tegula spp. Native H Ciona robusta NIS SF Ectopleura spp. (E. crocea & E. Larynx) NIS Hydractinia pacifica Native SF Fissurella spp. Native H Corella eumyota Cryptogenic SF Eudendrium sp. Unassigned Hydractinia sp. NIS SF Concholepas concholepas Native C Didemnum studeri Native SF Halecium sp. Unassigned Obelia spp. Cryptogenic SF Crepipatella fecunda Native H Diplosoma listerianum NIS SF Halecium sp2. Unassigned Orthopyxis sp. (O. mollis ) Native SF Thecacera darwini Native C Paramolgula sp. Native SF Hydractinia pacifica Native Phiaella cf. quadrata Cryptogenic SF Phidiana lottini Native C Pyura chilensis Native SF Hydractinia sp. NIS Plumularia setacea Cryptogenic SF Robsonella fontaniana Native C Bovichtus chilensis Native C Obelia geniculata Cryptogenic ** Sertularellidae Unassigned SF Xanthochorus cassidiformis Native C Genypterus chilensis Native C Obelia dichotoma Cryptogenic **** Cereus herpetodes Native C Crustacea Hypsoblennius sordidus Native C Orthopyxis sp. (O. mollis ) Native	Nov16 Apr17 Nov16 Apr17 Nov16 Apr17 Local Llico Chome Coliumo ***** ****** * Chome Coliumo Coronel San V. Lirquén ****** ****** * * * * ** * ** **** **** *** ** **** * ** * * * ** ** * * * **** ** * ** ** * * * ** **** ** * * * ** ** ** **** **** ** ***** *** **** * * * ** **** ****** ****** **** ***** ** ** * * * ****** ***** * ** *** *** * ** * **** ** * ** ** ** * * * ** ** **** **** * * **** ** ** * * **** ** * * ** * *** **** * * * ** *** * **** ***** ***** *** **** ***** ** * **** * ** * ***** * * * ***** *** ***** * * * * ** ***** ** **** **** **** **** **** * * *** * * * * *** * * * ** * ** *** * **** **** **** * * * ** * ** ** * ** ** * ****** **** ** * ** ***** ***** * * ** *** ** *** * **** ** * **** ** **** ** * ** *	***** ***** * Coronel * * Nov16 Apr17 Nov16 Apr17 Nov16 Apr17 International San Vicente Lirquén * * ** ** * *** * * * * ** ** * * * * * * * * * * * ** *** * *** *** *** ** ** *** * *** *** *** * **** * ** **** * * ** ** *** ** ** * * *** * * * * * * * *** ***** * * ****** ****** ** * * * *** ** * ** ** **** ***** ***** ***** **** ** **** ***** * *** *** * * ** *** *** * ****** ***** ** ***** ** ***** ** * ***** ***** * *** *** * * *** ** * ***** ***** * ***** **** * * ***** * ** * * * * * * * * * * * * * * * ** * * * * * * ** * ** ***** **** * *** * ***** ***** **** ***** **** ***** * * * * *** *** ** * * * * * * *** *** **** **** **** **** **** * *** **** ***** * * **** *** * **
	Phiaella cf. quadrata	Cryptogenic	*					**	***
	Plumularia setacea	Cryptogenic	****		*			******
	Cereus herpetodes	Native	*
	Acontiaria sp. 2	Native				****
		Bryozoa
	Alcyonidiodies mytilii	Cryptogenic	*			* *		* *	* *
	Amathia cf. gracilis	Cryptogenic	**		**	*		****	***
	Amathia sp. (A. imbricata )	NIS					*
	Bugula neritina	NIS				*				*
	Bugulina flabellata	NIS		****		*	***		**	*****
	Cauloramphus spiniferum	Cryptogenic		**
	Celleporella cf. hyalina	Cryptogenic	**	****	******				*
	Electra monostachys	Cryptogenic				*
	Exochella n. sp.	NIS			**	****			***
	Lagenicella variablilis	Native	**	*
	Schyzoporella maulina	Native			**
	Scruparia ambigua	Native	*					* *
	Tricellaria acuelata	Native	**
	Tubulipora sp.	Unassigned	*
	Turbicellepora sp.	Unassigned		*

Table S4 .

 S4 List and relative abundance of sessile taxa identified on settlement plates throughout the experiment. Relative abundances are expressed according to occurrences: ******: 80-100%, *****: 60-79%, ****: 40-59%, ***: 20-39%, **: 5-19%, *: 0-4%. Status are indicated. Local International

Method related to sediment conditions: In the laboratory, sediment samples were lyophilized and pulverized. From each samples, 3 replicates were taken as quality control.

The pH was measured in 1:2.5 sediment to water ratio using an electrode. The organic matter content (%OM) was determined after calcination at ca. 550ºC. Metal contents (of Al, Cr, Cu, Fe, Hg, Mn, Ni, Pb, V and Zn) were determined using total Ray X reflexion fluorescence (TXRF) analysis [START_REF] Towett | Quantification of total element concentrations in soils using total X-ray fluorescence spectroscopy (TXRF)[END_REF]. Initially, the samples were digested by aqua regia solutions (ISO 11466.2002) (1:3 HNO 3 /HCl) in a digester brand SCP SCIENCE, model DigiPrep. Samples were then siliconized: ten µL silicone solution in isopropanol were dropped onto the surface of the quartz carriers and dried upon a hot plate (110 °C). Then, ten microliters of each digestion sample containing 1000 µg L -1 Ga as internal standard were dropped onto the siliconized carriers. Finally, samples were drought at 80°C before TWRF analyses. The benchtop TXRF system (S4 TStar, Bruker® AXS Microanalysis GmbH, Berlin, Germany) is equipped with a 50 W X-ray tube with molybdenum (Mo) anode and a multilayer monochromator (17.5 keV). The characteristic radiation emitted by elements present in the sample is detected by a silicon drift detector with 60 mm 2 area and an energy resolution < 145 eV, working at 50 kV and 750 µA in air. Measurement live time is 500 s. Qualitative analysis and spectra deconvolution were performed using the spectrometer software (Tstar 1.0, Bruker). Elements are identified automatically referring to the software database. Deconvolution is done with the "super bias optimized fit" function of the SPECTRA software, which calculates it on basis of measured reference spectra present in the software database. The background correction is carried out in a maximum value of 1000 iterations. Supplement S6. Detailed richness of sessile taxa identified on panels within each site per sampling occasion.

Figure S6

. Sessile taxa (total, NIS, cryptogenic and native) richness on the experimental panels collected after 1, 3 and 7 months in all study sites at each sampling occasion (± S.E.). Legend is the same as in figure S5.

Supplement S7. Detailed abundance of sessile taxa identified on panels within each site per sampling occasion.

Figure S7

. Sessile taxa (total, NIS, cryptogenic and native) cover (%) on the experimental panels collected after 1, 3 and 7 months in all study sites at each sampling occasion (± S.E.). Legend is the same as in figure S5.

Supplement S8. Detailed multivariate structure of sessile taxa identified on panels within each site per sampling occasion.

Figure S8

. Principal coordinate analyses describing the assemblage structure of all, non-native, cryptogenic and native species on the panels collected after 1, 3 and 7 months in all study sites for each trial ( w : winter and s : summer). Vector plots of variables correlated with the PCO axes are indicated within the same panels, with r > 0.6 for all species and r > 0.4 for NIS, cryptogenic and native species.

Supplement S9. Summary of pairwise tests following univariate and multivariate PERMANOVAs.

Figure S9

. Summary of pairwise tests following univariate and multivariate PERMANOVAs testing for differences in sessile taxa richness, cover and community structure. Tests are presented for all, non-indigenous (NIS), cryptogenic (Cry.) and native (Nat.) species, separately. Results are illustrated as changes between levels of the factor 'time' (panels collected after 1, 3 or 7 months) within each site and at each sampling occasion (first and second trials are illustrated within left and right corners, respectively. Neutral (white), positive (pale green), negative (purple) and non-directional (grey) changes were considered at α = 0.05 (based on Monte Carlo procedure).