Melanie Bon
email: melanie.bon@gmail.com

Céline Labrune

Joao B Gusmao

Maritza Fajardo

Chris Harrod

François Le Loc'h

Aldo S Pacheco

Jacques Grall

Germán Amezaga

Assessing benthic ecological status with biotic indices: an upwelling ecosystem quality paradox?

To my friend Carlos Álvarez Mazú and my cousin Basile Verrier who shared my passion for the ocean and already left us A mi amigo Carlos Álvarez Mazú y mi primo Basile Verrier que compartían mi pasión por el océano y ya se nos fueron A mon ami Carlos Álvarez Mazú et mon cousin Basile Verrier qui partageaient ma passion pour l'océan et nous ont déjà quitté

RÉSUMÉ SUBSTANTIEL (in French)

Les écosystèmes marins côtiers La zone marine côtière est composée d'une grande diversité d'habitats abritant une richesse spécifique importante qui offre de nombreux biens et services écosystémiques (sensu [START_REF] Costanza | The value of the world ' s ecosystem services and natural capital[END_REF][START_REF] Costello | Marine Biodiversity, Biogeography, Deep-Sea Gradients, and Conservation[END_REF][START_REF] Leo | Coastal habitat squeeze: A review of adaptation solutions for saltmarsh, mangrove and beach habitats[END_REF] ayant la plus grande valeur globale de la planète malgré qu'ils ne couvrent que 6% de la superficie totale [START_REF] Costanza | Changes in the global value of ecosystem services[END_REF]. Ces bénéfices comprennent entre autres, des ressources alimentaires (83% des pêches mondiales; [START_REF] Worm | Impacts of Biodiversity Loss on Ocean Ecosystem Services[END_REF], la protection des côtes, le piégeage du carbone [START_REF] Ray | Coastal-Zone biodiversity patterns[END_REF], la régulation du climat, et l'amélioration de la qualité des eaux [START_REF] Douglas | Macrofaunal Functional Diversity Provides Resilience to Nutrient Enrichment in Coastal Sediments[END_REF]. Cette zone est cependant parmi les plus menacées par les activités humaines [START_REF] Lotze | Depletion degradation, and recovery potential of estuaries and coastal seas[END_REF][START_REF] Halpern | A global map of human impact on marine ecosystems[END_REF][START_REF] Jones | The location and protection status of Earth's diminishing marine wilderness[END_REF] notamment par l'apport de nutriments (eutrophisation) provenant de l'agriculture, des industries et des eaux usées qui favorise les producteurs primaires y compris la prolifération d'algues toxiques [START_REF] Beman | Agricultural runoff fuels large phytoplankton blooms in vulnerable areas of the ocean[END_REF][START_REF] Heisler | Eutrophication and harmful algal blooms: A scientific consensus[END_REF][START_REF] Van Beusekom | Eutrophication[END_REF]. Ceci augmente la quantité de matière organique qui sédimente et sa décomposition par les microorganismes peut provoquer de l'hypoxie ou de l'anoxie impactant les organismes vivants et pouvant provoquer des mortalités massives [START_REF] Grall | Marine eutrophication and benthos: The need for new approaches and concepts[END_REF][START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Rabalais | Dynamics and distribution of natural and human-caused hypoxia[END_REF][START_REF] Stauffer | An oceanographic, meteorological, and biological "perfect storm" yields a massive fish kill[END_REF]. L'extraction des ressources marines peut exacerber ces effets, notamment celle des suspensivores qui réduisent naturellement la matière organique de la colonne d'eau [START_REF] Jackson | Historical overfishing and the recent collapse of coastal ecosystems[END_REF]. Les milieux côtiers sont aussi impactés par les changements climatiques dûs à l'homme comme la diminution du pH, l'augmentation des températures et l'extension des zones hypoxiques depuis les années 1960 [START_REF] Diaz | Spreading dead zones and consequences for marine ecosystems[END_REF][START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF]. L'ensemble de ces pressions a un fort impact sur la biodiversité marine et altère les services écosystémiques au point de menacer sérieusement la sécurité alimentaire et le bien-être humain [START_REF] Jackson | Historical overfishing and the recent collapse of coastal ecosystems[END_REF][START_REF] Worm | Impacts of Biodiversity Loss on Ocean Ecosystem Services[END_REF]IPBES, 2019). Ces pressions viennent s'ajouter aux conditions environnementales naturelles qui façonnent déjà les écosystèmes côtiers. Les estuaires par exemple, sont des zones de transition entre les fleuves et l'océan qui présentent un gradient de salinité, de température et de luminosité ainsi que des variations d'oxygène, de matière organique et de turbidité qui varient avec les marées et permettent seulement aux organismes les plus tolérants de s'y développer.

Les systèmes d'upwelling

Les upwellings sont des remontés d'eau froide profonde souvent pauvre en oxygène et riche en nutriments qui, lorsqu'ils pénètrent la zone photique, boostent les productions primaires et secondaires formant des écosystèmes parmi les plus productifs de la planète (Kampf et Chapman, 2016;[START_REF] Chan | The dynamics and impact of ocean acidification and hypoxia[END_REF][START_REF] Largier | Upwelling Bays: How Coastal Upwelling Controls Circulation, Habitat, and Productivity in Bays[END_REF]. Les quatre principaux systèmes d'upwelling sont associés au courant du Benguela, de Californie, de Humboldt et des

Canaries [START_REF] Fréon | Functional group biodiversity in Eastern Boundary Upwelling Ecosystems questions the wasp-waist trophic structure[END_REF] et se caractérisent par les paramètres des sous-courants de chaque région, notamment la teneur en nutriments et en oxygène dissous. En Californie, la zone d'oxygène minimale (O 2 < 0.5 mL.L -1 ; [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF] se trouve à plus de 600 m de profondeur alors que dans les courants de Humboldt et du Benguela elle se situe entre moins de 100 m et 600 m [START_REF] Helly | Global distribution of naturally occurring marine hypoxia on continental margins[END_REF], ce qui varie en fonction de l'oscillation australe interannuelle d'El Niño. Le changement climatique devraient augmenter l'intensité des upwellings et des événements d'hypoxie et d'anoxie pouvant impacter de manière critique les pêcheries et les processus biogéochimiques et écologiques de ces écosystèmes [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Grantham | Upwelling-driven nearshore hypoxia signals ecosystem and oceanographic changes in the northeast Pacific[END_REF][START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF][START_REF] Sydeman | Climate change and wind intensification in coastal upwelling ecosystems[END_REF]. Environ 20% des poissons marins sauvages sont pêchés dans les zones d'upwellings alors qu'elles représentent moins d'1% de la surface des océans [START_REF] Pauly | Primary production required to sustain global fisheries[END_REF]Chavez et Messie, 2009). Ces pêches sont supportées par une forte production primaire et secondaire qui, lorsqu'elle se décompose, peut engendrer une importante réduction de l'oxygène dissous surtout au niveau du sédiment participant à la formation de la zone d'oxygène minimale [START_REF] Kämpf | Upwelling systems of the world[END_REF]. Les nutriments, qu'ils proviennent d'un phénomène naturel ou d'une activité anthropique, mènent à l'eutrophisation qui peut produire de l'hypoxie et un enrichissement en matière organique du sédiment (Levin et al., 2009a). La macrofaune benthique qui vit dans ces fonds est généralement dominées par quelques polychètes bien adaptés à ces conditions et contrôlées par des facteurs environnementaux comme l'oxygène et la concentration en matière organique [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Gutiérrez | Oxygenation episodes on the continental shelf of central Peru: Remote forcing and benthic ecosystem response[END_REF][START_REF] Soto | Influence of organic matter inputs and grain size on soft-bottom macrobenthic biodiversity in the upwelling ecosystem of central Chile[END_REF]. Cependant, le fonctionnement écologique de ces communautés benthiques est très peu connu [START_REF] Sivadas | Trait-based and taxonomic macrofauna community patterns in the upwelling ecosystem of the southeastern Arabian sea[END_REF].

Le système côtier du nord du Chili est un laboratoire naturel pour étudier le fonctionnement de ces communautés benthiques. Les précipitations sont négligeables le long de la côte du désert d'Atacama ce qui évite la confusion avec les nutriments apportés par les ruissellements terrestres [START_REF] Vargas | Aluviones históricos en Antofagasta y su relación con eventos El Niño/Oscilación del Sur[END_REF][START_REF] Guiñez | Variabilidad espacial y temporal de la materia orgánica sedimentaria, asociada a la Zona de Mínimo Oxígeno (ZMO), en un ambiente costero del norte de la corriente de Humboldt, bahía de Mejillones, Chile[END_REF][START_REF] Salazar | From the use of space to territorialisation during the Early Holocene in Taltal, coastal Atacama Desert, Chile[END_REF]. Les upwellings y sont présents quasiment tout au long de l'année et dans certaines baies où la circulation est réduite comme la baie de Mejillones, ils provoquent de l'hypoxie en dessous de 10 m de profondeur et de l'anoxie vers 40 ou 50 m [START_REF] Cerda | Variación espacial y temporal de las masas de agua, nutrientes y sedimentación de la materia orgánica e inorgánica en la bahía Mejillones del sur (23° S), Chile[END_REF][START_REF] Guiñez | Variabilidad espacial y temporal de la materia orgánica sedimentaria, asociada a la Zona de Mínimo Oxígeno (ZMO), en un ambiente costero del norte de la corriente de Humboldt, bahía de Mejillones, Chile[END_REF] avec occasionnellement un peu d'oxygénation [START_REF] Escribano | Understanding upwelling circulation off Antofagasta (northern Chile): A three-dimensional numerical-modeling approach[END_REF]Valdés et al. 2006).

Le fonctionnement des communautés benthiques qui résident à faibles profondeurs (10 m) est caractérisé par des voies d'alimentation variées et une forte bioturbation alors que les communautés plus profondes (50 m), là où la matière organique est plus importante présentent des voies d'alimentation moins variées ainsi que moins de structures biologiques et de bioturbations [START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. Dans cette thèse, ces communautés macrobenthiques ont été étudiées, y compris celle des communautés de profondeurs intermédiaires, le long d'un gradient environnemental naturel sur deux ans afin d'étudier leur réponse fonctionnelle à l'augmentation naturelle de l'hypoxie et de la concentration en matière organique liée au phénomène d'upwelling et de connaître les potentielles variations lors des épisodes d'oxygénation.

Etude du fonctionnement des communautés macrobenthiques

Pour étudier la réponse du fonctionnement de ces communautés, l'association de deux approches: les analyses des traits biologiques qui décrivent la structure de la communauté en terme de fréquence de traits biologiques [START_REF] Bremner | Methods for describing ecological functioning of marine benthic assemblages using biological traits analysis (BTA)[END_REF] et de la mesure de la diversité fonctionnelle qui examine les mécanismes qui influencent la réponse de la biodiversité au stress environnemental ainsi que son effet sur le fonctionnement de l'écosystème (Mason et Mouillot, 2013), ont montré leur efficacité dans des zones impactées par le déversement des eaux usées (ex. [START_REF] Gusmao | Functional diversity of macrobenthic assemblages decreases in response to sewage discharges[END_REF][START_REF] Llanos | Functional diversity in the intertidal macrobenthic community at sewage-affected shores from Southwestern Atlantic[END_REF]. L'analyse de traits biologiques et la diversité fonctionnelle permettent de compléter l'approche taxonomique en fournissant des informations sur le fonctionnement de l'écosystème et ses processus écologiques [START_REF] Naeem | Biodiversity and ecosystem functioning: Maintaining natural life support processes[END_REF][START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF].

L'analyse de traits biologiques décrit la structure de la communauté en termes de fréquence de traits biologiques qui permettent d'évaluer comment les traits des espèces répondent aux facteurs environnementaux [START_REF] Bremner | Assessing ecological functioning in marine benthic communities[END_REF]. La diversité fonctionnelle comme la diversité taxonomique requiert plusieurs indices tels que la richesse fonctionnelle (FRic), l'uniformité fonctionnelle (FEve) et la divergence fonctionnelle (FDiv) chacune étant liée à différents processus écologiques [START_REF] Mason | Functional richness, functional evenness and functional divergence: The primary components of functional diversity[END_REF][START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF][START_REF] Mouchet | Functional diversity measures: An overview of their redundancy and their ability to discriminate community assembly rules[END_REF]. FRic indique la diversité des traits fonctionnels de la communauté sans prendre en compte l'abondance des taxons [START_REF] Schuldt | Tree diversity promotes functional dissimilarity and maintains functional richness despite species loss in predator assemblages[END_REF]. FEve mesure si la distribution de l'abondance est régulière [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]. FDiv mesure l'écart entre le centre de gravité de l'espace fonctionnel et la distribution de l'abondance des taxons [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]. RaoQ peut aussi être utilisé pour estimer leur dispersion multivariée [START_REF] Botta-Dukát | Rao's quadratic entropy as a measure of functional diversity based on multiple traits[END_REF].

Fonctionnement des communautés benthiques sous l'influence d'upwellings

Dans cette thèse, neuf traits biologiques divisés en un total de 38 catégories ont été sélectionnés pour refléter le fonctionnement écologique des communautés de la baie de

Mejillones et sa sensibilité aux perturbations environnementales [START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. Ils ont permis de mettre en évidence la présence de trois types de communautés benthiques fonctionnelles différentes (Figure II.7). Le premier type de communauté caractérisée par des organismes de grande taille, à longue durée de vie, et aux structures biogéniques telles que les coquilles offrant de nombreux microhabitats est présent dans les zones peu profondes où il y a peu de matière organique et les concentrations d'oxygène sont proches de l'hypoxie. Les communautés benthiques des zones les plus profondes influencées par une hypoxie sévère et de fortes concentrations en matière organique sont associées à des organismes de petite taille, de cycles de vie courts et à la reproduction asexuée et donc possédant d'importantes adaptations à l'hypoxie. Elles sont similaires aux communautés benthiques décrites sous influence d'hypoxie saisonnière [START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF]. Les communautés des habitats intermédiaires sont associées à des catégories de traits qui font transition entre les deux autres types de communautés, par exemple dans les zones peu profondes les corps des organismes sont fortement protégés par des coquilles, dans les zones profondes ils ne sont pas protégés du tout alors que dans les zones intermédiaires, ils sont légèrement protégés par exemple par des tubes.

Dans ces communautés, les dépositivores et filtreurs bénéficient d'une importante quantité de matière organique et profitent de leur structures alimentaires ramifiées pour mieux capter l'oxygène (Levin et al., 2009b). La présence de carnivores n'a pas été reporté dans les études d'hypoxie d'origine anthropique (Wu, 1982;Weston, 1990) ce qui peut être due au faite que l'hypoxie naturelle dans cette baie est permanente permettant des stratégies alimentaires relativement diversifiées et la présence d'organismes sessiles bien adaptés.

Les indices fonctionnels ont permis de comprendre que les différences entre ces trois types de communautés sont dues à la perte de catégories de traits non adaptés aux conditions environnementales présentes [START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF] de la même façon que dans des zones de l'estran rocheux affectées par l'évacuation des égouts [START_REF] Garaffo | Assessing functional diversity of macrobenthic assemblages in sewage-affected intertidal shores[END_REF]. Cette étude montre aussi la présence d'un seuil à 2 mL.L -1 O 2 pour les richesses taxonomique et fonctionnelle en dessous duquel elles décroissent radicalement. Les catégories de traits sensibles disparaissent progressivement le long du gradient environnemental alors que les organismes spécialisés et opportunistes apparaissent ce qui peut être attribué au filtre environnemental et à la diminution des intéractions biologiques (Perronnet et al., 2017).

L'événement d'oxygénation qui a eu lieu pendant les deux années d'échantillonnage n'a pas été suivi par un changement fonctionnel détectable ce qui peut s'expliquer par des changements plutôt progressifs sur le long terme qui pourraient être observés lors d'oxygénations plus longues comme celles décrites pendant El Niño. L'étude de ces changements et les mécanismes qui les contrôlent permettraient de mieux prévoir les effets sur le long terme du nombre croissant d'écosystèmes impactés par l'hypoxie et l'eutrophisation ainsi que l'étude du fonctionnement des communautés benthiques si l'hypoxie naturelle est saisonnière ou épisodique comme dans le centre et sud du Chili. Plus d'information est nécessaire concernant la macrofaune de l'hémisphère sud pour améliorer la compréhension du fonctionnement de ces communautés. Une évolution de la méthode vers l'utilisation de traits quantitatifs mesurables permettrait d'inclure la diversité interspécifique et d'étudier des organismes peu connus.

Le macrobenthos marin côtier et les perturbations

Les espèces macrobenthiques des fonds meubles accomplissent de nombreuses fonctions telles que la reminéralisation, le stockage du carbone, les échanges entre la colonne d'eau et le fond [START_REF] Virnstein | Predation on estuarine infauna: Response patterns of component species[END_REF][START_REF] Reise | Sediment mediated species interactions in coastal waters[END_REF][START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF] qui peuvent être perturbées par des phénomènes anthropiques ou naturels comme l'hypoxie éliminant les espèces sensibles [START_REF] Villnäs | Consequences of Increasing Hypoxic Disturbance on Benthic Communities and Ecosystem Functioning[END_REF]. Le modèle de Pearson et Rosenberg (1978) décrit la succession écologique des communautés macrobenthiques en fonction de l'enrichissement en matière organique qui a une richesse spécifique et une biomasse relativement faible quand la concentration en matière organique est faible. Quand la concentration en matière organique est plus importante, les espèces sensibles disparaissent et les espèces opportunistes occupent l'espace, s'accaparent la totalité des ressources ce qui résulte en un pic d'abondance et de biomasse. Si la quantité de matière organique est trop importante et induit de l'hypoxie, elle peut être néfaste pour toutes les espèces.

L'évaluation des effets des perturbations

Grâce à leur mobilité réduite, leur durée de vie relativement longue et les différents niveaux de tolérance des taxons qui les composent [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF][START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF][START_REF] Vaquer-Sunyer | Thresholds of hypoxia for marine biodiversity[END_REF], les communautés benthiques peuvent servir d'indicateurs en complément de mesures abiotiques pour évaluer la perturbation d'un habitat donné [START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF][START_REF] Bustos-Baez | Using indicator species to assess the state of macrobenthic communities[END_REF][START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF] et sont incluses dans le suivi de qualités des écosystèmes dans de nombreuses régions du monde (ex., Water Framework Directive, 2000). La structure des communautés benthiques est simplifiée sous forme d'indices biotiques qui mesurent de manière quantitative le degré de perturbations anthropiques d'un habitat donné à un moment donné. Ils peuvent être comparés dans le temps et dans l'espace et sont facilement interprétables par les preneurs de décisions [START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. La plupart de ces indices ont été établis pour une région particulière principalement en Europe et aux Etats-Unis mais certains ont été utilisés avec succès dans d'autres régions comme l'index des polychètes opportunistes et des amphipodes (BOPA; [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]. Il existe plus de 30 indices basés sur le macrobenthos des fonds meubles (Tableau I.1) qui sont généralement composés de plusieurs mesures telles que la richesse spécifique, l'abondance et la biomasse et une estimation de la biodiversité. Les résultats de ces indices sont classés sur une échelle standardisée comprenant cinq statuts de qualité écologique (Tableau I.2) par la directive européenne cadre sur l'eau (Water Framework Directive, 2000). La plupart de ces indices reposent sur les espèces sensibles qui disparaissent lors de perturbations anthropiques comme l'enrichissement en matière organique [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF]Levin, 2009a) et sont très utiles dans de nombreux systèmes côtiers. Cependant dans les milieux naturellement stressés comme les estuaires où le gradient de salinité sélectionne naturellement les espèces opportunistes, peu sensibles aux perturbations, il est difficile d'évaluer l'impact des activités humaines [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF][START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF]. Les upwellings sont des milieux naturellement riches en matière organique et parfois hypoxique surtout au niveau des sédiments, ce sont donc les mêmes facteurs environnementaux qui sont influencés aussi bien par les activités humaines que par le phénomène naturel. Dans cette thèse, la capacité de différents indices biotiques à détecter l'effet potentiel de l'enrichissement en matière organique et de l'hypoxie associé à l'activité humaine est évaluée dans la baie de Mejillones au nord du Chili, qui est naturellement hypoxique et riche en matière organique dû au phénomène d'upwelling.

Evaluation du statut écologique basée sur les communautés macrobenthiques dans une baie influencée par les upwellings

La baie de Mejillones a une très riche productivité primaire qui supporte une pêche industrielle importante de petits poissons pélagiques et une pêche artisanale de faune benthique, surtout crustacés et mollusques due à sa proximité d'un centre de remontée d'eaux profondes [START_REF] Romero | Análisis de la macrofauna bentónica del sublitoral de la bahía de Mejillones del Sur[END_REF]. Cependant ces dernières années, des mortalités massives de poissons et d'invertébrés ont été observées à plusieurs reprises et associées à l'hypoxie sans connaître précisément son origine. Or depuis les années 1960, la partie centrale de la baie a connu un développement industriel rapide et est aujourd'hui une des zones industrielles les plus importantes du nord du Chili [START_REF] Valdés | Petrographic and geochemical study of organic matter in surficial laminated sediments from an upwelling system (Mejillones del Sur Bay, Northern Chile)[END_REF].

Différents indices biotiques ont donc été testés dans cette baie. Certains basés sur la tolérance et la sensibilité des taxons aux perturbations comme l'index marin biotique de l'AZTI (AMBI; [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. Cet indice utilise des catégories pré-établies en 5 groupes écologiques [START_REF] Grall | Using biotic indices to estimate macrobenthic community perturbations in the Bay of Brest[END_REF][START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. L'indice de qualité benthique (BQI) calcule la sensibilité de chaque espèce en se basant sur des données empiriques [START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF]. Deux indices basés sur les conditions de référence ont été évalués: l'AMBI multivarié qui se calcule à partir de l'AMBI, la richesse spécifique et l'indice de Shannon et inclut des valeurs de référence arbitraires [START_REF] Muxika | Using historical data, expert judgement and multivariate analysis in assessing reference conditions and benthic ecological status, according to the European Water Framework Directive[END_REF], et l'indice benthique d'utilisation générale (GPBI) qui compare la composition et la structure en macrofaune benthique avec une station de référence possédant une bonne condition écologique [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF]. Deux indices basés sur le fonctionnement de la communauté: FRic (volume du polygone convex de l'espace occupé par la communauté) et FDis (distance moyenne entre le centroid de tous les taxa et celui de chaque taxon pondérée par son abondance), ils ont été calculé en utilisant les catégories de traits et aussi les valeurs isotopiques des organismes présents [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF][START_REF] Laliberté | A distance-based framework for measuring functional diversity from multiple traits A distance-based framework for measuring from multiple traits functional diversity[END_REF][START_REF] Rigolet | Investigating isotopic functional indices to reveal changes in the structure and functioning of benthic communities 29[END_REF]. [START_REF] Fleischer | An adjustment of benthic ecological quality assessment to effects of salinity[END_REF][START_REF] Leonardsson | Theoretical and practical aspects on benthic quality assessment according to the EU-Water Framework Directive -Examples from Swedish waters[END_REF][START_REF] Villnäs | Evaluating the performance of benthic multi-metric indices across broad-scale environmental gradients[END_REF], leur utilisation est donc à éviter sur les catégories des traits biologiques permettent de mettre en évidence les mécanismes qui lient la biodiversité au fonctionnement de l'écosystème [START_REF] Casanoves | FDiversity: A software package for the integrated analysis of functional diversity[END_REF]. Ils sont moins influencés par la variation temporelle [START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF]

Les sentinelles

Les écosystèmes d'upwellings offrent un fort potentiel pour mieux comprendre les changements lents et multiples sur le long terme comme ceux qui sont attendus associés au changement climatique induit par l'être humain. Les activités humaines ont induits de nombreux facteurs de pression environnementale comme le réchauffement, l'acidification, la désoxygénation, la pollution, la destruction des habitats et les floraisons d'algues nocives qui ont rendu les régions côtières particulièrement vulnérables [START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF]. Les upwellings responsables d'environ 20% des pêcheries mondiales de poissons sauvages marins et essentiels à la biodiversité marine [START_REF] Pauly | Primary production required to sustain global fisheries[END_REF][START_REF] Block | Tracking apex marine predator movements in a dynamic ocean[END_REF] sont aussi menacés. L'exemple des mortalités massives observées dans le système d'upwelling de Californie à montrer que le suivi sur le long terme des paramètres physico-chimiques et biologiques peuvent permettre de comprendre les causes de ces mortalités [START_REF] Chan | The dynamics and impact of ocean acidification and hypoxia[END_REF]. Un suivi similaire devrait donc être rapidement mis en place dans les autres systèmes d'upwelling, particulièrement dans les systèmes de Humboldt et du Benguela où l'hypoxie sévère est plus répandue et plus fréquente. De plus, dans ces conditions le métabolisme bactérien peut générer du sulfure d'hydrogène qui est très toxique pour la plupart des organismes marins [START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF]. Il est probablement responsable de mortalités massives observées au large de la Namibie et de l'Afrique du Sud en 1997 [START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF]. De plus, le sulfure d'hydrogène peut se diffuser dans la colonne d'eau et impacter ainsi les organismes pélagiques, ce qui est régulièrement observé en Namibie [START_REF] Weeks | Massive emissions of toxic gas in the Atlantic[END_REF] et plus récemment au large du Pérou et du Chili [START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF][START_REF] Schunck | Giant Hydrogen Sulfide Plume in the Oxygen Minimum Zone off Peru Supports Chemolithoautotrophy[END_REF]. Les conséquences économiques de la diffusion du sulfure d'hydrogène peuvent être considérables et les mécanismes responsables ne sont toujours pas élucidés [START_REF] Bailey | Seasonal variation of oxygen deficiency in waters off southern South West Africa in 1975 and 1976 and its relation to the catchability and distribution of the Cape rock lobster Jasus lalandii[END_REF][START_REF] Currie | Winning ways with hydrogen sulphide on the Namibian shelf[END_REF] [START_REF] Bakun | Global climate change and intensification of coastal ocean upwelling[END_REF][START_REF] Sydeman | Climate change and wind intensification in coastal upwelling ecosystems[END_REF][START_REF] Wang | Intensification and spatial homogenization of coastal upwelling under climate change[END_REF] ce qui pourrait augmenter l'intensité et la fréquence de ces événements toxiques et hypoxiques ainsi que leur répartition spatial [START_REF] Sydeman | Climate change and wind intensification in coastal upwelling ecosystems[END_REF][START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF] engendrant des conséquences préoccupantes pour les pêcheries, l'écologie et les processus biogéochimiques de ces milieux [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Grantham | Upwelling-driven nearshore hypoxia signals ecosystem and oceanographic changes in the northeast Pacific[END_REF][START_REF] Bakun | Climate change and ocean deoxygenation within intensified surface-driven upwelling circulations[END_REF][START_REF] Salvatteci | Smaller fish species in a warm and oxygen-poor Humboldt Current system[END_REF]

LIST OF TABLES CHAPTER I -GENERAL INTRODUCTION

Marine coastal ecosystems 1.General description

The marine coastal zone is the intersection between aerial, terrestrial and marine realms.

Depending on their interaction and the influence of geomorphological (i.e., erosion or accumulation) and hydrographical processes (e.g., wave energy, tides, and terrestrial runoff), a multitude of habitats can be found in this area including rocky shores, sandy beaches, estuaries, lagoons, fjords, mudflats, and mangroves [START_REF] Mentaschi | Global long-term observations of coastal erosion and accretion[END_REF][START_REF] Leo | Coastal habitat squeeze: A review of adaptation solutions for saltmarsh, mangrove and beach habitats[END_REF][START_REF] Woodworth | Forcing Factors Affecting Sea Level Changes at the Coast[END_REF]. This diversity of habitats reflects different environmental factors and provides habitat for numerous life forms. The wider marine environment hosts about twenty times more phyla and classes compared to land despite that only 16% of named living species are marine [START_REF] Ray | Coastal-Zone biodiversity patterns[END_REF][START_REF] Costello | Global Coordination and Standardisation in Marine Biodiversity through the World Register of Marine Species (WoRMS) and Related Databases[END_REF]. Such a variety of the phyla represents many different body plans and life histories mainly concentrated in the coastal zone. Coastal areas are often characterised by increased primary and secondary productivity as well as species richness compared to the open ocean and high seas [START_REF] Costello | Marine Biodiversity, Biogeography, Deep-Sea Gradients, and Conservation[END_REF]. Each marine biological community, its physical and chemical environment and the dynamic interaction that link them in the coastal zone (i.e., marine coastal ecosystem) provides many goods and services (sensu [START_REF] Costanza | The value of the world ' s ecosystem services and natural capital[END_REF] from which humans benefit directly or indirectly.

Ecosystems functions and human benefits

Coastal areas provide the highest total global value of annual ecosystem services on earth (22%) despite representing only 6% of the Earth surface area [START_REF] Costanza | Changes in the global value of ecosystem services[END_REF].

Human benefits from marine coastal ecosystems include food provision, coastal protection, water purification, nutrient cycling, carbon sequestration, and recreational opportunities [START_REF] Buonocore | Trends and evolution in the concept of marine ecosystem services: An overview[END_REF]. They include some of the most productive ecosystems on earth (i.e., estuarine and upwelling) and produce about 20% of marine primary production. Most global fisheries yields (i.e., 83%) over the past 50 years have been produced by ecosystems located from coastal areas to the edge of the continental shelves [START_REF] Worm | Impacts of Biodiversity Loss on Ocean Ecosystem Services[END_REF].

Nursery and habitats for species subject to fisheries as well as areas of feeding and reproduction of oceanic species are found in coastal areas. They also host unique resources that are being exploited for genetic material and for medical purposes [START_REF] Barbier | The value of estuarine and coastal ecosystem services[END_REF].

Coastal ecosystems protect the coast against storms, flood and sea level rise by providing erosion control and sediment retention thanks to coastal wetlands, reefs and submerged vegetation. Carbon sequestration also occurs on continental margins which synthesise about 50% of organic carbon that is fixed in the world ocean [START_REF] Ray | Coastal-Zone biodiversity patterns[END_REF]. Coastal areas contribute to climate regulation e.g. of temperature and precipitation. They provide waste treatment, pollution control and detoxification of nutrients and other chemical products originating from riverine inputs of freshwater and sediments. Water filtering by coastal suspension feeders increase water quality and avoid algal blooms, oxygen depletion and death of shellfish and fishes [START_REF] Douglas | Macrofaunal Functional Diversity Provides Resilience to Nutrient Enrichment in Coastal Sediments[END_REF]. Other services include recreational activities and tourism (e.g., ecotourism, sport fishing and other outdoor activities), cultural, artistic, educational, spiritual and scientific values [START_REF] Costanza | The value of the world ' s ecosystem services and natural capital[END_REF]. Coastal areas have played key roles in the development of prehistorical and historical human societies [START_REF] Westley | Coastal environments and their role in prehistoric migrations[END_REF].

Despite providing this plethora of benefits, coastal marine ecosystems are also threatened by human activities.

Anthropogenic stressors

Coastal marine ecosystems are among the most severely impacted by human activities [START_REF] Lotze | Depletion degradation, and recovery potential of estuaries and coastal seas[END_REF][START_REF] Halpern | A global map of human impact on marine ecosystems[END_REF][START_REF] Jones | The location and protection status of Earth's diminishing marine wilderness[END_REF]. N utrient loads originating from agriculture, industry and sewage disposal are transported by rivers, terrestrial runoff or marine emissions to coastal waters. Such nutrient enrichment or eutrophication can promote growth of primary producers, including blooms of harmful algae [START_REF] Beman | Agricultural runoff fuels large phytoplankton blooms in vulnerable areas of the ocean[END_REF][START_REF] Heisler | Eutrophication and harmful algal blooms: A scientific consensus[END_REF][START_REF] Van Beusekom | Eutrophication[END_REF]. This production increases the biomass of organic matter that falls and deposits on the sea bottom, limiting light penetration in the water column and increasing oxygen consumption by microbiota during the decomposition of organic matter [START_REF] Diaz | Spreading dead zones and consequences for marine ecosystems[END_REF]. The resulting hypoxia or anoxia has deleterious effects for most living organisms and can lead to mass mortalities, especially if toxic hydrogen sulphide is produced [START_REF] Grall | Marine eutrophication and benthos: The need for new approaches and concepts[END_REF][START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Rabalais | Dynamics and distribution of natural and human-caused hypoxia[END_REF][START_REF] Stauffer | An oceanographic, meteorological, and biological "perfect storm" yields a massive fish kill[END_REF]. Simultaneously, strong harvesting pressure on suspension feeders which naturally reduce particulate organic matter present in the water column, can also exacerbate the effects of eutrophication including hypoxia [START_REF] Jackson | Historical overfishing and the recent collapse of coastal ecosystems[END_REF].

Extraction of marine resources for human consumption has an important impact on biodiversity (IPBES, 2019). Overfishing and ecological extinction have led to the collapse of entire ecosystems [START_REF] Jackson | Historical overfishing and the recent collapse of coastal ecosystems[END_REF]. The related loss of biodiversity is affecting marine ecosystem services [START_REF] Worm | Impacts of Biodiversity Loss on Ocean Ecosystem Services[END_REF][START_REF] Quijas | Biodiversity and Ecosystem Services[END_REF]. Significant alteration of nature around the globe related to human activities is now seriously threatening human food security and well-being (IPBES, 2019). Human induced climate change is also impacting coastal areas, resulting in reduction of water pH, increased temperature and the extension of hypoxic zones since the 1960s [START_REF] Diaz | Spreading dead zones and consequences for marine ecosystems[END_REF][START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF].

Natural pressures

Coastal ecosystems can also be shaped by naturally stressful environmental conditions. The most studied type of marine ecosystem under such conditions is probably the intertidal zone that is exposed to air during low tides and under seawater during high tides (e.g. [START_REF] Dial | Theory of marine communities: The intermediate disturbance hypothesis[END_REF][START_REF] Ricciardi | Global patterns of macroinvertebrate biomass in marine intertidal communities[END_REF][START_REF] Joseph | Resistance of benthic intertidal communities to multiple disturbances and stresses[END_REF]. Intertidal organisms have developed adaptations to cope with this stress related to drastic shifts of temperature and salinity and possibly withstand desiccation and/or strong waves action (e.g., Davison and Pearson, 1996). The location where biota can occur on the intertidal gradient depends on their ability to tolerate environmental stress [START_REF] Thompson | Rocky intertidal communities: Past environmental changes, present status and predictions for the next 25 years[END_REF].

Another coastal ecosystem that has recently attracted increased attention are estuaries, transitional waters influenced both by riverine freshwater and ocean water. A gradient of increasing salinity exists from the river to the sea which fluctuates with tides (and riverine discharge) and that creates variation in salinity, temperature and light, as well as shifts in the concentration of dissolved oxygen, organic materials and turbidity. Such variation restricts the use of estuarine habitats to tolerant organisms or those with such a mobility that they can avoid stressful conditions. Nutrient inputs derived from terrestrial habitats can also promote algal growth, increasing turbidity and limiting photosynthesis and oxygenation. Finally, upwelling ecosystems are another important type of coastal ecosystem that are subject to naturally stressful environmental conditions but have drawn little attention to date as natural stressors.

Eastern boundary upwelling systems (EBUS)

In this thesis, we will focus on Eastern boundary upwelling systems also referred to as major coastal upwelling regions which have year-round wind driven upwelling and are highly productive. However, seasonal upwelling systems are also wind driven upwelling usually due to monsoon winds or more episodically such as those from southwest India, the Gulf of Mexico or south Australia (Fig. 1). Other upwelling includes upwelling induced by boundary currents that interact with the seafloor inducing dynamic uplift due to shelf break as in the Malvinas and Agulhas banks or by tidal-mixing front as in the Yellow Sea (Kämpf and Chapman, 2016).

Physics of coastal EBUS

Eastern boundary upwelling systems (EBUS) are formed by coastal upwelling of deeper waters. Coastal upwelling is generated by equatorward winds blowing parallel to the coast which forms a lateral boundary. This process can last for several days and is then affected by the Coriolis effect, due to Earth rotation, producing an offshore flow of surface waters that is replenished by an onshore upward movement. Upwelling is defined as a significant upward water movement of at least 100 m maintained for several days to weeks [START_REF] Kämpf | Upwelling systems of the world[END_REF]. These waters are typically cold, nutrient-rich, oxygen-poor and high salinity. When reaching the euphotic zone, these nutrients fuel photosynthesis, enhancing primary and secondary productivity, and are recognized as some of the World's most productive systems [START_REF] Chan | The dynamics and impact of ocean acidification and hypoxia[END_REF][START_REF] Largier | Upwelling Bays: How Coastal Upwelling Controls Circulation, Habitat, and Productivity in Bays[END_REF]. There are four major eastern boundary upwelling systems. They are located on the eastern flanks of the major subtropical gyres in the Atlantic and Pacific oceans. The EBUS are associated with equatorward winds driving broad and slow eastern boundary currents: the Benguela, California, Iberia/Canary and Humboldt currents (the latter is also known as the Peru-Chile current) [START_REF] Fréon | Functional group biodiversity in Eastern Boundary Upwelling Ecosystems questions the wasp-waist trophic structure[END_REF]. Depending on local wind conditions in the region, upwelling can be either seasonal or quasi-permanent (Fig. 1). A distinct feature of these systems is the poleward undercurrents that play significant roles in the distribution of the nutrients and dissolved oxygen in these regions [START_REF] Kämpf | Upwelling systems of the world[END_REF]. In the California Current System (Fig. 2), the oxygen minimum zone (OMZ; where oxygen concentrations are < 0.5 mL.L -1 ; [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF] is located on the continental slope at depths greater than 600 m; while at the Humboldt and Benguela

Current systems the OMZ overlies the continental shelf between < 100 and about 600 m [START_REF] Helly | Global distribution of naturally occurring marine hypoxia on continental margins[END_REF]. The depth of these severe hypoxic conditions are impacted by the interannual El Niño Southern Oscillation warm phase (i.e., El Niño) when warm equatorial water masses poor in oxygen enter upwelling areas. El Niño can improve oxygenation in the southern hemisphere while it can cause shallower oxygen deficiency than usual off California [START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF]. Climate change is hypothesised to enhance upwelling-favourable winds in general and increase upwelling intensity [START_REF] Bakun | Global climate change and intensification of coastal ocean upwelling[END_REF][START_REF] Sydeman | Climate change and wind intensification in coastal upwelling ecosystems[END_REF]. The related increase in nutrient input will likely boost local productivity, and associated respiration leading to increased spatial and temporal coverages of hypoxia and anoxia in EBUS which would be critical for fisheries, ecological, and biogeochemical processes [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Grantham | Upwelling-driven nearshore hypoxia signals ecosystem and oceanographic changes in the northeast Pacific[END_REF][START_REF] Bakun | Climate change and ocean deoxygenation within intensified surface-driven upwelling circulations[END_REF][START_REF] Salvatteci | Smaller fish species in a warm and oxygen-poor Humboldt Current system[END_REF]. [START_REF] Chan | Emergence of anoxia in the California current large marine ecosystem[END_REF] reported severe hypoxia and the first records of anoxia over the California continental shelf that could lead to major perturbations of the structure and functioning of coastal marine ecosystems [START_REF] Doney | Climate change impacts on marine ecosystems[END_REF]. However, future scenarios at local scale are still debated in the northern Humboldt upwelling system [START_REF] Echevin | Physical and biogeochemical impacts of RCP8.5 scenario in the Peru upwelling system[END_REF].

Ecology of coastal upwelling systems

Upwelling input of nutrients from deep waters into the euphotic zones subsidises highly productive pelagic ecosystems [START_REF] Escribano | Physical-chemical processes and patterns of diversity of the Chilean eastern boundary pelagic and benthic marine ecosystems: An overview[END_REF][START_REF] Largier | Upwelling Bays: How Coastal Upwelling Controls Circulation, Habitat, and Productivity in Bays[END_REF]. Upwelling areas represent less than 1% of the global ocean but support about 20% of the world's wild marine fish harvest including mainly small pelagic fishes: anchovies and sardines (i.e., Engraulis sp.

and Sardinops sagax ) [START_REF] Pauly | Primary production required to sustain global fisheries[END_REF]Chavez and Messie, 2009). Pelagic habitats have been well studied given their economical and socio-ecological importance (e.g., [START_REF] Montecino | The Humboldt Current System: Ecosystem components and processes, fisheries, and sediment studies[END_REF][START_REF] Sydeman | Sixty-five years of northern anchovy population studies in the southern California Current: A review and suggestion for sensible management[END_REF]. Some pelagic-derived materials are transported into shallow waters, and can dominate the supply of organic material to subtidal communities [START_REF] Docmac | Habitat coupling writ large: pelagic-derived materials fuel benthivorous macroalgal reef fishes in an upwelling zone[END_REF][START_REF] Smith | Intraspecific variation and energy channel coupling within a Chilean kelp forest[END_REF]. A high proportion of this pelagic biomass sinks in the water column where it is degraded by microbiota participating in oxygen depletion and the formation of oxygen minimum zone such as in the eastern Pacific [START_REF] Kämpf | Upwelling systems of the world[END_REF]. However, less information is available on the ecology of the benthic communities and habitats that underlay areas of upwelling-enriched primary productivity [START_REF] Thiel | The Humboldt Current System of Northern and Central Chile: Oceanographic Processes , Ecological Interactions and Socioeconomic feedback[END_REF]. In those areas, benthic communities or assemblages are depauperate and show low species richness. They are typically dominated by taxa such as polychaetes that are well adapted to conditions of high organic matter content in the sediment [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Gutiérrez | Oxygenation episodes on the continental shelf of central Peru: Remote forcing and benthic ecosystem response[END_REF]. Their diversity is mainly controlled by environmental factors such as oxygen and organic matter concentrations [START_REF] Soto | Influence of organic matter inputs and grain size on soft-bottom macrobenthic biodiversity in the upwelling ecosystem of central Chile[END_REF].

However, very little is known about the ecological functioning of these benthic communities [START_REF] Sivadas | Trait-based and taxonomic macrofauna community patterns in the upwelling ecosystem of the southeastern Arabian sea[END_REF].

The coastal ecosystem of Northern Chile offers a valuable area to study macrobenthic community functioning subject to upwelling. Located in the Humboldt upwelling system, it is part of one of the most productive ecosystems on Earth [START_REF] Kämpf | Upwelling systems of the world[END_REF]. The average volume of upwelled water in the region is higher than in any other EBUS due to the very narrow continental shelf (i.e., continental margin is 10 to 15 km from the coast; [START_REF] Strub | Coastal ocean circulation off western South America coastal segment[END_REF] and the presence of almost continuous upwelling throughout the year. Upwelling events are stronger in wind-exposed capes, known as upwelling centres, such as those located adjacent to the Mejillones Peninsula [START_REF] Escribano | Understanding upwelling circulation off Antofagasta (northern Chile): A three-dimensional numerical-modeling approach[END_REF][START_REF] Thiel | The Humboldt Current System of Northern and Central Chile: Oceanographic Processes , Ecological Interactions and Socioeconomic feedback[END_REF]. In bays located downwind of a prominent headland, the upwelling jet separates, forming an eddy known as an upwelling shadow [START_REF] Marín | Upwelling shadows at Mejillones Bay (northern Chilean coast): a remote sensing in situ analysis[END_REF][START_REF] Largier | Upwelling Bays: How Coastal Upwelling Controls Circulation, Habitat, and Productivity in Bays[END_REF]. In these bays, currents are slow, allowing biological processes to continue with less physical disruption, and thus are usually highly productive (Chavez and Messie, 2009). However, the upwelling influence coupled with high retention of waters in some bays, creates hypoxic conditions (i.e.

<2 mL.L -1 O 2 ) to develop in the water column, even in waters very close to the water surface.

For example, hypoxic conditions are present in Mejillones Bay (23°S) below 10 m depth throughout the year, with infrequent oxygenation events such as seen in August 2016 (Fig.

3

) when dissolved oxygen concentrations rapidly increased through the water column. Since this bay is located on the arid coastline of the Atacama Desert [START_REF] Guiñez | Variabilidad espacial y temporal de la materia orgánica sedimentaria, asociada a la Zona de Mínimo Oxígeno (ZMO), en un ambiente costero del norte de la corriente de Humboldt, bahía de Mejillones, Chile[END_REF][START_REF] Salazar | From the use of space to territorialisation during the Early Holocene in Taltal, coastal Atacama Desert, Chile[END_REF], it is possible to discount the confounding influence of terrestrial run-off on coastal eutrophication, as the influence of precipitation is negligible (i.e., 4 mm yearly average during the last century; [START_REF] Vargas | Aluviones históricos en Antofagasta y su relación con eventos El Niño/Oscilación del Sur[END_REF].

A recent study highlighted the increasing dissimilarity of the macrobenthic community structure from shallow normoxic (10 m) to deeper hypoxic habitats (up to 50 m) [START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF]. Functioning of these shallow normoxic communities are characterised by diverse feeding-related energy pathways and high bioturbation [START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. However, the communities living in deeper hypoxic habitats with higher organic matter content displayed low biogenic structure and bioturbation and were supported by less diverse energy

Macrobenthic community functioning

Biological traits analysis

Biological trait-based approaches have been increasingly recognized as a useful complementary approach to the assessment of community structure and diversity, providing information on ecosystem functioning, i.e., ecological processes, their maintenance and regulation [START_REF] Naeem | Biodiversity and ecosystem functioning: Maintaining natural life support processes[END_REF][START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. This approach focuses on describing community structure in terms of biological traits frequencies, which allows the assessment of how species traits respond to environmental factors and gradients [START_REF] Bremner | Assessing ecological functioning in marine benthic communities[END_REF]. [START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF] found that multimetric indices based on general life history traits such as living habits are less influenced by temporal variation. Traits can be standardised to address all macrobenthic species, meaning that comparisons are possible among different systems or regions. However, studies on the functional diversity of marine benthic ecosystems remain scarce [START_REF] Gusmao | Functional diversity of macrobenthic assemblages decreases in response to sewage discharges[END_REF] despite their high analytical potential [START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. Further studies evaluating how macrobenthic functional traits change with different environmental disturbances will contribute to a better understanding of environmental impacts in benthic systems and the utility of trait-based approaches as reliable analytical tools. Additionally, very few studies assessing ecological functioning of macrobenthic communities include a temporal aspect [START_REF] Frid | Temporal variability in the benthos: Does the sea floor function differently over time?[END_REF][START_REF] Clare | Community variability and ecological functioning: 40 years of change in the North Sea benthos[END_REF].

A number of different trait databases have been developed, making it easier to collect information about numerous species such as the Biological Traits Information Catalogue (MarLIN, 2006) and Marine Species Traits (Marine Species Traits editorial Board, 2018).

Some attempts have also been made to standardise the vocabulary and trait classification [START_REF] Costello | Biological and ecological traits of marine species[END_REF]. Trait information can be collected from studies grouping relevant information as well, such as [START_REF] Fauchald | The diet of worms: a study of polychaete feeding guilds[END_REF] for feeding modes and [START_REF] Queirós | A bioturbation classification of European marine infaunal invertebrates[END_REF] for sediment reworking and mobility. The original source of the information provided is not always cited properly, making compilation more complicated (personal observation) and making it very difficult to find relevant information. Moreover, available trait information is mainly based on European species, with biological trait data from South American and Pacific taxa still being very limited. Therefore, an extensive literature review is required as well as consultation with local experts of the target taxa. Individual traits can also be measured in different ways, for example body size can refer to average mass (e.g., van

der [START_REF] Van Der Linden | Functional changes in polychaete and mollusc communities in two tropical estuaries[END_REF] or anatomical measures (e.g., [START_REF] Gusmao | Functional diversity of macrobenthic assemblages decreases in response to sewage discharges[END_REF]. In addition, stable isotope analyses can complement the information regarding the feeding strategies of given invertebrates. Commonly, δ 13 C is used to identify reliance on different energetic pathways (e.g. energy derived from particulate organic matter, macroalgae and organic matter from the surface sediment), δ 15 N values increase with trophic position (Hobson et al., 2002) and δ 34 S provides information on habitat and feeding mode.

Functional diversity indices

Functional diversity incorporates diverse biological components, in a manner similar to taxonomic diversity: as such, it needs to be estimated using several indices [START_REF] Mouchet | Functional diversity measures: An overview of their redundancy and their ability to discriminate community assembly rules[END_REF]. [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] demonstrated the complementarity of the following indices:

functional richness (FRich), functional evenness (FEve) and functional divergence (FDiv).

FRic represents the volume of functional trait space occupied by the community [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]. It is a proxy of the range of functional traits of a community and does not account for relative abundance of taxa [START_REF] Schuldt | Tree diversity promotes functional dissimilarity and maintains functional richness despite species loss in predator assemblages[END_REF]. FEve measures the regularity of the abundance distribution in the filled trait space by summing the branch lengths of the minimum spanning tree that connects all the community taxa, weighted by their abundance [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]. FDiv quantifies the divergence of the taxa abundance distribution from the centre of gravity in the functional space [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]. Each of these components of functional diversity help to reveal the different ecological processes driving the communities under study [START_REF] Mason | Functional richness, functional evenness and functional divergence: The primary components of functional diversity[END_REF]. RaoQ can also be used to estimate multivariate dispersion by summing the abundance-weighted pairwise distances between taxa in the functional trait space [START_REF] Botta-Dukát | Rao's quadratic entropy as a measure of functional diversity based on multiple traits[END_REF]. This index is affected by increases or decreases in the abundance (or occurrences) of taxa with unique trait combinations [START_REF] Botta-Dukát | Rao's quadratic entropy as a measure of functional diversity based on multiple traits[END_REF].

Marine coastal macrobenthos and disturbances

Importance of macrobenthos and effect of disturbances

Soft-bottom habitats cover much of the ocean seafloor and macrobenthos are an important component of these ecosystems. Macrobenthic species perform different functions such as remineralization and carbon storage; they also serve as food for higher trophic levels and some of them and their predators are of commercial interest [START_REF] Virnstein | Predation on estuarine infauna: Response patterns of component species[END_REF]. Some macrobenthos play essential roles in material exchange between the water column and the seabed through bioturbation and suspension-feeding [START_REF] Reise | Sediment mediated species interactions in coastal waters[END_REF][START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF]. Large-bodied macro-and mega-benthos can not only change sediment matrix and microtopography, but also promote oxygenation of deeper sediment layers, allowing smaller organisms to thrive inside the sediment at depths greater than 10 cm [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF][START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF].

Natural and anthropogenic disturbances, such as hypoxia caused by eutrophication, can cause local suppression of sensitive species or life stages, which can have direct and indirect implications on ecosystem functioning [START_REF] Villnäs | Consequences of Increasing Hypoxic Disturbance on Benthic Communities and Ecosystem Functioning[END_REF]. For example, introduced bivalves (e.g., Corbula amurensis ) changed San Francisco Bay phytoplankton dynamics by preventing seasonal phytoplankton blooms, until a shift in temperature allowed the immigration of bivalve predators (flatfishes and crustaceans) that reduced the density of suspension-feeders and induced the return of the blooms [START_REF] Thompson | One estuary, one invasion, two responses--Phytoplankton and benthic community dynamics determine the effect of an estuarine invasive suspension-feeder[END_REF][START_REF] Cloern | A cold phase of the East Pacific triggers new phytoplankton blooms in San Francisco Bay[END_REF]. The classical model of [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF] predicts ecological succession in macrobenthic communities as a function of organic matter enrichment (Fig. 4a). In this model, very high levels of sediment organic matter, causing hypoxia, is detrimental for all macrobenthic species. At slightly high to moderate levels of enrichment, small-bodied, opportunistic species are favoured by low competition and high availability of food, which cause a peak in benthic abundance and biomass. After the ecotone point, the transition zone starts with moderate levels of organic enrichment, other species are favoured to the detriment of opportunistic species, which cause a decrease in abundance and increase in species richness and biomass. Finally, at low levels of organic matter, the community reaches a mature phase, characterised by relatively lower biomass and richness as a product of increased importance of competition (Fig. 4b; [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF].

assessment studies [START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF]. For example, some species of polychaetes such as Capitella sp. (Capitellidae) and Paraprionospio pinnata (Cirratulidae) are known indicators of organic enrichment [START_REF] Tsutsumi | Population persistence of Capitella sp (Polychaeta; Capitellidae) on a mud flat subject to environmental disturbance by organic enrichment[END_REF][START_REF] Tomassetti | Polychaetes as indicators of marine fish farm organic enrichment[END_REF]; Díaz-Castañeda and de [START_REF] Díaz-Castañeda | Comunidades de anélidos poliquetos en Bahía Magdalena, Baja California Sur, México[END_REF]. They are opportunistic species with broad geographic distributions and well adapted to cope with high levels of organic matter. However, they also can be found in low abundance in areas with low organic matter concentration, therefore it is recommended to use a combination of species and abiotic measurements to evaluate the degree of disturbance in a given habitat [START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF].

Biotic indices

General aspects

When assessing ecological status, the use of biotic indices allows researchers to evaluate the effect of environmental conditions on living organisms. This is useful as benthic communities, by definition, are in direct relation with the seabed and thus, live in direct exposure to contaminants and oxygen depletion [START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF][START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. In addition, benthic taxa can rapidly respond to disturbances since they cannot avoid disturbed conditions due to their limited motility [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF][START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF]. They also have relatively long life spans (i.e., from days to years) that can allow the detection of effects of impacts that may be missed during physico-chemical monitoring, as well responses to chronic stresses [START_REF] Bilyard | The value of benthic infauna in marine pollution monitoring studies[END_REF]. Benthic communities are composed of assorted taxa with different levels of tolerance to stress, for example hypoxia [START_REF] Vaquer-Sunyer | Thresholds of hypoxia for marine biodiversity[END_REF]. The macrobenthos also includes species, or prey of species of commercial interest. That is why they have been incorporated in assessment and monitoring of ecosystem quality of coastal and transitional ecosystems in many parts of the world (e.g., Australian National Land and Water Resources Audit; [START_REF] Heap | Australian Estuaries & Coastal Waterways: A Geoscience Perspective for Improved and Integrated Resource Management[END_REF]Water Framework Directive, 2000; United States of America Clean Water Act; USEPA, 2002).

Biotic indices are quantitative tools that simplify information regarding benthic community structure that, together with other environmental metrics, measure the relative degree of disturbance in a given habitat at a point in time [START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. They are easily interpreted by non-scientists and are especially useful for marine managers and decision makers [START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. The main objectives of these indices are to compare different sites (spatial variation) and/or to examine putative change at a site over time (temporal variation). The aim is to evaluate the benthic community condition described by the indices' value, which could be in general or as a response to a specific anthropogenic disturbance.

For example, the Trawling Disturbance Index (TDI) considers groups with different levels of vulnerability to trawling using biological traits (de Juan and Demestre, 2012), while the AZTI's biotic index (AMBI) was developed to establish the ecological quality of the European coasts according to the sensitivity of macrobenthos to organic matter enrichment [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. Most biotic indices were developed for a specific location, mostly in the Northern hemisphere, and in particular, Europe and the USA. However, several indices have been successfully applied elsewhere. For example, the Benthic Opportunistic Polychaetes Amphipods Index, BOPA [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF] was developed in the English Channel, and has been successfully applied in Africa, Asia, Europe, Oceania and South America [START_REF] Dauvin | Twenty years of application of Polychaete/Amphipod ratios to assess diverse human pressures in estuarine and coastal marine environments: A review[END_REF]. To date, at least 28 different ecological indices have been proposed for soft-bottom marine macrobenthos (Table 1).

Table 1. List of biotic indices based on macrobenthos taxonomy and traits (updated from Díaz et al., 2004) Biotic indices usually incorporate community metrics such as taxonomic richness, abundance and/or biomass. They can be either based on estimation of biodiversity such as the Shannon-Wiener index [START_REF] Shannon | The mathematical theory of communication[END_REF], the Simpson diversity index [START_REF] Simpson | Measurement of diversity[END_REF] or Pielou's evenness index [START_REF] Pielou | An introduction to mathematical ecology[END_REF]. Some indices are based on the tolerance and sensitivity to disturbance of taxa, either at species level (i.e., AZTI's Marine Biotic Index: AMBI; [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Borja | The application of a Marine Biotic Index to different impact sources affecting soft-bottom benthic communities along European coasts[END_REF] or at higher taxonomic levels, for example in the Benthic Opportuntistic Polychaetes-Amphipods index (BOPA; [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]. These sensitivity categories can be predefined, as in the AMBI that assigns taxa to one of five ecological groups [START_REF] Grall | Using biotic indices to estimate macrobenthic community perturbations in the Bay of Brest[END_REF][START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. It can be difficult to assign species to a given tolerance group, as they may respond in different ways to different types of disturbances. [START_REF] Brauko | Spatial variability of three benthic indices for marine quality assessment in a subtropical estuary of Southern Brazil[END_REF] described how to include new species in predefined groups, however, the reaction of some species to novel contaminants may not be understood. One way to reduce the risk of group assignment error is to decrease the number of groups such as in the BENTIX index [START_REF] Simboura | Benthic indicators to use in Ecological Quality classification of Mediterranean soft bottom marine ecosystems, including a new Biotic Index[END_REF], where species are divided into three ecological groups. The first group includes species that are sensitive or indifferent to disturbance. The second group includes tolerant and opportunistic species that can increase their abundance under slight disturbances. The final group includes opportunistic species that are adapted to withstand strong disturbances. Sensitivity categories can also be calculated using empirical data, such as in the Benthic Quality Index (BQI; [START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF]). In the BQI, the sensitivity of each taxon is calculated as the 5% of the abundance frequency distribution using the value of the expected number of species among 50 individuals in this sample (ES50; [START_REF] Hurlbert | The non-concept of species diversity: a critique and alternative parameters[END_REF].

Biotic indices can also be based on reference conditions. The multivariate-AMBI integrates standardised AMBI, S, and H using factor analysis, including minimum and maximum reference values as arbitrary samples [START_REF] Muxika | Using historical data, expert judgement and multivariate analysis in assessing reference conditions and benthic ecological status, according to the European Water Framework Directive[END_REF]. The recently developed General-Purpose Benthic Index (GPBI; [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF] compares benthic macrofauna community composition and structure to one, or several reference station(s) with good ecological status. In the context of the European Union Water Framework Directive (Water Framework Directive, 2000), those indices are compared to a standardised scale including 5 different classifications of ecological quality status (Table 2). The Infaunal Quality Index [START_REF] Phillips | Infaunal quality index : Water Framework Directive classification scheme for marine benthic invertebrates[END_REF] differs by including confidence indicators of the classification obtained with values of "Risk of Misclassification" and the "Confidence of Class".

Naturally stressful environments

Benthic environmental quality indices have proved to be useful tools to detect anthropogenic disturbances in a range of coastal systems (e.g., [START_REF] Nilsson | Benthic habitat quality assessment of an oxygen stressed fjord by surface and sediment profile images[END_REF][START_REF] Roberts | Developing an efficient macrofauna monitoring index from an impact study -A dredge spoil example[END_REF][START_REF] Maurer | The Infaunal Trophic Index (ITI): Its suitability for marine environmental monitoring[END_REF]. However, some habitats have naturally stressful physico-chemical conditions and support well-adapted communities. Hence, it is difficult to detect possible anthropogenic impacts (such as organic enrichment) in such systems [START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF]. This is the case in estuarine habitats, which can naturally resemble disturbed habitats due to their natural highly variable characteristics: salinity, oxygen concentration, light regime and organic content that vary with tides and river flow (Whitfield et al., 2012). This ambiguity has been described as the Estuarine Quality Paradox [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF][START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF].

In upwelling areas, the ability to differentiate between natural and anthropogenic disturbances using biotic indices is expected to be even more challenging. This reflects the point that organic matter content and dissolved oxygen concentration are both influenced by both natural phenomena (i.e., upwelling) and anthropogenic activities (e.g., sewage water Multimetric indices are more robust than univariate metrics [START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF]Bonifacio, 2015). Abundance-based indices will be largely used, reflecting their increased sensitivity compared to those based on biomass [START_REF] Gusmao | Functional diversity of macrobenthic assemblages decreases in response to sewage discharges[END_REF], especially where perturbation levels are moderate [START_REF] Grall | Using biotic indices to estimate macrobenthic community perturbations in the Bay of Brest[END_REF]. In general, defining a reference state can be difficult due to the scarcity of information from sites, e.g., reflecting conditions prior to the impacts of anthropogenic disturbance. A reference site located away from the targeted disturbance can be used as an approximation of reference conditions, taking into consideration impacts of other putative types of disturbance. In the context of upwelling, it is expected that it will be challenging to distinguish between natural and anthropogenic impacts. As precipitation is extremely low in Northern Chile, run-off is negligible and terrestrial inputs are likely limited to anthropogenic influences such as sewage water. Thus, the inclusion of stable isotopes analysis could help to detect the origin of the nutrients incorporated under these conditions by the organisms. Stable isotope ratios of carbon (δ 13 C), nitrogen (δ 15 N) and sulphur (δ 34 S) in organisms reflect the origin of organic material they assimilate into their somatic tissues [START_REF] Peterson | Stable isotopes as tracers of organic matter input and transfer in benthic food webs: A review[END_REF]. Therefore, stable isotopes analysis will enhance our understanding of whether target taxa use organic material from terrestrial and/or marine origins (either benthic or pelagic) using mixing model analysis [START_REF] Parnell | Source partitioning using stable isotopes: Coping with too much variation[END_REF].

Research questions

The general objective of this thesis is to evaluate the potential of different multivariate, multimetric, and functional multivariate analytical approaches to assess disturbance related to organic enrichment on marine macrobenthic communities under upwelling influence.

In the chapter II "Macrobenthic community functioning under upwelling influence", the following specific objectives will be addressed according to the points developed in sections 2. and 3. reflecting knowledge gaps ( 1) and ( 2):

Specific objective 1:

Evaluate functional diversity of macrobenthic communities along an environmental depth gradient from normoxia to hypoxia.

Hypothesis 1:

Functional diversity differs between macrobenthic communities living in normoxic and permanent hypoxic habitats. However, there is a functional overlap in the benthic communities living in intermediary habitats oscillating between normoxia and hypoxia.

Prediction 1:

Functioning of macrobenthic communities will differ between normoxic and hypoxic habitats [START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF] and overlap in transitory habitats [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF].

Specific objective 2:

Study temporal variation of functional diversity of macrobenthic communities during episodes of oxygenation in a hypoxic environment.

Hypothesis 2:

Through time, functional diversity will increase during oxygenation events.

Prediction 2:

Oxygen is expected to be a limiting factor, thus increasing dissolved oxygen should allow benthic communities to develop to a more mature stage of succession.

In the chapter III "Ecological status assessment based on macrobenthic communities in a bay under upwelling influence" I examine the factors developed in sections 4. and 5. and knowledge gap (3). In this chapter, the following specific objective will be addressed:

Specific objective 3:

To evaluate the ability of different types of biotic indices to detect the possible impacts of organic matter enrichment and hypoxia associated with anthropogenic activity in a bay with naturally high organic matter concentration and hypoxia.

Hypothesis 3:

Benthic communities under naturally low dissolved oxygen and high organic matter concentrations will be classified as being of low ecological status by different biotic indices under and away from anthropogenic influence. Biotic indices will be unable to distinguish between natural and anthropogenic impacts.

Prediction 3:

The different biotic indices will be unable to distinguish between the different effects of natural and anthropogenic influences as they drive variation in the same oceanographic parameters (i.e., organic matter content and dissolved oxygen concentration).

Preamble

Organic matter enrichment and hypoxia are some of the most impacting disturbances on soft-bottom macrobenthic communities in coastal areas [START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF]. They alter their structure and function with different effects depending on regional history (e.g. [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF]Levin et al., 2009a). In the last decades, studies on the functioning of soft-bottom macrobenthic communities have increased, most of them focusing on seasonal or episodic impacts from anthropogenic origin within estuaries, fjords or enclosed seas (e.g., [START_REF] Villnäs | Consequences of Increasing Hypoxic Disturbance on Benthic Communities and Ecosystem Functioning[END_REF][START_REF] Gusmao | Functional diversity of macrobenthic assemblages decreases in response to sewage discharges[END_REF][START_REF] Llanos | Functional diversity in the intertidal macrobenthic community at sewage-affected shores from Southwestern Atlantic[END_REF]. However, hypoxia is not always punctual as some upwelling areas are subject to permanent hypoxia including in shallow waters. These systems are sheltering organisms highly adapted to these harsh conditions [START_REF] Escribano | Physical-chemical processes and patterns of diversity of the Chilean eastern boundary pelagic and benthic marine ecosystems: An overview[END_REF]Levin et al., 2009b). In this chapter, I studied the functioning of macrobenthic communities subject to a gradient of natural organic matter enrichment and hypoxia due to quasi-continuous upwelling in Mejillones Bay, Northern Chile.

In this bay, soft-bottom macrobenthic organisms were collected monthly, during two years, along 2 hypoxic gradients with increasing organic matter from 10 to 50 m depth. We recorded 150 different taxa from which I compiled information from the literature on nine different biological traits divided into 38 categories. These traits were selected to reflect ecological functioning of the community and their sensitivity to environmental disturbances [START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. Traits expression within the communities were analysed using fuzzy coding [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF] as well as using functional indices [START_REF] Mason | Functional Diversity Measures[END_REF] in order to better understand the functioning of these communities. This study has been published in Marine Pollution Bulletin [START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF].

This work highlighted for the first time the existence of three different functional communities along the depth gradient of increasing hypoxia and organic matter concentrations that were associated with different trait dominance patterns. From shallow to deep habitats, these communities were driven by decreasing biological interactions and increasing habitat filtering. I found a general reduction in taxonomic and functional richness along the environmental gradient with a sharp drop below 2 mL.L -1 of dissolved oxygen. Oxygen below this concentration is thus strongly impacting the benthic communities despite their adaptations to hypoxia and organic matter content (e.g., presence of predators and sessile organisms). In accordance with this threshold, the total anthropogenic organic matter inputs in the bay should be managed so that dissolved oxygen concentration remains above 2 mL.L -1 . In this region, monitoring of anthropogenic influence, especially eutrophication, is expected to be highly challenging due to confounding effects of natural and human induced hypoxia and organic matter enrichment.

Conclusions and perspectives

Biological traits analysis uses selected features of organisms that describe their life history, morphology and behaviour, to represent relevant ecological roles they play describing the functions of the ecosystem in which they live [START_REF] Bremner | Methods for describing ecological functioning of marine benthic assemblages using biological traits analysis (BTA)[END_REF]. Benthic organisms interact with their chemical and physical environment, maintaining and regulating ecosystem processes [START_REF] Snelgrove | The biodiversity of macrofaunal organisms\rin marine sediments[END_REF] which has been defined as ecological functioning [START_REF] Naeem | Biodiversity and ecosystem functioning: Maintaining natural life support processes[END_REF]. The search for relevant biological information involved accessing and analysing a large number of studies from the literature, most of which were generated from the northern hemisphere, mainly from Europe and USA. To counter the clear knowledge gap, I encourage further studies to be carried out in the southern hemisphere to increase the understanding of local species. Some apparently ubiquitous species such as Magelona phyllisae that have been reported in different oceans (Atlantic: [START_REF] Flint | Polychaete Ecology and Niche Patterns: Texas Continental Shelf[END_REF]Rabalais, 1980, Pacific: Moreno et al., 2008;and Indian: Sivadas et al., 2016) are very likely to be different species. This has been the case for many species, e.g. Marphysa sanguinea [START_REF] Lavesque | Guess who? On the importance of using appropriate name: Case study of marphysa sanguinea (montagu, 1803)[END_REF]. Furthermore, the same species living in different contexts can also display different characteristics [START_REF] Maurer | The Infaunal Trophic Index (ITI): Its suitability for marine environmental monitoring[END_REF][START_REF] Zettler | On the Myths of Indicator Species: Issues and Further Consideration in the Use of Static Concepts for Ecological Applications[END_REF]. Some efforts are indeed in progress to compile the existing information within databases (e.g., MarLIN, 2006;Marine Species Traits editorial Board, 2018). I suggest incorporating information on the source of the information (e.g., article citation, name of the expert), as well as the location and environmental conditions where the study was performed to facilitate the evaluation of the relevance of information present . The trait categories of species can change along environmental gradients [START_REF] Zettler | On the Myths of Indicator Species: Issues and Further Consideration in the Use of Static Concepts for Ecological Applications[END_REF]. I believe that future studies will move towards including more quantitative traits (e.g. biomass,

mouth gape, trophic level based on isotopic analyses) that would allow researchers to work on taxa with less available biological information and give the opportunity to include intraspecific variation in such studies. This approach has been developed so far mainly for terrestrial vegetation and aquatic fishes and has been shown to provide relevant information regarding species niche width and interactions, as well as incorporate ontogenetically driven variations, especially for species-poor communities (e.g., [START_REF] Mason | Does niche overlap control relative abundance in French lacustrine fish communities? A new method incorporating functional traits[END_REF][START_REF] Westerband | Intraspecific trait variation in plants: a renewed focus on its role in ecological processes[END_REF]. However, measuring quantitative traits in macrobenthic communities has revealed the existence of new challenges, i.e. measures must be applicable to the different life forms found in all the different habitats that they occupy (e.g., the deep sea) and must be also ecologically meaningful.

The results of this study confirmed the interest in combining biological trait analysis and functional diversity with a taxonomic approach to better understand marine ecosystems functioning [START_REF] Bremner | Methods for describing ecological functioning of marine benthic assemblages using biological traits analysis (BTA)[END_REF][START_REF] Cernansky | Biodiversity moves beyond counting species[END_REF]. Gradual decrease in taxonomic richness and variability of soft-bottom benthic communities along an environmental gradient of increasing depth, hypoxia and organic matter concentration were previously reported [START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF]. Community functioning could be impacted by a disappearance of a single taxon with unique trait values without notable changes in species composition [START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. Inversely, community functioning would not be impacted by the disappearance of several taxa if other taxon would fulfil the same functions (insurance hypothesis ;[START_REF] Frid | Temporal variability in the benthos: Does the sea floor function differently over time?[END_REF]. Here,the existence of three different types of benthic communities functioning were revealed, associated with specific functional trait categories along this environmental gradient (Fig. 7; [START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. Nonetheless, questioning remained regarding the temporal and/or spatial variability of the functioning response of fauna to hypoxia. Further studies are thus required to investigate if a similar pattern is observed when the disturbance (i.e., increasing organic matter) is seasonal or episodic. One way would be to repeat this study along the latitudinal gradient of the Chilean coast as upwelling is seasonal in central Chile and absent further south [START_REF] Escribano | Physical-chemical processes and patterns of diversity of the Chilean eastern boundary pelagic and benthic marine ecosystems: An overview[END_REF].

In this study, the benthic community patterns encountered were persistent in time despite an oxygenation event recorded in August 2016 when oxygenated waters were recorded at all depths (i.e., at least down to 50 m; [START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF][START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. Benthic community functioning can take several years to decades to recover from hypoxia and organic matter enrichment of anthropogenic origin in Europe and North America [START_REF] Steckbauer | Ecosystem impacts of hypoxia: Thresholds of hypoxia and pathways to recovery[END_REF][START_REF] Villnäs | Structural and functional shifts in zoobenthos induced by organic enrichment -Implications for community recovery potential[END_REF]. However, in Northern Chile, macrobenthic species richness increased and species dominance decreased over only six months after the shift to warmer, oxygenated waters associated with the El Niño event of 1997-1998 [START_REF] Moreno | Subtidal macrozoobenthos communities from northern Chile during and post El Niño 1997-1998[END_REF].

Long term monitoring in the region would allow to reveal if the functional pattern observed in this study is persistent or not over longer lasting fluctuations of environmental conditions such as the El Niño Southern Oscillation (ENSO) and the Pacific decadal oscillations that induce substantial changes in the community structure [START_REF] Pacheco | El Niño-Southern Oscillation cyclical modulation of macrobenthic community structure in the Humboldt Current ecosystem[END_REF]. This information would help to predict the long term consequences of hypoxia and eutrophication on the rising number of ecosystems affected by this phenomenon [START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF].

Despite the general patterns observed, spatial differences were recorded between Punta Chacaya and Playa Blanca. Indeed the western part of the bay, where Playa Blanca is located, may be subject to direct anthropogenic effects through urbanisation and industry runoffs, including possible organic matter inputs from Mejillones City. The potential effects of these activities is further studied in the following chapter through the use of a range of biotic indices, whose performance in this naturally stressed environment are assessed .

Preamble

Coastal systems are particularly impacted by anthropogenic activities and at the same time provide many benefits for human beings [START_REF] Worm | Impacts of Biodiversity Loss on Ocean Ecosystem Services[END_REF]. In the last decades, a need to evaluate the ecological conditions of these coastal ecosystems has arised with the emergence of many biotic indices based on macrobenthic communities, principally in Europe and USA [START_REF] Diaz | A review of approaches for classifying benthic habitats and evaluating habitat quality[END_REF][START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. Ecological quality status evaluation in naturally stressed environments appears to be especially challenging, for example in estuaries [START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF]. Upwelling systems are also naturally stressed environments, affected mainly by nutrients input triggering the same processes as nutrients input from anthropogenic origin which can lead to hypoxia and organic matter enrichment at the sediment interface (Levin et al., 2009b). The use of biotic indices depicting ecological status and relying on benthic fauna have been overlooked so far in these ecosystems. In this study, the suitability of a range of biotic indices were evaluated based on diversity, taxa sensitivity, reference conditions and functional diversity to assess the ecological quality status of a soft-bottom bay subject to marked upwelling influence in northern Chile.

In summary, the results revealed new limitations of some widely used biotic indices. These limitations are mainly related to the high adaptation of organisms to organic matter enrichment and hypoxic conditions, but also to the low taxonomic richness and organisms abundance encountered on the studied sites. Moreover, most indices showed decreasing ecological status along the natural environmental gradient of increasing hypoxia and organic matter. This allowed us to describe a new concept: the Upwelling Ecosystem Quality Paradox related to the confounding effect of natural hypoxia and organic matter enrichment.

This concept should be applicable to different upwelling areas where the natural eutrophication induced stress on ecosystems that can be confounded with anthropogenic stress, principally where natural hypoxia is present. One index stood out by being less influenced by the natural stress while another index detected early degradation of ecological quality status in the intermediate depths in the area of the city sewage water discharge.

Despite these discrepancies, a general pattern of decreasing ecological quality status from east to west in Mejillones Bay appeared consistently, related to a combination of natural and anthropogenic disturbances. This study raises further awareness on the difficulty of identifying anthropogenic impacts in naturally stressed environments. It also highlighted the importance of combining structural and functional approaches when assessing environmental conditions. Ecological quality status evaluation is essential in order to preserve the benefits provided by coastal ecosystems and it is becoming more and more relevant in the context of increasing human pressures on coastal areas, especially hypoxia. Keywords marine macrobenthic communities; upwelling system; hypoxia; organic matter; eutrophication; environmental quality assessment

Introduction

Marine coastal ecosystems provide numerous benefits to humankind, including food provision, coastal protection, water purification, nutrient cycling, carbon sequestration, and recreational opportunities [START_REF] Buonocore | Trends and evolution in the concept of marine ecosystem services: An overview[END_REF]. Provision of such benefits relies on ecosystem functioning and biodiversity that are currently threatened by increasing anthropogenic pressures such as overfishing, eutrophication, invasive alien species, habitat destruction, pollution, and climate change [START_REF] Worm | Impacts of Biodiversity Loss on Ocean Ecosystem Services[END_REF][START_REF] Barbier | The value of estuarine and coastal ecosystem services[END_REF][START_REF] Johnson | Biodiversity losses and conservation responses in the Anthropocene[END_REF] , 2002). Among the biological quality elements of evaluation, benthic macroinvertebrates are commonly used for assessing the ecological quality status (EcoQ) of coastal and transitional ecosystems. Macrobenthic communities play key roles in coastal ecosystems. They are essential in nutrient cycling and influence the chemical fluxes between sediment and water column through bioturbation and suspension-feeding [START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF]. Macrobenthos include species, or prey of species of commercial interest. Benthic communities are composed by several taxa with different levels of tolerance to stress and live in direct exposure to contaminants and oxygen depletion at the water-sediment interface, making them particularly suitable for EcoQ assessment [START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF][START_REF] Vaquer-Sunyer | Thresholds of hypoxia for marine biodiversity[END_REF][START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. In addition, benthic taxa can rapidly respond to disturbances since they cannot avoid disturbed conditions due to their limited motility and have relatively long life spans [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF][START_REF] Dauer | Relationships between benthic community condition, water quality, sediment quality, nutrient loads, and land use patterns in Chesapeake Bay[END_REF].

Macrobenthic taxa can be negatively impacted by substantial organic matter enrichment leading to hypoxia [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF]Levin et al., 2009a). At a community level, this impact is reflected by the disappearance of sensitive taxa, while few opportunistic species increase in abundance. Opportunistic species are characterised by small body size with high turn-over populations (fast-breeding) that allow them to thrive in conditions that are stressful for most taxa [START_REF] Warwick | A new method for detecting pollution effects on marine macrobenthic communities[END_REF]. Moreover, loss of taxa has direct and indirect impacts on ecosystem functioning [START_REF] Villnäs | Consequences of Increasing Hypoxic Disturbance on Benthic Communities and Ecosystem Functioning[END_REF]. For example, introduced bivalves (e.g., Corbula amurensis ) changed San Francisco Bay phytoplankton dynamics by preventing the seasonal phytoplankton blooms until a shift in temperature allowed the immigration of flatfishes and crustaceans (bivalve predators), that reduced the density of suspension-feeders, inducing the return of the blooms [START_REF] Thompson | One estuary, one invasion, two responses--Phytoplankton and benthic community dynamics determine the effect of an estuarine invasive suspension-feeder[END_REF][START_REF] Cloern | A cold phase of the East Pacific triggers new phytoplankton blooms in San Francisco Bay[END_REF].

In the search of reliable EcoQ assessment, a myriad of biotic indices based on macrobenthic communities has been developed especially in the last decades. Most such indices are based on the presence or abundance of sensitive species [START_REF] Diaz | A review of approaches for classifying benthic habitats and evaluating habitat quality[END_REF][START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. Several indices have been successfully applied across different geographical regions (e.g., the Benthic Opportunistic Polychaetes Amphipods Index in Africa, Asia, Europe, Oceania, and South America; [START_REF] Dauvin | Twenty years of application of Polychaete/Amphipod ratios to assess diverse human pressures in estuarine and coastal marine environments: A review[END_REF]. However, new indices are continually being developed, indicating that none is universally applicable and that all indices display limitations [START_REF] Diaz | A review of approaches for classifying benthic habitats and evaluating habitat quality[END_REF][START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF]. The EcoQ of regions with highly variable environments, such as estuaries, has been especially difficult to assess, as they are naturally subject to abiotic stress [START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF]. Especially in the mid and upper estuarine regions, large variation in salinity and often high natural organic matter inputs limit the number of taxa able to thrive. Most such taxa resemble those encountered in areas negatively influenced by anthropogenic activities (i.e., the small body size and high reproductive capacity typical of r-strategists). This makes it difficult to evaluate human impacts, especially using indices relying on sensitive species. Estuarine benthos show no obvious shift towards increased contribution of k-strategists and large individuals, and the food web remains dominated by organic/detritus-responsive invertebrates. This suggests that the variable state is paradoxically stable [START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF], a situation described as the Estuarine Quality Paradox [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF]Elliott and Quitino, 2007).

Beyond estuaries, other naturally marine eutrophic ecosystems exist, including those subject to upwelling.Upwelling brings cold, nutrient-rich, and oxygen-poor waters with high salinity from deeper waters to the surface [START_REF] Kämpf | Upwelling systems of the world[END_REF]. This input of nutrients to the euphotic zone drives high primary and secondary production [START_REF] Escribano | Physical-chemical processes and patterns of diversity of the Chilean eastern boundary pelagic and benthic marine ecosystems: An overview[END_REF][START_REF] Kämpf | Upwelling systems of the world[END_REF], and increases the supply of organic matter to underlying habitats.

Organic matter falls out of the water column, accumulates on the seafloor where associated high microbial oxygen demand can cause hypoxia or anoxia [START_REF] Grall | Marine eutrophication and benthos: The need for new approaches and concepts[END_REF][START_REF] Breitburg | Hypoxia, Nitrogen, and Fisheries: Integrating Effects Across Local and Global Landscapes[END_REF]. Such hypoxia and anoxia can result in massive mortalities of marine organisms, especially those with limited motility [START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF]. In affected areas, benthic communities are characterised by low species richness and are dominated by a few taxa with morphological and physiological adaptations to hypoxia [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF]. The structure of those communities is largely regulated by environmental factors such as dissolved oxygen (DO) and organic matter concentrations [START_REF] Soto | Influence of organic matter inputs and grain size on soft-bottom macrobenthic biodiversity in the upwelling ecosystem of central Chile[END_REF][START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF]. Therefore, assessing benthic EcoQ under these conditions is expected to be highly challenging and raises the question: is there an upwelling ecosystem quality paradox?

To answer this question, we undertook a study within the Humboldt Current System, one of the major upwelling zones of the world [START_REF] Kämpf | Upwelling systems of the world[END_REF]. Elevated pelagic production rates reflect high irradiance and relatively consistent nutrient supply by upwelling [START_REF] Anderson | Upwelling Ecosystems[END_REF]). An oxygen minimum zone extends through deeper waters and over the continental shelf, affecting benthic community structure. Those communities are characterised by low taxa richness and biomass but a high abundance of the few macroinvertebrate taxa, mainly polychaetes, that can live and thrive under hypoxic conditions [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF][START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF]. In Northern Chile, upwelling events are intense throughout the year, leading to very shallow hypoxia and natural organic matter enrichment, especially within sheltered bays [START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF]. Therefore, the region represents a natural laboratory to examine the effects of natural nutrient enrichment.

The often-confounding effect of terrestrial run-off is negligible, as the area is located on the extremely arid coastline of the Atacama Desert [START_REF] Vargas | Aluviones históricos en Antofagasta y su relación con eventos El Niño/Oscilación del Sur[END_REF]. These bays protected from the main winds provide conditions that support human activities, including harbours and industrial hubs, that simultaneously threaten the marine environment. In such a scenario, it is challenging to detect environmental impacts such as industrial and municipal water discharges and to differentiate them from those associated with natural eutrophication.

In this context, we aim to evaluate the ability of different types of biotic indices to detect the possible impacts of organic matter enrichment and hypoxia associated with anthropogenic activity in a bay with naturally high organic matter concentration and hypoxia. Most biotic indices have been created based on the Pearson and Rosenberg model (1978) describing particularly mollusks and crustaceans (Zúñiga et al., 1983). The Mejillones Peninsula is a marine mammal hotspot (e.g., sea-otters, sea-lions, whales, and dolphins) and other emblematic marine species such as ocean sunfish, marine turtles, and penguins are also commonly seen [START_REF] Pacheco | Presence of fin whales (Balaenoptera physalus) in Mejillones Bay, a major seaport area in northern Chile[END_REF][START_REF] Norambuena Ramírez | Actualización de información de objetos de conservación en Bahía Mejillones, comuna de Mejillones, región de Antofagasta[END_REF][START_REF] García-Cegarra | Citizen science as a tool to assess cetacean diversity in the Atacama Desert coast[END_REF]. However, in the last decade, mass mortalities of fishes and invertebrates have been repeatedly (every 2 to 3 years) reported by local newspapers (e.g., Timeline Antofagasta, 2016; El Diario de Antofagasta, 2019) and have been attributed to the rise of natural hypoxic conditions.

Since the 1990s, industrial development has rapidly occurred along the bay's shore. The bay is now a logistic, energy-production, and industrial hub with several port terminals and water-cooled power plants. It also includes a fishmeal and oil-processing factory, gas transport and processing, a sulfuric acid plant, an explosive manufacturer, and several projects in progress including a desalination plant [START_REF] Arroyo | Proyectos de apoyo logístico al negocio portuario en la Bahía de Mejillones[END_REF][START_REF] Arroyo | Infraestructura y Plataforma de Servicios Habilitantes en la Bahía de Mejillones para la Industria de Hidrógeno Solar[END_REF]. Mejillones Bay is now one of the most developed industrial zones in northern Chile, with its activities concentrated in the central part of the coastline [START_REF] Valdés | Petrographic and geochemical study of organic matter in surficial laminated sediments from an upwelling system (Mejillones del Sur Bay, Northern Chile)[END_REF] adjacent to the city of Mejillones (~14,000 inhabitants) and a mariculture facility growing the non-native macroalgae Agarophyton chilense . Development is such that Mejillones is often referred to as an environmental sacrificial zone.

Sampling strategy

Sampling was carried out in the bay during May, August, September, and October 2017

along 3 transects: one on each side of the bay (Fig. 1: EAST and WEST), where samples of sediment and associated taxa were collected at depths of 10, 20, 30 and 50 m. A third transect named OUTFALL was located between the EAST and WEST transects, directly adjacent to the city's sewage water outfall. Sediments and associated taxa were sampled at 10, 25, and 50 m (n.b. the mouth of the outfall is at 25 m).

At each sampling station, vertical profiles of the water column were recorded using a CTDO (Sea-Bird SBE 19 plus) to measure temperature (± 0.01 ºC), dissolved oxygen (± 0.07 mL/L), and salinity (± 0.03) from the sea surface to 0.5 m above the seabed. Sediment samples were collected using a van Veen grab sampler (0.1 m 2 ) with a subsample collected in a 50 mL falcon tube used for organic matter analysis and another for analysis of sediment carbon (C) and nitrogen (N) stable isotopes. On each sampling occasion, and at each depth, five grabs were collected and sieved through a 0.5 mm mesh to retain macrobenthic organisms. Macrobenthos was kept in a solution of seawater with ethanol and then frozen at -20ºC for subsequent identification. An additional grab sample of macrobenthic organisms was collected and sieved through a 1 mm mesh, transported in ice, and frozen at -20ºC at the laboratory for C and N stable isotope analysis. Samples of macrobenthic organisms were identified to the lowest possible taxon (normally species), counted, and weighed with shells after drying at 60ºC for 48 h. For stable isotope analyses, when possible, a muscle sample of one individual was used. Otherwise, chitinous parts were removed, and several individuals were pooled together to obtain the necessary biomass for analysis. The obtained samples were then homogenised and weighed into tin capsules for determination of δ 13 C and δ 15 N at the University of Antofagasta stable isotope facility. Organic matter was estimated as a percentage of mass loss by burning 10 g of dry sediment at 550 °C for four hours in a muffle furnace.

Ecological indices

We selected and calculated 16 indices, including community metrics, estimations of diversity, and indices based on taxa sensitivity, reference conditions, and community functioning.

Indices based on diversity

Benthic fauna abundance and dry biomass were recorded and summed for each sample to obtain total abundance (A), total dry biomass (B), and total number of taxa (i.e., taxonomic richness; S). Diversity was estimated using Shannon-Wiener index (H) with logarithm base 2 [START_REF] Shannon | The mathematical theory of communication[END_REF] and Simpson diversity index (D; i.e., 1-D; [START_REF] Simpson | Measurement of diversity[END_REF] using the "diversity" function from the R package "vegan" [START_REF] Oksanen | Community Ecology package vegan[END_REF]). Pielou's evenness index (J; [START_REF] Pielou | An introduction to mathematical ecology[END_REF] was also calculated (Table 1). All indices were calculated for each sample and then averaged (arithmetic mean) for each sampling site.

2.3.2 Indices based on taxa sensitivity

Four indices based on taxa sensitivity to disturbance were calculated, requiring different degrees of previous knowledge for taxa classification. The Benthic Quality Index (BQI; [START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF]) is based on an objective classification of benthic taxa according to their tolerance and sensitivity to organic enrichment and oxygen stress. The expected number of species among 50 individuals (ES50; [START_REF] Hurlbert | The non-concept of species diversity: a critique and alternative parameters[END_REF] is calculated for each sample, and the sensitivity of each taxon is evaluated using the ES50 value at 5% of the abundance frequency distribution. This procedure excluded samples with less than 50 individuals from the ES50 calculation. To include the maximum number of samples in the index calculation, we pooled the five samples instead of averaging them as [START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF] recommended and included all the recorded taxa. Thus, 35 of the 44 samples (i.e., 80%) were included in the calculation instead of 98 of the 220 (45%) otherwise. This allowed calculating ES50 0.05 values for 109 of the 110 taxa. The Azti Marine Biotic Index (AMBI) is based on the percentage of the abundance of macrobenthic taxa assigned into five ecological groups (EG) according to their sensitivity to disturbance [START_REF] Grall | Using biotic indices to estimate macrobenthic community perturbations in the Bay of Brest[END_REF][START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. Taxa absent from the Azti list from December 2020 were assigned to the EG of a closely related taxon if there was no possible ambiguity. However, 16 of the 110 taxa could not be assigned to any EG, resulting in < 5% of the total abundance being excluded from analyses. The AMBI was calculated for each sample following [START_REF] Sigovini | M-AMBI revisited: looking inside a widely-used benthic index[END_REF]. The benthic index BENTIX [START_REF] Simboura | Benthic indicators to use in Ecological Quality classification of Mediterranean soft bottom marine ecosystems, including a new Biotic Index[END_REF] reduced taxa into two EGs: one sensitive and one tolerant. Thus, 100 of the 110 taxa could be assigned (i.e., six additional taxa compared to the AMBI), reducing the non-assigned taxa to 4% of total abundance. Finally, the Benthic Opportunist Polychaetes-Amphipods (BOPA) index relies on relative frequency rather than abundances [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]. It further reduces the need for taxonomic classification as it uses only amphipods and opportunist polychaetes.

Our dataset included ten opportunist polychaetes and seven amphipod taxa (without Jassa sp. that were excluded, as recommended by [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]. For calculation of BOPA, 61 of the 220 samples had less than 20 individuals but were included in the calculation regardless. All indices were calculated for each sample, then averaged by month for each of the 11 sampling sites, except for the BQI where the five samples were summed.

2.3.3 Indices based on reference conditions

Two indices based on reference conditions were calculated. The multivariate-AMBI (M-AMBI) integrates standardised AMBI, S, and H by a factor analysis, including minimum and maximum reference values as arbitrary samples [START_REF] Muxika | Using historical data, expert judgement and multivariate analysis in assessing reference conditions and benthic ecological status, according to the European Water Framework Directive[END_REF]. It was calculated following [START_REF] Sigovini | M-AMBI revisited: looking inside a widely-used benthic index[END_REF] for each sample (i.e., without pooling the samples together).

The "bad" references used were S = 0, H = 0, and AMBI = 7. We replaced the AMBI default value of six with a value of seven, as this value is assigned to samples without fauna, and we had several in our dataset. We used two different references, since [START_REF] Muxika | Using historical data, expert judgement and multivariate analysis in assessing reference conditions and benthic ecological status, according to the European Water Framework Directive[END_REF] define reference conditions as "undisturbed (= pristine) conditions" and such conditions were not available. The EAST transect is located away and up-current from the anthropogenic activities, so we could assume that anthropogenic pressure on benthic communities was minimum at this location, and stations of this transect were used as reference. To account for potential autocorrelation between the parameters from Mejillones Bay, we compared the data with a reference site located further south, in Antofagasta Bay. We used data of benthic communities collected in February 2020 from another project studying the impact of climate change on this region, selecting the sampling station further away from anthropogenic activities at Lenguado (LEN; 23°46'S, 70°29'W; Fig. 1; unpublished data). This station is located ~70 km to the south of Mejillones Bay and was sampled at 10, 30, and 50 m depth following a strictly identical sampling method used for the other stations in this study.

M-AMBI was calculated for each sample using the full dataset as for the other indices, using EAST as reference. Also, it was calculated for each sample at shallow (10 m), intermediate (20 to 30 m), and deep (50 m) separately month by month using each of the two reference sites to reduce the variation within each dataset. Finally, the recently developed General-Purpose Benthic Index (GPBI; [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF] compares benthic macrofauna community composition and structure to one or several reference station(s) with good ecological status. We calculated this index for each sample using the full dataset at once with EAST as reference. Then, we calculated it at shallow, intermediate, and deep for each month separately, using each of the two reference sites, one by one, as for the M-AMBI.

2.3.4 Indices based on community functioning

Functional diversity indices were also included as proxies of communities' ecological niches (sensu [START_REF] Rigolet | Investigating isotopic functional indices to reveal changes in the structure and functioning of benthic communities 29[END_REF]. Two different facets of functional diversity were estimated from the multidimensional trait space. The functional richness index (FRic) estimates the convex hull volume of functional space occupied by the community [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]. The functional dispersion index (FDis) represents the abundance-weighted mean distance of individual taxa to the abundance-weighted centroid of all taxa [START_REF] Laliberté | A distance-based framework for measuring functional diversity from multiple traits A distance-based framework for measuring from multiple traits functional diversity[END_REF]. In our study sites, these indices were calculated as in [START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF], based on nine biological traits divided into 38 categories reflecting the ecological functioning of the community and their sensitivity to environmental disturbances (Table S1; [START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. Each taxon was assigned a score ranging from 0 (no affinity) to 3 (complete affinity) for each trait category according to the fuzzy coding procedure [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF]. If no relevant information was available for a specific trait for a particular taxon, values of 0 were assigned to all categories for this trait so the taxon made no contribution to this trait in the analysis [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF]. This was the case of 26 traits of 17 different taxa (i.e., less than 1% of the total). This biological trait matrix was used together with the matrix of taxa abundance to calculate FRic and FDis for each sample using the R package "FD" [START_REF] Laliberté | FD: Measuring functional diversity (FD) from multiple traits, and other tools for functional ecology[END_REF].

These two indices were also calculated based on stable isotope ratios of δ 13 C and δ 15 N replacing biological traits [START_REF] Rigolet | Investigating isotopic functional indices to reveal changes in the structure and functioning of benthic communities 29[END_REF]. Functional dispersion based on stable isotope ratios (FDis ISO ) was also weighted by abundance. To obtain sufficient stable isotope data to calculate those indices, we included the stable isotopes dataset from another project that collected samples during 2016 at the same locations at EAST and WEST (unpublished data). Thus, isotope values for 68 of the 110 taxa (i.e., 87% of the total abundance) were available. However, we still could not separate the isotope values for each site. Functional indices were calculated for each sample and then averaged.

Data analysis

Each environmental variable was fitted in a linear model to test the effect of the factors "sampling station" (depth nested in site) and "month" using an analysis of variance. DO, organic matter, temperature, and macroalgal biomass data were all square root transformed to fulfil the assumption of homogeneity of variance for "sampling station". ANOVA was applied even if the variables did not display a normal distribution, as ANOVA is robust to type I error under violation of normality [START_REF] Blanca | Non-normal data: Is ANOVA still a valid option?[END_REF]. After detecting significant effects, pairwise comparisons of the estimated marginal means (least-squares means) tests with Tukey adjustment (α = 0.05) were applied. For salinity, homogeneity of variance could not be reached after transformation for sampling location. Thus, the non-parametric test, Kruskal-Wallis, was used.

To include the maximum information available and to use the same dataset for the different analyses, when the calculation was not possible due to the presence of samples with no fauna, the worst possible index value was assigned following the recommendations for AMBI and BENTIX (i.e., BOPA = log10(2); H, J, FRic and FDis = 0). For FRic, values of zero were also assigned for samples with less than 3 functionally singular taxa and one for J when there was only 1 taxon. Each sampling site was then classified into ecological quality status (EcoQ) from the European Union's water framework directive (WFD) according to each index average value. A range of values used for classification were taken from the literature for AMBI, M-AMBI, BENTIX, BOPA, BQI and GPBI when available. For the rest of the indices, the EcoQ "high" was assigned to the highest encountered values (i.e., the 80 percentiles), "good" to the percentiles 80 to 60, "fair" to the 40 to 60, "poor" to the 40 to 20

and "bad" to the lower 20 percentiles.

Redundancy analysis was applied to examine potential relationships among the 16 indices, environmental variables (i.e., organic matter, DO, temperature, salinity, macroalgal biomass, and measured depth), and the macrofaunal community structure. Index values and environmental variables were previously centred and scaled without previous transformation, and site was used as a factor. To evaluate which variable explained most variation in each index, variation partitioning was calculated for each index and each explanatory variable separately. Then, an analysis of deviance using 999 permutations was conducted to test the significance of the contribution of each explanatory variable for each index. All analyses were performed using R version 3.6.2 (R Core Team, 2019) managed through Rstudio version 1.2.5033 (RStudio Team, 2019) and graphs using the R package "ggplot2" [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF].

Results

Environmental parameters

The main variation in environmental parameters was observed in DO and organic matter concentrations (Fig. 2) rather than temperature, salinity, and macroalgal biomass (Fig. S1).

Dissolved oxygen decreased along each transect, with hypoxic conditions (i.e., < 0.2 mL/L) present below 10 m and anoxic conditions at 50 m depth (Fig. 2). However, DO did not vary significantly through time (Table 2). Organic matter concentrations increased slightly with depth and showed high concentrations at 50 m, especially in WEST and OUTFALL but did

Macrobenthic communities

The 220 sediment samples collected contained 24 022 individuals from 110 different taxa, mainly polychaetes (69%), crustaceans (19%, dominated by amphipods), and mollusks (9%, dominated by gastropods). Thirty taxa were represented by less than five individuals.

Thirty-one samples had no fauna, eleven from OUTFALL at 50 m, two from WEST at 30 m, and eighteen from WEST at 50 m depth.

Ecological quality status and indices performance

Overall, the indices values indicated that the lowest ecological quality status was recorded from the deepest sites at WEST and OUTFALL. Conversely, EAST deepest site displayed relatively good conditions (Table 3). The ecological quality status of WEST 30 m was mainly The triplot from the redundancy analysis (Fig. 3; R 2 adj = 50.3%; permutation test: df = 8, 211, F = 28.67, p < 0.05) revealed that most indices were highly co-correlated and that they showed similar relationships with depth either positive (AMBI and BOPA) or negative e.g., BQI, BENTIX, and M-AMBI. However, GPBI correlated with abundance, especially when EAST was used as the reference station (Fig. 3). It was associated negatively with organic matter concentrations that separate the centroids of the stations OUTFALL and WEST from EAST. The EAST stations followed the depth gradient of decreasing DO and temperature.

The stations at WEST and OUTFALL were separated from EAST and associated with higher organic matter content spreading into three groups (Fig. 3). A first group with the shallowest stations associated with higher DO and temperature and high variability was observed for the stations of 10 m WEST. The second group with the stations of intermediate depths of

Discussion

In this study, a range of different biotic indices was used to evaluate the benthic ecological quality status (EcoQ) of Mejillones Bay, and to examine whether the impacts of anthropogenic activities could be distinguished against a background of natural nutrient enrichment. Although some indices were able to detect anthropogenic impact under natural stress, most of them also indicated natural conditions were such that benthic EcoQ was degraded (i.e., away from direct anthropogenic pressure).

Benthic ecological quality status of the upwelling ecosystem

Benthic communities under naturally low oxygen and high organic matter concentrations were not always classified as low ecological quality by the biotic indices examined here. In the deepest habitats (i.e., 50 m), most indices classified the EAST station as having good benthic ecological quality despite low DO and high organic matter concentrations. This station stood out from the other deep stations with higher taxonomic richness and abundance. Several thousand individuals from 27 different taxa have been collected at this station, and samples were highly dominated by two polychaetes: Magelona phyllisae and Paraprionospio pinnata. These species are characteristic of benthic habitats in the SE Pacific oxygen minimum zone [START_REF] Palma | Macrobenthic animal assemblages of the continental margin off Chile (22° to 42°S)[END_REF]. The deep EAST station also displayed a moderately wide functional space (i.e., intermediate values of functional richness), indicating some unique combination of trait categories. Previous studies have associated a reduction of functional richness with environmental filtering and competitive dominance [START_REF] Perronne | How to design trait-based analyses of community assembly mechanisms: Insights and guidelines from a literature review[END_REF]. In EAST, the depth gradient of hypoxia and organic matter are expected to be the main factors reducing the number of viable trait categories. High levels of specialisation due to trait displacement reflecting interspecific competition or other biological interactions may also occur [START_REF] Perronne | How to design trait-based analyses of community assembly mechanisms: Insights and guidelines from a literature review[END_REF]. This agrees with previous studies in Mejillones Bay revealing that macrobenthic communities are highly adapted to hypoxic conditions [START_REF] Laudien | Sublittoral soft bottom communities and diversity of Mejillones Bay in northern Chile (Humboldt Current upwelling system)[END_REF][START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF][START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF][START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. Even during short-term oxygenation or inter-annual events such as El Niño, that bring well-oxygenated and nutrient poor waters, these benthic communities have shown a highly persistent and stable taxonomic and functional structure in this region [START_REF] Carrasco | Long-term dynamics (1990 to 2004) of the polychaete fauna from the sublittoral soft-bottoms off Punta Coloso (Antofagasta), northern Chile[END_REF][START_REF] Pacheco | El Niño-Southern Oscillation cyclical modulation of macrobenthic community structure in the Humboldt Current ecosystem[END_REF][START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF][START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. A similar pattern of stability has been reported from estuarine fauna, indicating a paradoxical stable state under variable and stressed conditions [START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF]. The relatively diverse macrobenthic fauna observed at EAST is natural, as this site is located away from direct anthropogenic impact and could thus be used as reference.

The central and western parts of the bay differed considerably from the eastern part. The deepest stations of these two sites mainly displayed poor ecological quality. Only small numbers (tens of individuals) of eight taxa were collected at the OUTFALL station and a few individuals from two taxa at WEST. Several samples were free of macrofauna at those sites, in marked contrast with communities from the eastern part of the bay. Also, the functional space was reduced (i.e., lower functional richness based on biological traits). This possibly indicates that some of the resources potentially available to the community remain unused at those sites. It can also be related to a loss of taxa with trait categories poorly adapted to the existing environmental conditions [START_REF] Mason | Functional richness, functional evenness and functional divergence: The primary components of functional diversity[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. For example, filter feeders were absent from many samples, increasing particulate matter sedimentation and disturbing larval settlement and thus the community structure [START_REF] Douglas | Macrofaunal Functional Diversity Provides Resilience to Nutrient Enrichment in Coastal Sediments[END_REF].

Burrowers also decreased, which changes local bioturbation potential and hence the denitrification processes that provide resilience to eutrophication [START_REF] Douglas | Macrofaunal Functional Diversity Provides Resilience to Nutrient Enrichment in Coastal Sediments[END_REF].

Thus, the loss of trait categories can contribute to a feedback loop that intensifies eutrophication impacts at those sites (Fig. S2). Moreover, a reduction in functional dispersion indicated fewer taxa with a unique combination of trait categories. These two sites are most likely affected by environmental filtering as taxonomic richness and abundance are extremely reduced, so biological interactions are not expected to play an important role in structuring communities at these parts of the bay [START_REF] Perronne | How to design trait-based analyses of community assembly mechanisms: Insights and guidelines from a literature review[END_REF]. The observed relationship between functional diversity indices and benthic environmental health is similar to the pattern observed along the Mar del Plata coastline in Argentina. There, the functional richness of rocky shore benthic communities was reduced in sites exposed to sewage discharges [START_REF] Garaffo | Assessing functional diversity of macrobenthic assemblages in sewage-affected intertidal shores[END_REF].

The degradation of EcoQ in the central and western parts of the bay compared to the east was mainly related to the organic matter content, with other environmental parameters showing little evidence for varying within sites. This confirmed previous results revealing that organic matter mainly controls macrobenthic taxonomic richness variation in this bay [START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. Organic matter concentrations in the sediment reported here are similar to those reported previously in the bay [START_REF] Moraga | Prokaryotic community composition involved production of nitrogen in sediments of Mejillones Bay[END_REF] and reflect organic sedimentation associated with extremely high local primary production [START_REF] Valdés | Petrographic and geochemical study of organic matter in surficial laminated sediments from an upwelling system (Mejillones del Sur Bay, Northern Chile)[END_REF][START_REF] Valdés | Interplay between sedimentary organic matter and dissolved oxygen availability in a coastal zone of the Humboldt Current System; Mejillones Bay, northern Chile[END_REF]. We hypothesise that the high accumulation of organic matter recorded in this part of the bay results from both natural processes and anthropogenic activities. Nutrient inputs from permanent upwelling are responsible for high primary production rates (3170 mg.C.m -2 per day; [START_REF] Palma | Abundancia estacional y distribución vertical del zooplancton gelatinoso carnívoro en una área de surgencia en el norte del sistema de la corriente de Humboldt[END_REF]. Phytoplankton is transported by the main current from east to west at a relatively slow rate (about 0.2 m.s -1 ; [START_REF] Marín | Upwelling shadows at Mejillones Bay (northern Chilean coast): a remote sensing in situ analysis[END_REF]Rojas and Landeata, 2014). Organic detritus from this high productivity falls through the water column and accumulates naturally in the deeper part of the bay's western side [START_REF] Valdés | Interplay between sedimentary organic matter and dissolved oxygen availability in a coastal zone of the Humboldt Current System; Mejillones Bay, northern Chile[END_REF].

However, measurements of organic matter alone would not be sufficient to classify the ecological quality status of the different sites.

The bay is also subject to significant anthropogenic inputs. In the central part of the bay's coastline, liquid wastes from a fishmeal and oil factory, that processed ~100,000 tonnes of fish in 2017 (SERNAPESCA, 2017;CORPESCA, 2018), and sewage waters from the city are discharged via submarine pipes at intermediate depths (i.e., 25-30 m) in addition to the organic matter that is naturally present. Several industrial installations also use seawater for cooling. These activities result in the discharge of large volumes (several hundreds of millions of cubic metres per year) of seawater that is warmed by up to 10°C above ambient temperature back in the bay, and that may include antifouling compounds (e.g., ENGIE, 2019). Such cooling systems have been reported to have a strong impact on plankton communities, killing up to 64% of the phytoplankton and 93% of the zooplankton present at the uptake due to temperature, pressure, biocide, and mechanical effects [START_REF] Jebakumar | Impact of coastal power plant cooling system on planktonic diversity of a polluted creek system[END_REF][START_REF] Lee | Comparison of mesozooplankton mortality impacted by the cooling systems of two nuclear power plants at the northern Taiwan coast, southern East China Sea[END_REF][START_REF] Rajeev | Thermal discharge-induced seawater warming alters richness, community composition and interactions of bacterioplankton assemblages in a coastal ecosystem[END_REF]. In Mejillones Bay, this can potentially represent a significant amount of plankton detritus that precipitates at the bottom due to the very high plankton concentration in the bay [START_REF] Marín | Effects of coastal upwelling on the spring primary productivity of Mejillones del Sur Bay (Antofagasta, Chile)[END_REF][START_REF] Rojas | Retención de larvas de peces asociada a una batimetría abrupta en Bahía Mejillones (norte de Chile) durante eventos de surgencia costera[END_REF]. This organic detritus is most probably carried westward by the main current and sediments.

Together with the natural organic matter, this enhances organic matter accumulation in the central and western areas compared to what would occur naturally. The increased organic matter flux towards the seabed represents a higher disturbance impacting the resident macrobenthic fauna. The accumulation of organic matter originating from industrial cooling systems in this part of the bay concurs with increased concentrations of heavy metals (Zn, Pb, and Ni) recorded from sediments in this area over recent years. These have been directly attributed to the impact of anthropogenic activities [START_REF] Valdés | Heavy metal distribution and enrichment in sediments of Mejillones Bay (23° S), Chile: a spatial and temporal approach[END_REF]. Between 2000 and 2005, Zn mean concentrations increased from 29.7 to 57.3 mg.kg -1 , Ni from 20.6 to 34.6 mg.kg -1 and Pb from about 2 to 19.9 mg.kg -1 [START_REF] Valdés | Distribution and enrichment evaluation of heavy metals in Mejillones Bay (23°S), Northern Chile: Geochemical and statistical approach[END_REF][START_REF] Valdés | Heavy metal distribution and enrichment in sediments of Mejillones Bay (23° S), Chile: a spatial and temporal approach[END_REF]. Moreover, concentrations of three heavy metals (Cu, Ni and Cd) have also been reported to reach potential adverse effects on marine biota, as well as impacting microbial communities living in the sediment [START_REF] Valdés | Heavy metal distribution and enrichment in sediments of Mejillones Bay (23° S), Chile: a spatial and temporal approach[END_REF][START_REF] Zárate | Benthic microbial diversity trends in response to heavy metals in an oxygen-deficient eutrophic bay of the Humboldt current system offshore the Atacama Desert[END_REF]. These studies showed that heavy metal enrichment in recent years most likely derives from rapid urbanisation and industrial activity [START_REF] Valdés | Distribution and enrichment evaluation of heavy metals in Mejillones Bay (23°S), Northern Chile: Geochemical and statistical approach[END_REF][START_REF] Valdés | 250 years of sardine and anchovy scale deposition record in Mejillones Bay, northern Chile[END_REF][START_REF] Valdés | Heavy metal distribution and enrichment in sediments of Mejillones Bay (23° S), Chile: a spatial and temporal approach[END_REF]. Indeed, Mejillones Bay has controversially been declared a "sacrificial zone" in Chile. That is a geographic location of high industrial concentration where industrial developments are prioritised over human wellbeing and environmental quality status. Industrial development in this area is ongoing: the volume of materials handled by port facilities in Mejillones tripled between 2005 and 2019, a new seawater cooled power generation plant was inaugurated in 2019, and a desalination plant for the mining industry is currently under construction. Anthropogenic pressure in the bay is expected to further increase in the near future, underlining the urgent need to develop monitoring that will allow reliable assessment of Mejillones Bay ecosystem health.

Indices performance in a bay influenced by upwelling

This study described a consistent pattern of lower benthic ecological quality at the central and western parts of the bay compared to the east, especially at intermediate and deeper habitats as described previously. However, different biotic indices often resulted in contrasting benthic ecological status classifications. Previous studies have highlighted this inconsistency (e.g., [START_REF] Labrune | Characterization of the ecological quality of the coastal Gulf of Lions (NW Mediterranean). A comparative approach based on three biotic indices[END_REF][START_REF] Puente | Is it possible to assess the ecological status of highly stressed natural estuarine environments using macroinvertebrates indices?[END_REF]Sivadas et al., 2016). In the upwelling context of the present study, three main limitations of biotic indices were related to i) the important natural environmental stress, ii) the limited knowledge available for some taxa in the area, and iii) the low taxonomic richness and abundance in some samples.

i) Influence of the natural environmental stress

The environmental parameter that explained most of the variation in the indices by itself was depth: a natural gradient (Table 5). Upwelling brings cold waters, poor in oxygen and rich in nutrients towards the surface, drastically increasing primary production, resulting in a marked reduction of DO due to microbial degradation of organic matter in the water column, especially above the sediment [START_REF] Kämpf | Upwelling systems of the world[END_REF]. Following a depth gradient of increasing hypoxia and organic matter content, taxonomic richness and abundance decreased, as well as interactions between organisms [START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. Biotic indices rely on the assumption that benthic macrofauna inhabits environmental conditions where they can thrive [START_REF] Pinto | Review and evaluation of estuarine biotic indices to assess benthic condition[END_REF], and tolerant species may not be specific to different kinds of stress [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF]. Thus, as expected, natural upwelling conditions were reflected in the results provided by those indices, indicating degrading ecological status with depth, which was related to the loss of sensitive taxa.

Previous studies have revealed that most biotic indices based on metrics derived from taxonomy respond to any disturbance, whether it is caused by natural or anthropogenic processes [START_REF] Dauvin | The estuarine quality paradox: Is it possible to define an ecological quality status for specific modified and naturally stressed estuarine ecosystems?[END_REF]Sivadas et al., 2016;[START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF]. This study confirmed this for indices based on biological traits and stable isotopes of macrobenthic communities under upwelling influence. This probably reflects the fact that natural stressors are the same environmental parameters than those impacted by human activities, i.e., increased organic matter production. Nutrient inputs from upwelling indirectly drive significant decreases in DO and increases in organic matter. These natural processes are exacerbated by the addition of organic matter from industrial related processes and sewage water discharge. Detecting the impact of anthropogenic activities in this naturally stressed environment is therefore extremely challenging. We propose to consider the phenomenon described in this study as an "Upwelling Ecosystem Quality Paradox", in line with the "Estuarine Quality Paradox'' described for estuaries [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF][START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF]. In estuaries, natural stress is related to strong currents, high turbidity, anoxia, and important variations in salinity [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF]. Salinity variations from fresh to marine waters have been incorporated in indices such as the Benthic Condition Index, in which Shannon diversity index values are corrected according to the expected values in the observed salinity conditions [START_REF] Engle | Refinement, Validation, and Application of a Benthic Condition Index for Northern Gulf of Mexico Estuaries[END_REF]. A more recent approach classifies stations according to their habitat characteristics, such as sediment type and intertidal or subtidal location, which influences EcoQ classification [START_REF] Blanchet | Use of biotic indices in semi-enclosed coastal ecosystems and transitional waters habitats-Implications for the implementation of the European Water Framework Directive[END_REF]. It is more similar to what we have proposed here, calculating GPBI and M-AMBI separately for each depth habitat.

GPBI accounts for the loss of individuals for each taxon present in reference sites compared to the abundance of the same taxon at study sites [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF]. It could not detect the higher increase in opportunistic polychaetes abundance at OUTFALL compared to WEST at intermediate depths. However, GPBI was the only multimetric index that displayed less influence from the depth gradient (10% or less). The strength of this index is the comparison of abundance at reference and study sites at a taxon-by-taxon level that allowed a substantial reduction of the "background noise". The reference station must be carefully selected with similar benthic communities in terms of the taxa and comparable environmental conditions, which is not always feasible. [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF] Indicated that GPBI required further testing in different habitats and pressures. We successfully applied this index here, calculating values for each sampling month and distinguishing shallow, intermediate, and deep habitats. This approach allowed to reduce the variability of the GPBI references within each calculation and help to bring a clearer pattern. Similarly, in transitional waters such as estuaries and lagoons, several studies have highlighted the need for adequate reference conditions for the natural gradient to correctly assess the ecological quality [START_REF] Blanchet | Use of biotic indices in semi-enclosed coastal ecosystems and transitional waters habitats-Implications for the implementation of the European Water Framework Directive[END_REF][START_REF] Dauer | Depth-related patterns in benthic community condition along an estuarine gradient in Chesapeake Bay, USA[END_REF][START_REF] Teixeira | Ecological indices tracking distinct impacts along disturbance-recovery gradients in a temperate NE Atlantic Estuary -Guidance on reference values[END_REF]Borja et al., 2012a;).

The GPBI values obtained were lower when using the reference site from outside the bay, i.e., Lenguado, Antofagasta Bay. However, using the two different reference sites allowed to rank the stations globally in the same order according to their ecological quality status.

Lenguado is located further south (Fig. 1), which limits the local character of the communities as using local reference conditions could account for autocorrelation leading to overestimation of the EcoQ. To our knowledge, spatial variability has not been identified as an important issue when selecting reference stations for biotic indices contrary to what have been reported for experimental studies or impact assessments. This likely reflects that most indices used reference stations exclusively to set minimum and/or maximum limits rather than compare them taxon by taxon with the study site. This further supports the complexity of selecting an appropriate reference (Borja et al., 2012) beyond the use of pristine or least disturbed areas for reference, as recommended by the European WFD and the US Environmental Protection Agency (EPA; [START_REF] Gibson | Estuarine and Coastal Marine Waters: Bioassessment and Biocriteria Technical Guidance[END_REF].

As described for GPBI, M-AMBI results were improved by calculating the index for each depth group and month separately rather than by pooling data. Such an approach also resulted in higher values for the EAST stations. However, M-AMBI was still highly influenced by the depth gradient which explained between 30 and 50% of its variability. This is because, in the calculation of the M-AMBI, the reference station is used only to set the maximum limits for taxonomic richness, Shannon diversity, and AMBI values. The reference station is not directly compared to the tested stations but rather used to set the maximum limits of the factorial analysis dimensional space. Thus, the results obtained still depended heavily on the natural depth gradient, and this index could not distinguish "environmental noise" from anthropogenic impact. In addition, M-AMBI is based on the AMBI classification, and in this study, we observed incoherence in the ecological group's assignment of several taxa which can skew calculations and the interpretation of the index.

ii) Taxonomic and ecological knowledge requirements Sensitivity classifications of benthic taxa for AMBI and BQI were both set to reflect their sensitivity to organic matter enrichment and relies on the model of [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF]. However, important differences and even contradictions in their rankings are reported in our results (see Table S2 in supplementary material; [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF]. For example, the polychaete Magelona phyllisae is classified in ecological group 2 (EG), according to the AMBI list of December 2020, which includes the species indifferent to enrichment that are always present in low densities [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF].

However, the density of M. phyllisae varies significantly along the depth gradient of hypoxia and organic matter. We found thousands of individuals between 50 and 30 m and only one individual between 20 and 10 m along the EAST transect. Moreover, taxa from the EG 2 of AMBI should include suspension feeders, less selective carnivores, and scavengers, contrasting with this subsurface deposit feeding polychaete that is small and has a short life span [START_REF] Moreno | Subtidal macrozoobenthos communities from northern Chile during and post El Niño 1997-1998[END_REF][START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. According to these characteristics, M.

phyllisae would be qualified as a second-order opportunistic species and, therefore, belongs to the AMBI EG 4. Other studies in Chile and the Gulf of Mexico confirmed our classification describing M. phyllisae as a generalist species that can be very abundant and is commonly found together with Paraprionospio pinnata , a species from AMBI EG 4 [START_REF] Carrasco Vázquez | Estimación de la producción secundaria de Paraprionospio pinnata (Spionidae, Polychaeta) frente a Bahía de Concepción, Chile[END_REF][START_REF] Flint | Polychaete Ecology and Niche Patterns: Texas Continental Shelf[END_REF]. This highlights the need to refine the AMBI species list considering species geographical occurrence and environmental context. For example, changing only the classification of M. phyllisae from EG 2 to 4 results in the ecological quality of EAST at 30 and 50 m switching from good to fair. About one-third of the taxa encountered are facing similar issues (e.g., Capitellidae, Phoxocephalidae, Polinices uber , Ampelisca sp., among others), leading to important changes in the ecological status of the stations from this study. As such, we suggest caution when interpreting results obtained from indices based on the AMBI classification (i.e., AMBI, BENTIX, and M-AMBI) in Mejillones

Bay. AMBI has been used worldwide in many contexts, including hypoxia and organic matter enrichment. However, to our knowledge, it has only been applied once in an upwelling ecosystem. In a study from India, Magelona sp. increased suddenly after the upwelling season and was therefore considered as EG 3 (Sivadas et al., 2016). This also highlights the issue of potentially misleading results that can be obtained when using a pre-classified list of sensitive/tolerant species.

iii) Low taxonomic richness and abundance

The presence of samples without fauna or very low taxonomic richness and abundance in the most impacted stations is highly informative regarding the evaluation of benthic ecological status at these stations. Yet, most indices cannot be calculated for samples without fauna because they are based on logarithm transformations (e.g., Shannon-Wiener diversity and Pielou's evenness) or on proportions and no constant has been added. This is the case of all the calculated indices in this study except GPBI, since its divisor is the sum of species' abundances at the reference station, and BOPA only for the frequency of amphipods and opportunist polychaetes. Also, functional richness cannot be calculated if there are less than three functionally singular species. To include the information provided by those samples without fauna, the value representing the worst ecological quality has been assigned to the samples without fauna as recommended for AMBI and BENTIX [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Simboura | Benthic indicators to use in Ecological Quality classification of Mediterranean soft bottom marine ecosystems, including a new Biotic Index[END_REF].

Operational limits have been set for several indices. For BOPA, they were assigned at less than 20 individuals per sample [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF][START_REF] Dauvin | Twenty years of application of Polychaete/Amphipod ratios to assess diverse human pressures in estuarine and coastal marine environments: A review[END_REF], which included 61 of the 220 samples in this study, all collected from the stations of WEST and OUTFALL, and none from EAST. Those samples gave similar BOPA values than those at the same sampling locations and were thus included in the calculation. BOPA's strength relies on the reduced need for taxonomic knowledge, saving time and expenses. However, sensitivity to pollution differs between species within a taxonomic group. Therefore, a low taxonomic resolution is not recommended in areas of high environmental stress or when the knowledge on local fauna is limited (Sivadas et al., 2016;Santibañez-Agauscaliente et al., 2018).

Nevertheless, the results obtained in this study are coherent with most of the other indices, indicating decreased ecological quality in WEST and OUTFALL and along the depth gradient. For BQI, [START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF] recommended that species encountered in less than 20 samples should not be included in the calculation of ES50. We did not follow this recommendation, because only four taxa were present in more than 20 samples. However, we followed the suggestion to remove samples with less than 50 individuals to calculate the ES50 (9 out of 44). These results should also be interpreted with caution as ES50 validation is based on the randomly distributed individuals for each species, which is not always the case [START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: A proposed new protocol within the European Union Water Framework Directive[END_REF]. Using taxonomic richness instead of ES50 might be a good alternative when samples are of the same type (i.e. grab samples of the same surface).

Low abundances of benthic fauna on the western and central parts of the bay were also an issue for the functional diversity indices based on stable isotopes. Isotopic values for taxa from the different transects and sites were pooled for calculation and could not be separated as [START_REF] Rigolet | Investigating isotopic functional indices to reveal changes in the structure and functioning of benthic communities 29[END_REF] recommended. Benthic food webs in this region are mainly fueled by pelagic-derived energy, even in macroalgae-dominated rocky reefs [START_REF] Docmac | Habitat coupling writ large: pelagic-derived materials fuel benthivorous macroalgal reef fishes in an upwelling zone[END_REF].

We thus considered that the different sites in the bay were subsidised by the same energy source and used stable isotope values for each taxon without separating the different transects to include the highest number of taxa possible in the calculation of those functional indices. These indices revealed that isotopic variation was reduced below 20 m depth in the central and western parts of the bay indicating a simplification and reduction in the size of consumer isotopic niches, which are largely driven by trophic factors. It also indicates that potentially available resources were unused in those habitats and that fewer functions or ecological processes are achieved [START_REF] Petchey | Integrating methods that investigate how complementarity influences ecosystem functioning[END_REF][START_REF] Rigolet | Investigating isotopic functional indices to reveal changes in the structure and functioning of benthic communities 29[END_REF]. Buffering capacity against environmental fluctuations is thus reduced in these areas [START_REF] Mason | Functional richness, functional evenness and functional divergence: The primary components of functional diversity[END_REF].

Conclusions

Evaluating the performance of biotic indices in Northern Chile has allowed us to propose a new concept: the upwelling ecosystem quality paradox. This has highlighted the importance of taking into account natural stress when assessing upwelling ecosystems. Most of the tested indices detected natural ecological stressors rather than anthropogenic perturbations, except the GPBI, which compares the study site to reference conditions taxon by taxon. This index needs further testing and probably habitat specific adjustments of the ranges of EcoQ categories as stated by [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF]. These results emphasise the importance of choosing appropriate reference conditions for each habitat and using them to analyse the changes from each taxon in each sample, rather than to set the limits of the index values. As we look to the future, coastal industrial expansion may require evolving spatial replication of the reference station.

This study also highlights the interest of using complementary functional diversity indices.

Habitats under naturally high organic matter concentrations and near anoxia may harbour true communities of interacting species highly adapted to hypoxia away from direct perturbations from industrial activities. Therefore, indices using pre-classified taxa groups according to their sensitivity/tolerance might be inappropriate in areas with benthic macrofauna adapted to strong natural disturbances. Efficient monitoring combining structural and functional assessments of macrobenthic communities would then be essential to ensure the integrity and sustainability of naturally stressed coastal ecosystems. Our results on marine macrobenthic communities under upwelling influence will become crucial in the framework of increasing coastal development and hypoxic zones global expansion impact assessment.

Conclusions and perspectives

In this study, I propose the new concept of the "Upwelling Ecosystem Quality Paradox" that highlights the challenge of disentangling the effects of anthropogenic and natural disturbances in upwelling areas. When impacts of disturbances due to human activities on benthic communities are confusable with the effects of natural stress, the best way to determine whether the disturbances originate from anthropogenic activities is to compare the surveyed (and presumably impacted) ecosystem with a very similar ecosystem under natural conditions free from direct human influence (i.e., a pristine environment). However, pristine environments are rarely available, given that human activities (such as human induced climate change) impact the whole planet.

Data available prior to any anthropogenic impact could be used, which stresses the importance of archiving data (or samples) for future analyses. Indeed in this study, some relevant historical data do exist but part of the information is lost as data was not properly stored for future use. These data provide information on the macrobenthic communities and organic matter and dissolved oxygen conditions before the settlement of the port and the Although potentially very useful, the utility of historical data to act as reference conditions can be limited as researchers must take into account temporal variation in the study area, which may prevent the comparison and identification of causal factors [START_REF] Labrune | Long-term comparison of soft bottom macrobenthos in the Bay of Banyuls-sur-Mer (north-western Mediterranean Sea): A reappraisal[END_REF].

Further discussion on the difficulty of selecting appropriate reference conditions have been reviewed (e.g., Borja et al., 2012a). The best approximation available for reference conditions is therefore the comparison of the least impacted similar areas within a similar temporal period, i.e., with minimum human intervention, selected along a gradient of anthropogenic pressure [START_REF] Elliott | Integrating benthic habitat indicators: Working towards an ecosystem approach[END_REF]. In this study, two reference sites were used i) the eastern side of the bay that is not directly under anthropogenic pressure and ii) a sampling station located further south within a similar ecosystem to avoid potential autocorrelation between parameters from Mejillones Bay. By using GPBI, an index that compares differences in abundance at a taxon by taxon level between reference and study sites, I highlighted the importance of spatial variation.

The degradation of ecological quality status identified by the biotic indices in the western part of the bay was related to organic matter content. At this location, organic matter in the sediment potentially originates from both natural and anthropogenic origins: settlement of natural detritus from the water column, sewage waters from the city and industrial inputs from the fish factory and cooling water systems. Organic sedimentation is associated with extremely high local primary production and naturally accumulates in the deeper part of the bay's western side [START_REF] Valdés | Petrographic and geochemical study of organic matter in surficial laminated sediments from an upwelling system (Mejillones del Sur Bay, Northern Chile)[END_REF][START_REF] Valdés | Interplay between sedimentary organic matter and dissolved oxygen availability in a coastal zone of the Humboldt Current System; Mejillones Bay, northern Chile[END_REF]. Indeed, organic matter coming from the sewage discharge of the city was not clearly detectable in the sediment using C/N ratio and stable isotopes analyses of carbon and nitrogen. However, organic detritus coming from the fish factory and industrial cooling systems (due to temperature increase and use of chlorine)

have the same origin as natural organic matter; they get their energy from nutrients brought by the upwelled waters. Therefore, further studies are necessary to determine the proportion of organic matter coming from those anthropogenic activities. I thus recommend the construction of an organic matter budget, quantifying organic matter coming from these human activities (i.e., sewage water input, fish factory and industrial cooling water discharges). Quantification of the organic matter input by the sewage water from the city and fish factory is quite straightforward measuring organic matter content and volume of the discharges at the emissary outputs. For organic matter derived from the industrial cooling systems, the quantity of living and dead organisms at the entrance and at the exit of the cooling systems should be assessed using a methodology similar to the one used by [START_REF] Lee | Comparison of mesozooplankton mortality impacted by the cooling systems of two nuclear power plants at the northern Taiwan coast, southern East China Sea[END_REF]. Knowing the concentrations of organic matter and the volume discharged from the different sources, it could be determined how significant is the anthropogenic organic matter input in the bay compared to the total organic matter present. If the quantity of organic matter produced by the industries are significant, industries output would need to be reduced or evacuated outside of the bay. This is crucial as overload of organic matter can induce anoxia and the production of hydrogen sulphide toxic for most marine life responsible which can lead to mass mortalities [START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF][START_REF] Bremner | Species' traits and ecological functioning in marine conservation and management[END_REF]. Such mass mortalities have been reported repeatedly in the last years in this bay by the local newspapers (e.g., Timeline Antofagasta, 2016; El Diario de Antofagasta, 2019). This should be taken into consideration in order to preserve the benthic species present.

Mejillones Bay provides important habitat for benthic species that have worldwide commercial interest, such as chilean scallops ( Argopecten purpuratus ) and chilean abalones ( Concholepas concholepas ) and others with more local commercial interest such as mussels ( Aulacomya atra ), sea urchins ( Loxechinus albus ), crabs ( Pseudograpsus setosus ) and octopus ( Octopus mimus ). These species not only have an importance for artisanal fisheries and mariculture in the bay (the word Mejillones means mussels in spanish) but also support endangered species such as marine otters ( Lontra felina ) and smaller oceanic sunfishes that feed on them [START_REF] Phillips | Bulk tissue and amino acid stable isotope analyses reveal global ontogenetic patterns in ocean sunfish trophic ecology and habitat use[END_REF][START_REF] Valqui | Lontra felina. The IUCN Red List of Threatened Species[END_REF] present in this bay. In the western part of Mejillones Bay, sea turtles including leatherback, pinnipeds, sea birds and 19 species of cetaceans including humpback whales have been reported [START_REF] García-Cegarra | Citizen science as a tool to assess cetacean diversity in the Atacama Desert coast[END_REF]. The presence of these emblematic species in the bay depends on the environmental conditions and food availability that respond to complex and dynamic processes.

The hypoxia in Mejillones Bay is not only limiting for the distribution of benthic species, it also affects the vertical distribution of some zooplankton species. The endemic krill that plays a key role in the food web of the Humboldt Current System ( Euphausia mucronata ), is more abundant above 50 m due to limited dissolved oxygen below this depth [START_REF] Escribano | Distribution of Euphausia mucronata at the upwelling area of Peninsula Mejillones, northern Chile: The influence of the oxygen minimum layer[END_REF][START_REF] Antezana | Euphausia mucronata: A keystone herbivore and prey of the Humboldt Current System[END_REF]. Dissolved oxygen concentration around 0.75 mL.L -1 can limit vertical distribution of zooplankton such as Engraulis ringens (i.e., anchovy larvae) and

Calanus chilensis , one of the most abundant copepod species off the Chilean coast [START_REF] Morales | Anchovy larval distribution in the coastal zone off northern Chile: The effect of low dissolved oxygen concentrations and of a cold-warm sequence (1990-95)[END_REF][START_REF] Gonzalez | Ldh activity in Euphausia mucronata and Calanus chilensis: Implications for vertical migration behaviour[END_REF]. Together with other zooplankton species, including fish larvae, they undertake diel vertical migration to escape predators and avoid the effects of offshore transport to benefit from the onshore nursery areas and from the food availability [START_REF] Landaeta | Spring spawning and early nursery zone of the mesopelagic fish Maurolicus parvipinnis at the coastal upwelling zone off Talcahuano, central Chile[END_REF][START_REF] Rojas | Retención de larvas de peces asociada a una batimetría abrupta en Bahía Mejillones (norte de Chile) durante eventos de surgencia costera[END_REF]. Any increase in the hypoxic zone in the bay could thus modify the pelagic food web and have disastrous consequences on the pelagic fisheries as well.

Ongoing research on understanding the benthic energy pathway from stable isotopes in Mejillones Bay may shed light on interactions between hypoxic and anoxic benthic environments and the productive fishing zones. Another study on the impact of heavy metals on the macrobenthic communities is in progress and will further contribute to local stressors and dynamics understanding in the bay. I stress the importance of implementing a long term and continuous monitoring of physico-chemical and biological parameters such as dissolved oxygen, sediment and particulate organic matter concentrations, carbonate chemistry, planktonic and macrobenthic communities. Such monitoring has been successfully implemented in California upwelling systems and enabled to understand the dynamics and processes that influence the upwelling ecosystem including hypoxic events leading to mass mortalities [START_REF] Chan | The dynamics and impact of ocean acidification and hypoxia[END_REF]. This example is further discussed in the next chapter. In

Mejillones Bay, such a monitoring programme could be achieved with limited additional resources counting on the collaboration between stakeholders, scientists and society.

Sampling efforts have already been carried out on a regular basis by scientists, the Chilean Navy, local industries, fishermen and the municipality separately. Organising and standardising the sampling protocols, sharing data and knowledge would help to benefit all by optimising costs and efforts. This knowledge would enable a better understanding of the processes and dynamics ruling the bay in order to take the appropriate management decisions. Hopefully, this monitoring could also be implemented at a regional scale. These appropriate management decisions would help to limit adverse effects such as severe hypoxia and anoxia, mass mortalities of marine organisms, fisheries collapse, jellyfish and harmful algal bloom, health hazard for local population and elevated costs for remediation.

Mejillones Bay hosts an unique and impressive ecosystem that has all the resources to be transformed from a sacrifice zone into an international example of successful coastal management.

Using biotic indices in naturally stressed environments 1.Naturally stressed environments

In this study, we propose the new concept of the "Upwelling Ecosystem Quality Paradox" that highlights the challenge of disentangling effects of anthropogenic and natural disturbances in upwelling areas. Upwelling brings cold waters, poor in oxygen and rich in nutrients towards the surface, drastically increasing primary production, resulting in a marked reduction of dissolved oxygen due to microbial degradation of organic matter in the water column, especially above the sediment [START_REF] Kämpf | Upwelling systems of the world[END_REF]. Nutrients originating either from natural processes, like upwelling, or anthropogenic activities (e.g., sewage water discharges and industrial activities) lead to eutrophication that can induce hypoxia and organic matter enrichment on the seabed (Levin et al., 2009a). Those macrobenthic fauna able to live in upwelling conditions have thus similar characteristics than those from areas negatively influenced by anthropogenic activities (i.e., the small body size and high reproductive capacity typical of r-strategists). This makes it difficult to evaluate human impacts as it has been reported for estuaries [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF]Elliott and Quitino, 2007). In estuaries, natural stress is related to strong currents, high turbidity, anoxia, and important variations in salinity [START_REF] Dauvin | Paradox of estuarine quality: Benthic indicators and indices, consensus or debate for the future[END_REF]. The similarities in organisms and communities influenced by natural and anthropogenic stress have been reviewed in the context of estuaries [START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF]. The related difficulties in assessing ecological status in those naturally stressed environments are likely present in other ecosystems as well. For example, in fjords where environmental stress is created by freshwater melt from glaciers diluting seawater resulting in high turbidity and sedimentation which define diversity patterns of benthic communities in northern and southern hemispheres [START_REF] Włodarska-Kowalczuk | Benthic response to chronic natural physical disturbance by glacial sedimentation in an Arctic fjord[END_REF][START_REF] Quiroga | Progress in Oceanography Geographic patterns of soft-bottoms benthic communities in Chilean Patagonian fjords (47ºS-54ºS) -influence of environmental stress on diversity patterns and stable isotope signatures[END_REF].

In this study, natural eutrophication from upwelling increases with depth, and correlates with hypoxia and organic matter content. Most of the tested biotic indices in this study were influenced principally by the natural depth gradient present in the bay.

Biotic indices limitations

The most widely used biotic indices are based on predefined classification of the taxa according to their sensitivity to human pressures, such as the Benthic Opportunist

Polychaetes-Amphipods (BOPA; [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]) and AZTI's marine biotic index (AMBI; [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Dauvin | Twenty years of application of Polychaete/Amphipod ratios to assess diverse human pressures in estuarine and coastal marine environments: A review[END_REF][START_REF] Muxika | The suitability of the marine biotic index (AMBI) to new impact sources along European coasts[END_REF]. However, we found ambiguities and incoherences in these classifications when applying these indices to macrobenthic communities in a bay influenced by natural hypoxia and organic matter enrichment due to upwelling. In the BOPA index, all amphipods are considered particularly sensitive to organic matter enrichment and other pollutants with the exception of the genus Jassa [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF] as they belong to the ecological group 1 of the AZTI's marine biotic index (AMBI; [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. In the present study, amphipods from the genus Caprella were observed but absent from the site with the lowest organic matter content. The family Caprellidae is not mentioned in the study by [START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF] although it is in the seminal study on which BOPA is based (Gómez Gesteira and [START_REF] Gómez Gesteira | Amphipods are good bioindicators of the impact of oil spills on soft-bottom macrobenthic communities[END_REF]. This family belongs to the ecological group 2 of the AMBI gathering the species indifferent to enrichment [START_REF] Grall | Using biotic indices to estimate macrobenthic community perturbations in the Bay of Brest[END_REF][START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. These ambiguities raise whether some groups (such as Caprellidae) would need to be excluded from BOPA calculation. The BOPA index presented ambiguities in the classification of the encountered taxa despite being highly simplified as it uses only two classes. AMBI uses five classes and presents incoherences discussed in chapter III. For example, the polychaete Magelona phyllisae , a species that has the characteristics of second order opportunistic species (i.e., proliferating in high organic matter content) according to our results and other studies in Chile, the Gulf of Mexico and India [START_REF] Flint | Polychaete Ecology and Niche Patterns: Texas Continental Shelf[END_REF][START_REF] Moreno | Subtidal macrozoobenthos communities from northern Chile during and post El Niño 1997-1998[END_REF]Sivadas et al., 2016) is considered as indifferent to organic enrichment according to the AMBI list from December 2020. Changing only this abundant polychaete species of ecological group would drastically change the ecological status results of several stations and thus have consequences on management decisions. The classification of all taxa should thus be carefully reassessed according to local biogeographic conditions before using indices based on taxon sensitivity to human pressure [START_REF] Gillett | Effect of ecological group classification schemes on performance of the AMBI benthic index in US coastal waters[END_REF] for management purposes.

In fact, sensitivity of taxa can change with environmental conditions. [START_REF] Zettler | On the Myths of Indicator Species: Issues and Further Consideration in the Use of Static Concepts for Ecological Applications[END_REF] showed how the tolerance of some taxa to organic matter content can change with different salinity regimes. A taxon can even display flexible feeding strategies depending on environmental conditions [START_REF] Maurer | The Infaunal Trophic Index (ITI): Its suitability for marine environmental monitoring[END_REF]) so its AMBI ecological group would need to be changed according to the location where it is observed. This is not the recommended AMBI approach so far as the provided software automatically assigns the ecological group to species according to a global classification list (Borja et al., 2012b). Taxa may have different reactions to disturbances depending on the environmental conditions where they are encountered, either due to adaptation capabilities or depending if they are in their optimal environmental conditions or at the edges of their tolerance range. Further studies debated the use of indices based on pre-existing sensitivity classifications [START_REF] Fleischer | An adjustment of benthic ecological quality assessment to effects of salinity[END_REF][START_REF] Leonardsson | Theoretical and practical aspects on benthic quality assessment according to the EU-Water Framework Directive -Examples from Swedish waters[END_REF][START_REF] Villnäs | Evaluating the performance of benthic multi-metric indices across broad-scale environmental gradients[END_REF], arguing these tools should be avoided in naturally stressed environments. Calculating biotic indices without making sure the concept and classification they rely on is applicable to the location under study is meaningless and could lead to inappropriate advice for management decisions, with subsequent implications for the environment. Indices should be easily interpretable by decision makers in order to be useful, however their applicability should first be evaluated by qualified scientists. In the same way a new index is evaluated and debated, the application of any index to a new location should be conscientiously evaluated by researchers before being implemented for management purposes.

Biotic indices assets

As described in the previous paragraphes, assessing ecological quality of ecosystems under natural stressors is a significant challenge and this work highlighted weaknesses in some existing biotic indices in this context. However, some indices displayed strengths such as the General Purpose Biotic Index (GPBI; [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF] that was the only multimetric index displaying less influence from the natural gradient of hypoxia and organic matter enrichment. This was possible because this index accounts for the loss of individuals for each taxon present in carefully chosen reference sites, away from direct anthropogenic impact, compared to the abundance of the same taxon at the study sites. Moreover, this calculation was performed for each depth and month separately which allowed a substantial reduction of the "background noise" related to the natural stress. This recent index is based on the general assumption that the most sensitive species will disappear first when a site becomes impacted and that stronger impact will lead to more important losses [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF]. Our study gave promising results for the GPBI to assess ecological quality status of naturally stressed environments; it will need to be further tested in other habitats such as estuaries.

Functional diversity indices are based on selected biological trait categories rather than taxonomy and provide information on the mechanisms linking biodiversity patterns to ecosystem functioning [START_REF] Casanoves | FDiversity: A software package for the integrated analysis of functional diversity[END_REF]. These biological traits can be selected to reflect ecosystem functions such as productivity, resilience to perturbations or invasion and regulation in the flux of organic matter (Villéger et al., 2008 and citations within). They are increasingly being recognized as a useful complementary approach to the assessment of community structure and diversity, providing information on the maintenance and regulation of ecological processes, i.e., ecosystem functioning [START_REF] Naeem | Biodiversity and ecosystem functioning: Maintaining natural life support processes[END_REF][START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. They are less influenced by temporal variation [START_REF] Reiss | Seasonal variability of benthic indices: An approach to test the applicability of different indices for ecosystem quality assessment[END_REF] and can be standardised to address all macrobenthic species so potentially applicable to all systems and regions.

In the results presented in the third chapter (Table 3 in Bon et al., in revision), functional diversity indices were able to detect a degradation of ecological quality status in the intermediate depths where the sewage water discharge from the city is located. The reduced values of functional richness at this site are possibly related to a loss of taxa with trait categories poorly adapted to the existing environmental conditions [START_REF] Mason | Functional richness, functional evenness and functional divergence: The primary components of functional diversity[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. For example, filter feeders were absent from many samples, increasing particulate matter sedimentation and disturbing larval settlement and thus the community structure [START_REF] Douglas | Macrofaunal Functional Diversity Provides Resilience to Nutrient Enrichment in Coastal Sediments[END_REF]. The loss of trait categories can thus contribute to a feedback loop that intensifies eutrophication impact at those sites. Therefore, a functional approach might be very useful for management and conservation purposes [START_REF] Bremner | Species' traits and ecological functioning in marine conservation and management[END_REF][START_REF] Garaffo | Assessing functional diversity of macrobenthic assemblages in sewage-affected intertidal shores[END_REF]. Indices based on taxonomic structure of the benthic community do not consider changes in functional structure central to understanding ecosystem functioning [START_REF] Darr | Functional changes in benthic communities along a salinity gradient-a western Baltic case study[END_REF].

These considerations strengthen the importance of combining structural and functional approaches when studying benthic communities. However, the functional diversity indices calculated in chapter 3 were highly influenced by depth: a natural gradient (Table 5 in Bon et al., in revision). Functional diversity indices might thus respond to any disturbance, whether it is caused by natural or anthropogenic processes as previously reported for most biotic indices based on metrics derived from taxonomy [START_REF] Dauvin | The estuarine quality paradox: Is it possible to define an ecological quality status for specific modified and naturally stressed estuarine ecosystems?[END_REF]Sivadas et al., 2016;[START_REF] Elliott | The Estuarine Quality Paradox Concept ☆[END_REF]. A functional index that is not heavily influenced by natural stress, similar to the GPBI, could fill this gap.

New index proposition

From the understanding generated in this thesis, I propose a new index, the Functional Biotic Index (FBI). This is based on the GPBI approach [START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF] and the assumption that a disturbance has an impact on community functioning as soon as the most sensitive trait categories suffer a reduction in their abundance. It assesses the deviation of macrofauna functional composition and structure from a valid reference. It is calculated using the GPBI formula :

R quant { r ; t } = 1 -(Σ Fr i =1 max( r i -t i ;0)/Σ Fr i =1 r i ) (1)
where F r is the total number of functional trait categories at the reference station, r i is the abundance of the trait category i at the reference station, and t i is the abundance of the trait category i at the tested station. The species richness is thus replaced by the total number of functional trait categories and the taxonomic abundances by the abundances of macrobenthic biological trait categories. If there is a single reference station, the index FBI for the tested station t is computed as :

FBI { t } = R quant { r ; t } (2)
If there are multiple reference stations, FBI { t } is computed as follows: S1 from chapter 3; [START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems : a comparison of approaches[END_REF][START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. The FBI values were calculated for each sample, for shallow (10 m), intermediate (20, 25 and 30 m) and deep (50 m) separately in each sampling month. I used EAST stations as references because this transect is located away and up-current from the anthropogenic activities, so I could assume that anthropogenic pressure on the benthic community was minimal at this location. In the same manner, I calculated the GPBI that compares benthic macrofauna community composition and structure to the EAST transect. I also calculated the functional richness index (FRic) that estimates the convex hull volume of the functional space occupied by the community [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] and the functional dispersion index (FDis) that represents the abundance-weighted mean distance of individual taxa to the abundance-weighted centroid of all taxa [START_REF] Laliberté | A distance-based framework for measuring functional diversity from multiple traits A distance-based framework for measuring from multiple traits functional diversity[END_REF]. These two functional indices were calculated for each sample separately using the R package "FD" [START_REF] Laliberté | FD: Measuring functional diversity (FD) from multiple traits, and other tools for functional ecology[END_REF]. In this assessment, the FBI seems to better detect the decrease in ecological quality status at the intermediate depths in outfall and west when calculated for each depth and month separately, as the functional richness and functional dispersion indices (Table 1). The FBI was also able to detect lower ecological quality status in the shallower depths at the outfall and west compared to east (Table 1).

FBI { t } = 1 -|R ̅ ref -R ̅ quant { r ; t } | (
The FBI thus appears more sensitive than the GPBI to detect a decline in ecological quality status. To evaluate which environmental variable explained most variation in each index, variation partitioning was calculated for each index and each explanatory variable separately. Sediment organic matter content explained the greatest proportion of variation for the FBI as for GPBI especially when calculated by depth and month separately (Table 2).

The natural gradient of depth only explained 14% of the FBI variation whereas it explained 39% of the functional diversity indices. The FBI thus appears as a new functional index that is less subject to the influence of natural stress, and could be more sensitive to eutrophication impacts than the GPBI. Moreover, the FBI provides information on the functional structure and composition on which the mechanisms linking biodiversity to ecosystem functioning rely, as abundance is grouped by function and not by taxa [START_REF] Casanoves | FDiversity: A software package for the integrated analysis of functional diversity[END_REF][START_REF] Darr | Functional changes in benthic communities along a salinity gradient-a western Baltic case study[END_REF]. It should also be able to provide earlier detection of system degradation in ecological functioning as it is based on organisms' abundances of trait categories rather than their taxonomic abundances, as in [START_REF] Rodil | Tracking environmental stress gradients using three biotic integrity indices: Advantages of a locally-developed traits-based approach[END_REF]. In addition, the FBI is adaptable as traits can be selected according to the functions of interest. Clearly, this new index requires further evaluation on larger datasets giving measured human pressures, in other environments and geographical locations, including in other naturally-stressed ecosystems, such as estuaries where it could be highly useful.

Functioning of macrobenthic communities in stressed environments

In this thesis, I revealed the existence of three types of functioning benthic communities associated with specific functional trait categories along an environmental gradient of hypoxia and organic matter enrichment (Fig. 7 in [START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. The shallow habitat was associated with larger organisms with long life spans, indicating communities that can reach advanced succession stages [START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF]. Biogenic structures such as shells and larger-bodied organisms provided multiple microhabitats, refuges and diverse food sources.

In fact, low organic matter content and dissolved oxygen close to the hypoxia limit allowed diversified functioning in these benthic communities. The deep habitats with severe hypoxia and high organic matter content were dominated by opportunistic organisms (i.e., small size and short life span), a common pattern in areas subject to seasonal hypoxia [START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF]. Asexual reproduction was recorded, a mechanism that enables body size to be reduced size, increasing area-to-volume ratios that enhance diffusion to satisfy aerobic metabolic demands [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Quiroga | Biomass size-spectra of macrobenthic communities in the oxygen minimum zone off Chile[END_REF]. We also observed an increase in permanent burrowers and subsurface deposit feeders, life-history strategies that could provide advantages when exposed to hypoxia [START_REF] Hagerman | Physiological flexibility; a necessity for life in anoxic and sulphidic habitats[END_REF]. The communities from these deep hypoxic habitats self-maintained over time thanks to a limited number of taxa well-adapted to severe hypoxia. In intermediate habitats, a transitional functional community was described associated with transitional trait categories, such as soft protected bodies, while "soft bodies" were rather found in the deeper habitats and "shells and exoskeleton" occured in shallower habitats. Intermediate habitats were also associated with surface deposit feeders and filter feeders which involve extended structures that increase surface contact with water enhancing oxygen acquisition (Levin et al., 2009b).

In the Baltic Sea, the absence of sessile taxa has been observed in areas with abrupt changes in salinity and strong anthropogenic impacts inducing eutrophication and hypoxia (Törnross et al., 2015). In this upwelling gradient, the proportion of swimmers decreased with hypoxia, which can be explained by the lack of need for species to be able to escape from hypoxic events since hypoxia is a permanent condition. However, the general pattern of functional benthic communities presented here agrees with the description of macrobenthic community response to sediment organic matter enrichment gradient as reviewed in [START_REF] Grall | Marine eutrophication and benthos: The need for new approaches and concepts[END_REF]. It can then appear as a potential complement to the model proposed by [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF]Fig. 1).

I believe however that these results should be generalised with similar studies that include a functional approach of benthic communities in other upwelling ecosystems having shallow hypoxia such as the Benguela upwelling ecosystem [START_REF] Mohrholz | The seasonal variability of the northern Benguela undercurrent and its relation to the oxygen budget on the shelf[END_REF]. There, hypoxia was determined as the main factor driving macrobenthic communities and their taxonomic richness [START_REF] Zettler | Macrozoobenthos diversity in an oxygen minimum zone off northern Namibia[END_REF][START_REF] Zettler | On the Myths of Indicator Species: Issues and Further Consideration in the Use of Static Concepts for Ecological Applications[END_REF]; however, little is known about the functioning of these communities. Further studies would also be required to investigate if this pattern is applicable to other areas subject to natural (e.g., land enclosed seawater bodies such as hypoxic fjords) and anthropogenic shallow hypoxia and organic matter enrichment. It could also be compared to other gradients of increasing stress such as salinity decrease where the switch from large, long-lived species with benthic larvae towards smaller, short-living species with planktonic larvae [START_REF] Darr | Functional changes in benthic communities along a salinity gradient-a western Baltic case study[END_REF]. Indeed in the most impacted areas, only a few species provide most of the functions [START_REF] Törnroos | Developing the multitrait concept for functional diversity: Lessons from a system rich in functions but poor in species[END_REF].

Finally, despite an increasing number of studies on community functioning, very few studies describe intermediate functional communities (e.g. [START_REF] Pacheco | Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile[END_REF] which are necessary to identify the conditions in which functions are lost. The increasing information available on the variations of the different trait categories along gradients with increasing and measured disturbances will enable a better understanding of the link between biodiversity and ecosystem functioning which are necessary to ensure an efficient management and conservation of those ecosystems.

the best studied upwelling ecosystem [START_REF] Kämpf | Upwelling systems of the world[END_REF]. The Humboldt and the Benguela upwelling systems that are more impacted by hypoxia and where more extreme events are occurring remain less understood.

In the Humboldt upwelling system, shallow benthic hypoxia is permanent in some coastal areas, particularly in bays [START_REF] Cerda | Variación espacial y temporal de las masas de agua, nutrientes y sedimentación de la materia orgánica e inorgánica en la bahía Mejillones del sur (23° S), Chile[END_REF][START_REF] Guiñez | Variabilidad espacial y temporal de la materia orgánica sedimentaria, asociada a la Zona de Mínimo Oxígeno (ZMO), en un ambiente costero del norte de la corriente de Humboldt, bahía de Mejillones, Chile[END_REF]. Benthic communities have thus lower taxonomic richness but can show high abundance of taxa well adapted to hypoxic conditions and are quite stable overtime [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Fajardo | Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem[END_REF][START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]. However, in the last decade, fish and invertebrate mass mortalities have been recurrently reported mainly by local newspapers (e.g., Madri+d 2016; Timeline Antofagasta 2016; El Diario de Antofagasta, 2019). The cause of these mortalities was assigned to hypoxia and hydrogen sulphide by a local researcher but, to my knowledge, no study has been published accordingly so far. We currently do not know which mechanisms lead to more severe hypoxic conditions or anoxia that trigger mass mortalities in communities adapted to hypoxic conditions. It is therefore key that information on the relevant physico-chemical and biological parameters are monitored and made public.

Also, one important aspect is that in severe hypoxic or anoxic conditions, bacterial metabolism generates hydrogen sulphide (H 2 S) which is highly toxic for most macroorganisms [START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF]. Extensive mats of sulphide-oxidising bacteria such as Thioploca and Beggiatoa have been reported from surface sediments in shallow areas of upwelling ecosystems [START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF][START_REF] Flood | Giant sulfur bacteria ( Beggiatoaceae ) from sediments underlying the Benguela upwelling system host diverse microbiomes[END_REF]. These giant bacteria detoxify the seabed and enable bottom dwelling communities to live in sulphidic and severely hypoxic areas [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF]. Such observations have been reported in both the Humboldt and the Benguela upwelling systems [START_REF] Fossing | Concentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca[END_REF][START_REF] Schulz | Dense populations of a giant sulfur bacterium in Namibian shelf sediments[END_REF], however, the associated macrobenthic communities still need to be characterised [START_REF] Currie | Winning ways with hydrogen sulphide on the Namibian shelf[END_REF]. Further studies are needed to understand the interactions between macrobenthic communities and these giant bacteria and determine possible relations with the mass mortalities. Hydrogen sulphide must be considered an important potential factor structuring communities in the Benguela upwelling system [START_REF] Laudien | Survivorship of juvenile surf clams Donax serra (Bivalvia, Donacidae) exposed to severe hypoxia and hydrogen sulphide[END_REF]. It may also be responsible for mass mortalities observed off Namibia and South Africa in 1997 [START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF]. High concentrations of H 2 S emerging from the sediment also occur regularly in the water column off the coast of Namibia [START_REF] Weeks | Massive emissions of toxic gas in the Atlantic[END_REF]. They severely affect pelagic organisms and cause considerable economic damage, however, the mechanisms responsible for such release from the benthic system are still not well understood [START_REF] Bailey | Seasonal variation of oxygen deficiency in waters off southern South West Africa in 1975 and 1976 and its relation to the catchability and distribution of the Cape rock lobster Jasus lalandii[END_REF][START_REF] Currie | Winning ways with hydrogen sulphide on the Namibian shelf[END_REF]. Such a sulphidic plume has been recorded for the first time off Long term and continuous monitoring of physico-chemical and biological parameters, such as dissolved oxygen, carbonate chemistry, planktonic and macrobenthic communities, are necessary to understand upwelling dynamics due to complex interactions of atmospheric, oceanographic, biogeochemical and biological processes as testified by the Californian upwelling system [START_REF] Chan | The dynamics and impact of ocean acidification and hypoxia[END_REF]. This knowledge is also essential to understand and predict the impact of the human-induced environmental change that is already affecting them [START_REF] Currie | Winning ways with hydrogen sulphide on the Namibian shelf[END_REF]. Similar monitoring should thus be implemented in other upwelling areas (e.g., Benguela, Canary, Humboldt, Arabian Sea, etc.) together with international collaborations between scientists, stakeholders and society in order to better understand these ecosystems, preserve them and the exceptional life they support as well as the many benefits that humans are receiving from them.

General conclusion

The evaluation of ecological conditions in highly stressed environments is very challenging, a new concept of an "Upwelling Ecosystem Quality Paradox" has been proposed in this thesis. The suitability of several existing biotic indices have been tested highlighting weaknesses and strengths. The ecological quality of upwelling ecosystems can be successfully assessed without being too influenced by natural conditions using biotic indices that compare taxonomic abundances of the studied stations with carefully selected reference stations. A new and complementary index that compares functional abundances of the studied stations with reference stations is proposed here, the Functional Biotic Index. This work has led to a better understanding of soft-bottom macrobenthic communities functioning along a gradient of hypoxia and organic matter enrichment in natural conditions. It has also stressed the interest of combining taxonomic and functional approaches to study and assess these communities. Macrobenthic communities functioning are able to link the biodiversity pattern to the ecosystem properties and services [START_REF] Casanoves | FDiversity: A software package for the integrated analysis of functional diversity[END_REF]. The conservation of upwelling ecosystems is critical as they are some of the most productive and valuable areas of the planet. Moreover, studying these challenging ecosystems provides information highly relevant in the context of global climate change and hypoxia increasingly impacting the coastal areas [START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF]. Finally, filling gaps of knowledge on benthic communities, sentinels of upwelling areas, and implementing a long term and continuous interdisciplinary monitoring in upwelling regions is urgent to provide the information necessary for studies that will permit a better understanding of observed and future long term impacts on upwelling and other marine systems.

  pour ne pas aboutir à des prises de décisions inadéquates. Les échantillons sans faune ne sont pas pris en compte par certains indices, qui utilisent par exemple une formule avec logarithmes ou divisions sans ajout de constante, alors que ces échantillons apportent une information importante. Dans ce cas, il est fortement recommandé d'attribuer à ces échantillons la valeur correspondant à la pire condition écologique. Tous les indices biotiques testés ont été influencés principalement par la profondeur (gradient naturel) plutôt que par la concentration en matière organique ou en oxygène à part le GPBI. Cet indice comptabilise la perte d'individus de chaque taxon présent dans un site de référence avec celle de ce même taxon dans le site étudié (Labrune et al., 2021), calculé pour chaque mois et chaque profondeur séparément la variabilité des stations de références est considérablement réduite. Le GPBI a été principalement influencé par la matière organique et permet donc de réduire de manière significative le bruit de fond lié au stress naturel. Il démontre que les stations de référence peuvent permettre de prendre en compte les variations naturelles du milieu. Cet indice récent a ainsi attiré l'attention et mérite d'être testé dans d'autres environnements naturellement stressés. Les indices fonctionnels basés

Figure 1 .Figure 2 .Figure 1 .Figure 2 .Figure 4 .Figure 1 .Figure 2 .Figure 3 .Figure 1 .

 121241231 Figure 1. World map showing the locations of significant coastal upwelling regions Figure 2. Drivers of hypoxia in the Californian upwelling system, off the Oregon coast Figure 3. Oceanographic conditions during a two-year period in two sites of Mejillones Bay, northern Chile Figure 4. Diagram representing a) the changes in number of species (S), abundance (A) and total biomass (B) along a gradient of organic enrichment CHAPTER II -MACROBENTHIC FUNCTIONING Figure 1. Sampling sites in Mejillones Bay, northern Chile (a). Bottom slope profiles of Playa Blanca (b) and Punta Chacaya (c) Figure 2. Boxplots representing the 1st quartile, the median, and the 3rd quartile of environmental conditions approximately one metre above seabed of dissolved oxygen (a) and organic matter (b) concentrations, temperature (c) and salinity (d) at four depths at Playa Blanca and Punta Chacaya in Mejillones Bay, northern Chile Figure 3. Correspondence analysis ordination plots based on monthly trait categories abundance (replicate averaged) at Playa Blanca (PB) and Punta Chacaya (PC): main biplot (a) and plots for each trait (b) Figure 4. Taxonomic richness (a) and functional diversity indices (b,c,d,e) of macrobenthic communities from Playa Blanca and Punta Chacaya Figure 5. Relationship of taxonomic richness (a,b,c), functional richness (d,e,f) and Rao's quadratic index based on abundance (g,h,i) with the environmental variables: dissolved oxygen, organic matter and depth respectively Figure 6. Functional richness (a) and Rao's quadratic index based on abundance (b) of macrobenthic communities from Playa Blanca and Punta Chacaya at 10, 20, 30, and 50 m depth in Mejillones Bay, northern Chile, during a two-year period Figure 7. Conceptual model of changes in functional diversity along a depth gradient, associated with naturally increasing hypoxia and concentrations of organic matter Figure S1. Stable isotopes variation in mean ± 95% CI of δ13C and δ15N in sediment in Playa Blanca and Punta Chacaya Figure S2. Time series of DO concentration (a) organic matter (b) and temperature (c) measurements one metre above seabed in Playa Blanca (PB) and Punta Chacaya (PC) at 10, 20, 30, and 50 m depth in Mejillones bay, Northern Chile, over two years CHAPTER III -ECOLOGICAL QUALITY Figure 1. Maps showing locations sampled in Mejillones Bay, northern Chile Figure 2. Boxplots of dissolved oxygen (a) and organic matter (b) concentrations at each sampling location Figure 3. Redundancy analysis (RDA) triplot (scaling 2) of the relationship among the indices, environmental variables, and macrofaunal community data Figure S1. Boxplots showing variation in temperature, salinity and macroalgal biomass at each sampling location from May to October 2017 Figure S2. Relative contribution of mean trait categories of macrobenthic communities of Mejillones Bay, Chile CHAPTER IV -GENERAL DISCUSSION Figure 1.Changes in benthic macrofauna functioning along an organic enrichment gradient according to[START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF] and[START_REF] Bon | Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system[END_REF]

Figure 1 .

 1 Figure 1. World map showing the locations of significant coastal upwelling regions (from Kämpf and Chapman, 2016).

Figure 2 .

 2 Figure 2. Drivers of hypoxia in the Californian upwelling system, off the Oregon coast[START_REF] Gewin | Dead in the water[END_REF]

pathways.

  Studying community biological traits along a depth gradient from normoxic to hypoxic conditions would allow a better understanding of the functioning of these communities in transitional habitats from this very shallow natural hypoxic environment (1). Additionally, a time series would determine if the pattern displayed is consistent in time or varies during oxygenation events (2). This is the subject of the first article of this thesis.

Figure 3 .

 3 Figure 3. Oceanographic conditions during a two-year period in two sites of Mejillones Bay, northern Chile (taken from Fajardo et al., 2018). Blank spaces indicate months with no sampling. https://doi.org/10.1371/journal.pone.0200349.g002

  discharge). In the second article of this thesis, different biotic indices are tested and compared to assess their relative strengths and weaknesses regarding their capacity to detect anthropogenic perturbation under the challenging conditions of the continuous upwelling of the Northern Chilean coast (Kämpf and Chapman, 2016) (3).

  This work was supported by the CONICYT Fondecyt project 1150042 granted to ASP, "Beneficio Complementario Gastos Operacionales del Proyecto de Tesis Doctoral" by CONICYT by the Comisión Nacional de Investigación Científica y Tecnológica de Chile and "Beca de Apoyo a Tesis Doctoral" by Universidad de Antofagasta granted to MB. MB's PhD studies are funded by CONICYT-PFCHA/Doctorado Nacional/2019-21190740, partially funded by "Beca Doctorado en Ciencias Aplicadas Mención Sistemas Marinos Costeros" and by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program "Investissements d'Avenir". CH is supported by ANID -Millennium Science Initiative Programmes NCN16_034 & NCN19_153.AbstractRecent decades have seen the development of numerous biotic indices based on macrobenthic communities to assess the impact of human activities on marine ecosystems, and to respond to environmental legislation in some countries. The interpretation of those indices to assess ecological quality status (EcoQ) is especially challenging in naturally stressed ecosystems. Highly productive upwelling ecosystems are characterised by natural nutrient enrichment which supports enhanced productivity and results in high amounts of organic matter deposited on the seafloor, causing hypoxia and/or anoxia. However, EcoQ assessments in such systems have been overlooked. Benthic communities in these soft-bottom ecosystems have low taxonomic richness and are highly adapted to hypoxic conditions. As most biotic indices have been created to detect the impact of anthropogenic eutrophication, here we examine whether these indices can detect such impacts under conditions of natural organic matter enrichment. This study evaluated the suitability of a range of different biotic indices based on diversity, taxa sensitivity, reference conditions and functional diversity (e.g., Benthic Quality Index, Benthic Opportunistic Polychaetes Amphipods index, General-Purpose Biotic Index, multivariate-AZTI Marine Biotic Index, functional richness and dispersion) to assess the EcoQ of a soft-bottomed bay subject to marked upwelling influence in northern Chile. We found evidence of high adaptation of macrobenthic communities to high organic matter concentrations and hypoxia. However, ecological quality decreased at sites under the combined influence of natural processes and industrial development. Our results highlighted several limitations of widely used indices under this natural stress, including low taxonomic richness and abundance or the pre-classification of taxa according to their putative sensitivity. Functional diversity indices performed best in detecting the degradation of EcoQ in the intermediate depths. The General-Purpose Biotic Index, comparing the abundance of each taxon to reference conditions, detected a reduced ecological quality in the shallower depths and was the least influenced by natural stress. Our results highlight the importance of combining structural and functional approaches, as well as the crucial need to identify confounding effects of natural and anthropogenic stressors in benthic communities. This is particularly important in the context of increasing coastal hypoxia due to climate change and coastal development.

  categorised as bad or poor, WEST 20 m and OUTFALL 25 m as fair or poor, and EAST 20 and 30 m as good. At the shallowest depth, the three sites showed high ecological quality status with high taxa richness and abundance and the highest dry biomass. The community metrics (i.e., S, A, B) decreased sharply along the transects, especially at OUTFALL and WEST. Indices based on diversity values (Shannon diversity index (H), Pielou's evenness index (J), and Simpson diversity index (D)) generally followed the same trend. H almost classified the whole EAST as having good ecological status, with only the deepest sampling point classified as fair. In general. D varied between 0 and 1, with values tending towards zero in polluted locations. However, due to its formula (1-D), it is equal to 1 when there is no fauna which leads to unexpected values at 50 m in WEST and OUTFALL. Indices based on taxa sensitivity (i.e., BQI, AMBI, BENTIX and BOPA), followed the general pattern of decreased EcoQ at WEST and OUTFALL compared to EAST. However, BQI decreased along the depth transect at all sites. AMBI and BENTIX revealed good or high ecological status at all depths for EAST. BENTIX gave similar results as AMBI despite a higher classification of WEST and OUTFALL 10 m and a worse one for OUTFALL 50 m. Nine of the 220 samples had more than 20% of taxa not assigned for the AMBI calculation: seven were from 10 m depth at WEST and OUTFALL. EcoQ classification from BOPA decreased with depth for WEST and OUTFALL, being lower at these two sites than at EAST. Indices based on reference conditions revealed similar patterns of higher ecological status at EAST than at WEST and OUTFALL. M-AMBI provided a very similar ecological quality status classification despite the use of different reference sites, with only a slight difference observed at OUTFALL 10 m and WEST 20 and 30 m depth. However, GBPI revealed different classifications at all depths if using one or the other reference site, except at the 50 m sites. Clear differences were recorded in both indices if they were calculated for all samples at once or by month and the depth categories (i.e., shallow, intermediate, and deep). Functional diversity indices followed the general trend of decreasing with depth with higher values at EAST than at WEST and OUTFALL.

  industrial development of the bay in the 90s. Mejillones Bay was studied between april 1975 and january 1977 from 3 to 130 metres depth[START_REF] Romero | Análisis de la macrofauna bentónica del sublitoral de la bahía de Mejillones del Sur[END_REF] and provides important information of the bay, including the abundance and biomass of the natural resources present at that time. However, information such as abundance and biomass are presented as mean values by sampling date and the detailed information for each sample has been lost (personal communication with the authors). Comparison with our data along the depth gradient was therefore not possible. Digitalization of the data and recent efforts for data archiving and open access in Chile (see http://datoscientificos.cl/) give hope that similar loss of data will be less common in the future.

  3)where R ̅ quant { r ; t } is the average of the R quant { r ; t } for station t in comparison to each reference station and R ̅ ref is the average of all the values in the R quant similarity matrix between reference stations, including its diagonal. An R function available from the supplementary material S1 in[START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF] can be used to calculate the FBI. The abundance of the different macrobenthic biological trait categories is obtained by summing the taxonomic abundances of each taxon weighted by its fuzzy coding score for each trait category in each sample separately.FBI accounts for the decrease in abundance of each trait category from the reference site(s) in the studied sites, with biological traits selected to reflect the ecological functioning of the community and their sensitivity to environmental disturbances. It could thus indicate deterioration in community functioning. To test if the FBI could be used to evaluate ecological quality status, I used the data from chapter 3 based on the three transects from Mejillones Bay, Northern Chile sampled in May, August, September and October 2017 (Fig.1in chapter 3). The same nine biological traits divided into 38 categories reflecting the ecological functioning of the community and their sensitivity to environmental disturbances were used (Table

  En général, les indices ont détecté une meilleure qualité écologique à l'est de la baie qu'au centre et à l'ouest, en relation avec la quantité de matière organique présente dans le sédiment due à un dépôt des détritus naturels de la colonne d'eau ainsi qu'aux eaux usées de la ville, à la contribution de l'usine de poissons et aux systèmes de refroidissements industriels. Un bilan de l'apport en matière organique de chaque source est donc nécessaire pour déterminer la contribution des activités humaines. Ceci a permis de mettre en évidence la difficulté à distinguer les effets anthropiques des perturbations naturelles dans les zones d'upwellings côtiers et a été nommé le paradoxe de qualité des écosystèmes d'upwellings.

Cependant, des différences ont été observées suivant les indices utilisés. Les indices qui classifient les taxons suivant leur sensibilité se contredisent, ce qui peut être dû à la connaissance limitée des certaines espèces, et au particularité du milieu, notamment quand une classification préliminaire est utilisée et n'est pas adaptée aux différents contextes environnementaux et géographiques (AMBI, BENTIX, M-AMBI, BOPA). Plusieurs études critiquent l'utilisation de ces indices dans des milieux naturellement stressés

  Ici, ils ont mis en évidence une dégradation de la qualité écologique autour de l'émissaire qui n'a pas été détectée par les indices basés sur la taxonomie. Ils renseignent sur les changements fonctionnels qui ne sont pas forcément visibles au niveau taxonomique. Un suivi incorporant une évaluation taxonomique basée sur des stations de

	référence et une évaluation fonctionnelle est donc recommandé pour s'assurer de la
	conservation de l'intégrité de ces écosystèmes côtiers naturellement stressés. Ces résultats
	prennent leur importance dans le cadre du développement du littoral et de l'augmentation
	mondiale des zones hypoxiques.
	Nouvel indice fonctionnel (FBI)
	Cependant les indices fonctionnels calculés ont été principalement influencés par le gradient
	naturel de profondeur. C'est pourquoi, basé sur les connaissances acquises lors de cette

et peuvent être standardisés pour toutes les espèces et donc potentiellement applicables partout dans le monde. thèse, un nouvel indice fonctionnel est proposé, l'indice biotique fonctionnel (FBI). Il est basé sur l'approche du GPBI (Labrune et al., 2021) et mesure la déviation de la structure et composition fonctionnelle de la macrofaune par rapport à une station de référence valide c'est-à-dire, la diminution de l'abondance des catégories de traits biologiques sur le site étudié. Testé comme les autres indices de cette thèse, il détecte la diminution de la qualité écologique au niveau de l'émissaire même dans les faibles profondeurs et est principalement influencé par la matière organique et non par le gradient naturel de profondeur. De plus, il est adaptable suivant les traits choisis en fonction des fonctions d'intérêt de l'écosystème; il est donc potentiellement applicable dans d'autres milieux notamment les milieux naturellement stressés et demande à être testé dans d'autres environnements et d'autres emplacements géographiques.

  . Le benthos des zones d'upwelling joue donc un rôle de sentinelle car il est le premier impacté par les événements d'hypoxie qui peuvent ensuite avoir des conséquences importantes sur tout le reste de l'écosystème. De

plus, il est prévu que le changement climatique stimule les vents responsables des upwellings de Californie, du Humboldt et du sud du système du Benguela

Table 1 .

 1 List of biotic indices based on macrobenthos taxonomy and traits

Table 2 .

 2 Examples of indices values categories according to the Ecological Quality Status defined by the European Union Water Framework Directive

CHAPTER II -MACROBENTHIC FUNCTIONINGTable 1 .

 1 Biological

	traits and categories used to describe the functional diversity of soft bottom macrobenthic communities in Mejillones Bay, northern Chile
	Table S1. Results of Kruskal-Wallis tests comparing the metrics of the environmental gradient among the different depths for each sampling site
	Table S2. Five most dominant taxa in abundance at each site and depth with their relative abundance in percentage (data pooled across sampling dates)
	Table S3. Results of Kruskal-Wallis tests comparing the ecological indices among the different depths for each sampling site
	Table S4. Results of Generalised Additive Models (GAMs) showing the effect of the environmental variables and sampling month on taxa richness (a), functional richness (b) and Rao's quadratic index (c)
	CHAPTER III -ECOLOGICAL QUALITY

Table 1 .

 1 Biotic indices and the formula used for their calculation

Table 2 .

 2 Results of ANOVA comparing environmental conditions among sampling locations and months

Table 3 .

 3 Mean index values at each sampling location

Table 4 .

 4 Ranges for each EcoQ category

Table 5 .

 5 Variation partitioning of each index

Table S1 .

 S1 Biological traits and categories used to describe the functional diversity of soft bottom macrobenthic communities in Mejillones Bay,

northern Chile Table S2. Sensitivity classification of the 110 taxa observed in Mejillones bay CHAPTER IV -GENERAL DISCUSSION Table 1. Mean index values at each sampling location Table 2. Variation partitioning of each indexTable 3 .

 3 Numbers of articles published according to different keyword searches on the Web of Science website

Table 2 .

 2 Examples of indices values categories according to the Ecological Quality Status defined by the European Union Water Framework Directive (EU WFD)

			Ecological Quality Status (EU WFD)
		High	Good	Moderate/Fair	Poor	Bad
	AMBI	0.0 -1.2	1.2 -3.3	3.3 -4.3	4.3 -5.5	5.5 -7.0
	BENTIX	6.0 -4.5	4.5 -3.5	3.5 -2.5	2.5 -2.0	0
	BHQ	15 -11	11 -7	7 -4	4 -2	2 -0
	ISI	> 8.75	8.75 -7.5	7.5 -6.0	6.0 -4.0	4.0 -0
	P-BAT	> 0.77	0.77 -0.53	0.53 -0.41	0.41 -0.20	< 0.20
	M-AMBI	> 0.82	0.82 -0.62	0.61 -0.41	0.40 -0.20	< 0.20
	IQI	1.00 -0.75	0.75 -0.64	0.64 -0.44	0.44 -0.24	0.24 -0.00

  . To ensure the sustainability of those benefits, appropriate assessment, and monitoring of ecosystem quality, including physical, chemical, and biological elements have been established worldwide (e.g., Australian National Land and Water Resources Audit;

	Heap et al., 2001; European Union Water Framework Directive, 2000; United States of
	America Clean Water Act; USEPA

Table 1 .

 1 Biotic indices and the formula used for their calculation : relative abundance of taxa I; EG: ecological groups that differ for AMBI and BENTIX; f p : opportunistic polychaete frequency (ratio of the total number of opportunistic polychaete individuals to the total number of individuals in the sample); f a : amphipod frequency (ratio of the total number of amphipod individuals excluding the opportunistic Jassa sp. to the total number of individuals in the sample); S r : taxonomic richness at the reference station; r i : abundance of taxa i at the reference station; t i : abundance of taxa i at the study site; R ref : average of all values in the R quant similarity matrix between reference stations and R quant{r;t} : average of the R quant{r;t} for station t in comparison to each reference station.

	Biotic indices			Formula		Reference
	H	Shannon-Wiener diversity index	-Σ p i log2 p i		Shannon and Weaver (1964)
	J	Pielou's evenness index	H / log(S)		Pielou (1969)
	D	Simpson diversity index	1 -Σ p i	2		Simpson (1949)
	BQI	Benthic Index	Quality	(Σ (ES50 0.05i *p i ))*log10(S+1)		Rosenberg (2004)	et	al.
	AMBI	Azti Marine Biotic Index	(1.5*EG2+3*EG3+4.5*EG4+6*EG5)/100 Borja et al. (2000)
	BENTIX			(6*EG1+2*EG2)/100		Simboura Zenetos (2002)	and
	BOPA	Benthic Opportunist Polychaetes-Amphipods index	log10((f p /((f a +1))+1)		Dauvin and Ruellet (2007)
	GPBI	General-Purpose Benthic Index	R quant{r;t} = 1 -(Σ i=1 Sr max(r i -t i ;0)/Σ i=1 GPBI = 1 -|R ref -R quant{r;t} |	Sr r i )	Labrune et al. (2021)

i

Table 2 .

 2 Results of ANOVA comparing environmental conditions among sampling locations and months. Values in bold are statistically significant at p < 0.05. For salinity among sampling locations, Kruskal-Wallis test was used due to heterogeneity of variance, and its results are shown in italics.

			Sampling stations		Months
			df	F	p-value		df	F	p-value
	Dissolved oxygen	site site:depth residuals	2 8 33	1.83 9.66	> 0.05 < 0.001	months residuals	3 40	0.957	> 0.05
	Organic matter	site site:depth residuals	2 8 33	40.75 46.28	< 0.001 < 0.001	months residuals	3 40	0.015	> 0.05
	Temperature	site site:depth residuals	2 8 33	0.03 0.22	> 0.05 > 0.05	months residuals	3 40	14.048	< 0.01
	Macroalgal biomass	site site:depth residuals	2 8 33	14.20 3.58	< 0.01 < 0.01	months residuals	3 40	2.165	> 0.05
	Salinity	sitedepth 10 H 14.48 > 0.05	months residuals	3 40	4.99	< 0.01

Table 3 .

 3 Mean index values at each sampling location. Colours represent the highest values

			shallow			intermediate			deep
		WEST	OUT-FALL	EAST	WEST	OUT-FALL	EAST	WEST	OUT-FALL	EAST
	Indices	10m	10 m 10 m 20 m 30 m 25 m 20 m 30 m 50 m 50 m 50 m
	Taxonomic richness (S)	12.7	11.3	12.7	6.7	2.7	7.9	13.2 13.2	0.2	1.0	9.0
	Total abundance (A)	115.3 43.6 331.0 35.0 31.0 133.6 65.4 231.1	0.2	1.6	213.6
	Total dry biomass (B)	10.0	12.8	19.3	0.8	0.4	0.4	7.8	3.2	0.0	0.0	0.8

(> 80 percentiles of all samples: blue; 80-60 percentiles: green), middle (60-40 percentiles: yellow), and the lowest values (40-20 percentiles: orange; < 20 percentiles; red) for each row separately except for BQI, AMBI, BENTIX, BOPA, M-AMBI and GPBI where values for each ecological quality status (EcoQ) are according to references from their respective article (Table

4

).

Table 4 .

 4 Ranges for each EcoQ category. The last column indicates the reference used: either an article or the category of percentiles(i.e., > 80, 80 -60, 60 -40, 40 -20, < 20).

				EcoQ (WFD 200/60/EC)
			HIGH	GOOD	FAIR	POOR	BAD	Ranges from:
		Taxonomic richness (S)	> 13	13 -10	10 -7	7 -2	< 2	Percentiles
	Community metrics	Total abundance (A)	> 171.2	171.2 -62.4	62.4 -34	34 -8.6	< 8.6	Percentiles
		Total biomass (B) dry	> 4.3	4.3 -1.2 1.2 -0.3 0.3 -0.01	< 0.01 Percentiles

Table 5 .

 5 Variation partitioning of each index. Variation explained is expressed as a percentage (%) based on adjusted R 2 . Shaded cells show the contributions of explanatory variable(s) with significant (p < 0.05) relationships.

		Depth	OM	DO	Tempe-rature	Salinity	Algal biomass	Site Month	Depth + OM + site + DO + sal + temp
	S	0.37	0.37 0.16	0.11	0.17	0.00	0.26	0.0	0.63
	A	0.03	0.08 0.00	0.00	0.04	0.00	0.22	-0.01	0.25
	B	0.14	0.03 0.12	0.06	0.01	0.01	0.02	0.01	0.19
	H	0.44	0.35 0.20	0.14	0.21	0.00	0.13	0.03	0.57
	J	0.21	0.18 0.08	0.05	0.13	0.02	0.02	0.02	0.25
	D	0.00	0.05 0.02	0.02	0.00	0.00	-0.01	0.02	0.17
	BQI	0.57	0.39 0.33	0.21	0.28	0.02	0.14	0.01	0.73
	BENTIX	0.38	0.41 0.18	0.08	0.16	0.00	0.20	-0.01	0.60
	AMBI	0.30	0.38 0.16	0.05	0.14	0.00	0.21	0.00	0.57
	BOPA	0.33	0.23 0.23	0.09	0.08	0.00	0.15	0.00	0.51
	M-AMBI EAST	0.43	0.42 0.19	0.12	0.20	0.00	0.26	0.00	0.69
	M-AMBI EAST by depth & month	0.30	0.33 0.16	0.07	0.22	0.00	0.25	0.01	0.60
	M-AMBI LEN by depth & month	0.47	0.38 0.27	0.15	0.22	0.00	0.21	0.00	0.70
	GPBI EAST	0.07	0.28 0.00	0.00	0.06	0.00	0.20	0.02	0.36
	GPBI EAST by depth & month	0.10	0.45 0.01	0.00	0.06	0.00	0.44	0.01	0.67
	GPBI LEN by depth & month	0.07	0.05 0.15	0.06	0.00	0.00	0.37	0.01	0.57
	FRic	0.39	0.33 0.17	0.18	0.18	0.01	0.15	0.03	0.55
	FDis	0.39	0.34 0.19	0.16	0.25	0.01	0.10	0.04	0.53
	FRic ISO	0.43	0.23 0.38	0.24	0.19	0.03	0.05	0.01	0.55
	FDis ISO	0.25	0.22 0.15	0.09	0.14	0.03	0.06	-0.01	0.33

Table 1 .

 1 Mean index values at each sampling location. Colours represent the ecological quality status (EcoQ) assigned for the highest values (> 80 percentiles of all samples: blue is high; 80-60 percentiles: green is good), middle (60-40 percentiles: yellow is fair), and the lowest values (40-20 percentiles: orange is poor; < 20 percentiles; red is bad) for each row separately except for GPBI where values for each EcoQ are according to[START_REF] Labrune | A general-purpose biotic index to measure changes in benthic habitat quality across several pressure gradients[END_REF].

			shallow				intermediate			deep
		WEST	OUT-FALL	EAST	WEST	OUT-FALL	EAST	WEST	OUT-FALL	EAST
		10 m 10 m 10 m 20 m 30 m 25 m 20 m 30 m 50 m 50 m 50 m
	GPBI EAST	0.85	0.84	0.90	0.86	0.87	0.93	0.93	0.89	0.80	0.80	0.90
	GPBI EAST by depth & month	0.50	0.49	0.92	0.71	0.70	0.79	0.93	0.91	0.26	0.26	0.86
	FRic	31.14 22.12 28.53 14.29 6.07 16.20 25.44 25.89 0.00	0.22 16.30
	FDis	4.99	5.40	4.30	3.76	1.79	3.35	4.50	4.75	0.17	1.08	3.61
	FBI EAST	0.79	0.73	0.78	0.68	0.61	0.77	0.81	0.87	0.40	0.41	0.87
	FBI EAST by depth & month	0.59	0.49	0.88	0.64	0.55	0.72	0.85	0.84	0.18	0.18	0.85

Table 2 .

 2 Variation partitioning of each index. Variation explained is expressed as a percentage based on adjusted R 2 . Shaded cells show the contributions of explanatory variable(s) with significant (p < 0.05) relationships.

		Depth	Organic Matter	Dissol-ved O 2	Tempe-rature	Salinity	Site	Month	All
	GPBI EAST	0.07	0.28	0.00	0.00	0.06	0.20	0.02	0.36
	GPBI EAST by depth & month	0.10	0.45	0.01	0.00	0.06	0.44	0.01	0.67
	FRic	0.39	0.33	0.17	0.18	0.18	0.15	0.03	0.55
	FDis	0.39	0.34	0.19	0.16	0.25	0.10	0.04	0.53
	FBI EAST	0.20	0.48	0.07	0.02	0.15	0.26	0.00	0.61
	FBI EAST by depth & month	0.14	0.45	0.04	0.00	0.09	0.38	0.02	0.66

Table 3 .

 3 Numbers of articles published according to different keyword searches on the Web of Science website ( www.webofscience.com accessed on the 14th of May 2022).

	Keywords	Number of articles
	benthic communities AND upwelling…	493
	…AND California	74
	…AND Humboldt	45
	…AND Benguela	28
	…AND Canary	8
	benthic communities AND estuary…	2,363
	…AND Chesapeake	178
	…AND Yangtze	74

Acknowledgements

Abstracts (in Spanish, French, and English)

Acknowledgements

We thank Maritza Malebran, Diego Andrade, and Evelyn Araya, who worked on many aspects of this project and Karina Gonzalez, Felipe Docmac, and Pablo Perez from the Harrodlab that helped by sharing their experience on stable isotope sample processing and interpretation of the results. We also acknowledge the team of Proyecto MEL "Impacto del Cambio Climático sobre la biodiversidad costera" for sharing the dataset used as an external reference station.

ACKNOWLEDGEMENTS

This thesis was financed principally through the competitive scholarship from ANID (Chilean National Agency for Research and Development, CONICYT-PFCHA/Doctorado Nacional/2019-21190740) that covered part of this research, most of my travels between Chile and France, and my stays in both countries. I received partial funding from the "beca Doctorado en Ciencias Aplicadas mención Sistemas Marinos Costeros" from the Universidad de Antofagasta as well as the ISblue project. This work was supported by the CONICYT Fondecyt project 1150042 (Chile) granted to Aldo S. Pacheco.

There are so many people that contributed in one way or another to help me reach this milestone. First of all, I would like to deeply thank Jacques Grall for guiding me through the construction of this thesis and for his continuous support. I also thank Aldo Pacheco for leading the project on which this thesis is based and his supervision in the first years of the PhD. Thank you Chris Harrod for your advice during the last years of this thesis and the opportunities for new collaborations. Thanks to François Le Loc'h as well.

My sincere gratitude to all the members of my thesis committee, PhD proposal and monitoring committees for your interest in my research, your comments that contributed to improve this work, and your encouragements: Julie Bremner; François Charles, Griselda Garaffo, Marcelo Oliva, Ricardo Guiñez, Céline Labrune and Daniel Dauer.

Agradezco sinceramente al director del programa de doctorado Marcelo Oliva por ayudarme a navegar por los meandros de la administración de la Universidad de Antofagasta así como las secretarías Gladys Ramos Seida (Facultad de ciencias del mar y recursos biológicos), Isabel Campos Notari (Escuela de posgrado) y Katherine Maureira Tavilo (Programa de doctorado en Ciencias Aplicadas). Gracias a Ricardo Guiñez por su disponibilidad y paciencia en compartir sus conocimientos en estadística y diseño de muestreo. Gracias a Joao Gusmao por ayudarme a domar el R.

Muchas gracias a todos mis compañeros del programa de doctorado y especialmente a mis amigos David, Karina, Ana Garcia, Liliana, Juan, Lucho por la compañía en los momentos de felicidad y los más difíciles. Gracias también a Maritza Fajardo, Maritza Malebran que siempre fueron presente cuando necesitaba ayuda o algún dato en el cuaderno del laboratorio y me encontraba en otro continente… Gracias al equipo del Harrod Lab, especialmente a Karina, Felipe, Varua y Pablo. Agradezco también a Evelyn, Diego y Espiri por su ayuda.

Gracias al Océano Pacífico por mostrarme sus maravillas, a Carlos, sus padres y hermano, Cata, Vladi, Oscar y los buzos de Buceo Oceano por ofrecerme tiempos de relajación profunda y compartir mi pasión por el buceo y la exploración de los fondos marinos. Quiero agradecer también a Marcela por sus clases de yoga que me ayudaron a bajar la presión. Muchísimas gracias a Max Penoy, Leticia, los abuelitos, la familia Comicheo Herrera por hacerme sentir en casa y parte de su familia además de descubrir su fantástica isla. A mi amiga Maca que me dio la bienvenida y la despedida de Chile además de momentos hermosos con su familia y amigos. Agradezco también a Juan Carlos y Eli por compartir su hogar conmigo y guardar mis cosas cuando tuve que irme de repente de vuelta a Francia. Gracias también a Juan Pancho, Mauro, Ljubi, Fernanda, Manuel, Diego, Nano, Juan,

Funding

LIST OF FIGURES CHAPTER I -GENERAL INTRODUCTION

 [START_REF] Arntz | El Niño and similar perturbation effects on the benthos of the Humboldt, California, and Benguela Current upwelling ecosystems[END_REF][START_REF] Schunck | Giant Hydrogen Sulfide Plume in the Oxygen Minimum Zone off Peru Supports Chemolithoautotrophy[END_REF].

Considering the above, there is an urgent need to further understand the physical and biogeochemical dynamics that allow recycling of the high amount of organic matter produced in upwelling systems that can lead to hypoxia and hydrogen sulphide. Because macrobenthic communities have limited motility and are in direct contact with the sediment, they can act as sentinels of these conditions likely to impact the whole ecosystem. Climate change is predicted to strengthen winds that drive upwelling intensity in the California, Humboldt and southern Benguela upwelling systems, that will further stimulate primary production [START_REF] Bakun | Global climate change and intensification of coastal ocean upwelling[END_REF][START_REF] Sydeman | Climate change and wind intensification in coastal upwelling ecosystems[END_REF][START_REF] Wang | Intensification and spatial homogenization of coastal upwelling under climate change[END_REF]. Such process will potentially lead to an increase in the intensity and frequency of hypoxia and H 2 S events, as well as a possible increase in their spatial extent over shelf habitats [START_REF] Sydeman | Climate change and wind intensification in coastal upwelling ecosystems[END_REF][START_REF] Breitburg | Declining oxygen in the global ocean and coastal waters[END_REF] which would be critical for fisheries, ecological, and biogeochemical processes [START_REF] Levin | Oxygen minimum zone benthos: adaptation and community response to hypoxia[END_REF][START_REF] Grantham | Upwelling-driven nearshore hypoxia signals ecosystem and oceanographic changes in the northeast Pacific[END_REF][START_REF] Bakun | Climate change and ocean deoxygenation within intensified surface-driven upwelling circulations[END_REF][START_REF] Salvatteci | Smaller fish species in a warm and oxygen-poor Humboldt Current system[END_REF].

Understanding oceanographic processes and their impact on the living organisms in upwelling regions is highly challenging. The use of benthic communities studies plays a key role (Thiel et al., 1978) in this objective. However, relatively limited information is available today on the benthic communities from upwelling environments compared to other coastal ecosystems such as estuaries. The California upwelling system has the highest number of publications on benthic communities: however, this is only equal or less than those derived from studies on some individual estuaries (Table 3). Basic information is missing from most taxonomic groups in the Benguela and Canary systems [START_REF] Konar | Surveying nearshore biodiversity[END_REF]. A recent study describes the intertidal benthic communities of the Canary upwelling ecosystems [START_REF] Lourenço | Strong upwelling conditions drive differences in species abundance and community composition along the Atlantic coasts of Morocco and Western Sahara[END_REF] while the subtidal macrobenthic communities remain largely unexplored.

Focusing research on benthic communities of upwelling systems is thus highly recommended.