Fellag Mustapha

Loukil Ahmed

Drancourt Michel
email: michel.drancourt@univ-amu.fr

Professeur Michel Drancourt

VERSION REVISEE Le puzzle de l'histoire naturelle évolutive de la tuberculose

Keywords: Histoire naturelle, Mycobacterium, Mycobacterium tuberculosis, tuberculose

INTRODUCTION

La tuberculose est l'une des principales causes infectieuses de décès dans le monde et l'OMS estime qu'environ un quart de la population mondiale est infectée de manière latente par Mycobacterium tuberculosis, et en particulier, une forte prévalence de tuberculose latente est rapportée dans les pays endémiques de tuberculose active [START_REF] Cohen | The global prevalence of latent tuberculosis: a systematic review and meta-analysis[END_REF][START_REF]Global tuberculosis report 2019[END_REF]. Cette maladie infectieuse mortelle touchant les mammifères dont l'homme est causée par 13 espèces de mycobactéries étroitement apparentées formant le complexe Mycobacterium tuberculosis (MTC) [START_REF] Brites | A New Phylogenetic Framework for the Animal-Adapted Mycobacterium tuberculosis Complex[END_REF]. En raison de leur proximité génomique, le statut taxonomique exact de ces espèces est débattu et certains auteurs considèrent les espèces MTC comme des variants écotypes de M.

tuberculosis [START_REF] Riojas | Phylogenomic analysis of the species of the Mycobacterium tuberculosis complex demonstrates that Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium microti and Mycobacterium pinnipedii are later heterotypic synonyms of Mycobacterium tuberculosis[END_REF]. Cependant, ces espèces ou écotypes présentent des différences génomiques, génétiques et phénotypiques qui peuvent expliquer les différences dans les réservoirs et les modes de transmission. M. tuberculosis stricto sensu ne cause pas la tuberculose uniquement chez l'homme, étant décrite chez divers animaux, principalement des mammifères [START_REF] Ghodbane | Non-human sources of Mycobacterium tuberculosis[END_REF].

L'issue pour les personnes infectées par M. tuberculosis est très variable, allant de l'élimination totale de l'agent pathogène par le système inflammatoire et immunitaire, au transport asymptomatique à long terme pendant la tuberculose dite dormante, en passant par la tuberculose symptomatique et finalement la mort [START_REF] Bhavanam | Understanding the pathophysiology of the human TB lung granuloma using in vitro granuloma models[END_REF]. Le rôle des microbiotes respiratoire et digestif émerge dans la modulation de l'expression de la tuberculose alors que le principal facteur du maintien de l'incidence élevée de cette maladie est la transmission continue de M. tuberculosis [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF].

Les mycobactéries du complexe M. tuberculosis.

Parmi les treize espèces ou écotypes actuellement décrites dans MTC, seules 09 espèces ont été associées à la tuberculose humaine, incluant le vaccin bacille bilié de Calmette et Guérin (BCG) [START_REF] Coscolla | Novel Mycobacterium tuberculosis Complex Isolate from a Wild Chimpanzee[END_REF][START_REF] Parsons | Novel Cause of Tuberculosis in Meerkats, South Africa[END_REF]. Les MTC ont des séquences génomiques hautement conservées (> 99.9 % d'identité nucléotidique) mais des phénotypes différents. Il a été suggéré que les MTC ont évolué d'un ancêtre commun via des suppressions / insertions successives d'ADN leurs conférant leurs différences de pathogénicité [START_REF] Jia | The Bioinformatics Analysis of Comparative Genomics of Mycobacterium tuberculosis Complex (MTBC) Provides Insight into Dissimilarities between Intraspecific Groups Differing in Host Association, Virulence, and Epitope Diversity[END_REF]. L'évolution génomique de ces mycobactéries se fait par diminution progressive de la capacité de codage de leur génome qui est exclusivement chromosomique, avec perte de fragments génomiques appelés régions de délétion et invasion du génome par des séquences d'insertion (IS) [START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF].

Mycobacterium canettii est l'espèce la plus proche de l'ancêtre commun au MTC, présentant un génome de 4,48 ± 0,05 Mb [START_REF] Osman | Smooth Tubercle Bacilli: Neglected Opportunistic Tropical Pathogens[END_REF] et codant pour des fonctions uniques telles que le gène cob de la Trans cobalamine. Responsable d'une tuberculose non contagieuse [START_REF] Osman | Smooth Tubercle Bacilli: Neglected Opportunistic Tropical Pathogens[END_REF], M. canettii présente une répartition géographique limitée à la Corne de l'Afrique. Sur le plan microbiologique, M. canetti présente un temps de dédoublement de 17 heures d'un tiers plus court que celui de M. tuberculosis (25 heures) et produit des colonies lisses contrairement M. tuberculosis qui présente un morphotype rugueux [START_REF] Osman | Smooth Tubercle Bacilli: Neglected Opportunistic Tropical Pathogens[END_REF]. M. tuberculosis stricto sensu est constitué de six grands lignages phylogénétiques (lignages 1, 2, 3, 4, 7 et lignage 8 récemment proposé) [START_REF] Ngabonziza | A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region[END_REF], inégalement répartis à travers le monde ; les lignages L1 et L2 prédominent en Asie de l'Est et du Sud-Est, tandis que les lignages L1 et L3 prédominent dans le sous-continent indien, les lignages L3 et L4 prédominent en Asie centrale et en Russie, alors que le lignage L4 prédomine en Europe, Amériques, Afrique du Nord et au Moyen-Orient [START_REF] Brites | The Nature and Evolution of Genomic Diversity in the Mycobacterium tuberculosis Complex[END_REF]. C'est en Afrique sub-saharienne que l'on retrouve la plus grande variété de lignages, car en plus des lignages L1, L2, L3 et L4, deux nouveaux lignages ont récemment été identifiés, le lignage L7 en Ethiopie et le lignage L8 au Rwanda et en Ouganda [START_REF] Ngabonziza | A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region[END_REF][START_REF] Brites | The Nature and Evolution of Genomic Diversity in the Mycobacterium tuberculosis Complex[END_REF][START_REF] Comas | Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans[END_REF]. Mycobacterium africanum comporte deux lignages (L5 and L6) exclusivement distribuées en Afrique de l'Ouest, responsable d'une tuberculose indistinguable de celle provoquée par M. tuberculosis [START_REF] Jong | Mycobacterium africanum Elicits an Attenuated T Cell Response to Early Secreted Antigenic Target, 6 kDa, in Patients with Tuberculosis and Their Household Contacts[END_REF][START_REF] Sharma | Tuberculosis Caused by Mycobacterium africanum, United States, 2004-2013[END_REF][START_REF] Yeboah-Manu | The Biology and Epidemiology of Mycobacterium africanum[END_REF]. La virulence des MTC est variable et diffère au sein des souches de la même espèce par exemple : les lignages modernes de M. tuberculosis (L2-L4) sont plus virulents et responsables de la grande majorité des cas de tuberculose dans le monde [START_REF] Jia | The Bioinformatics Analysis of Comparative Genomics of Mycobacterium tuberculosis Complex (MTBC) Provides Insight into Dissimilarities between Intraspecific Groups Differing in Host Association, Virulence, and Epitope Diversity[END_REF].

Spécifiquement, les souches Beijing (L2) sont considérées comme étant les MTC les plus virulentes pour l'homme avec une prédisposition à développer davantage de résistance aux antituberculeux et disposent d'une forte capacité de propagation, illustrée par le fait que ces souches ont été décrites chez les bovins et le chien [START_REF] Ghodbane | Non-human sources of Mycobacterium tuberculosis[END_REF][START_REF] Jia | The Bioinformatics Analysis of Comparative Genomics of Mycobacterium tuberculosis Complex (MTBC) Provides Insight into Dissimilarities between Intraspecific Groups Differing in Host Association, Virulence, and Epitope Diversity[END_REF][START_REF] Parsons | Pulmonary Mycobacterium tuberculosis (Beijing strain) infection in a stray dog[END_REF]. Cette virulence des souches Beijing est corrélée à des délétions dans le gène pks15/1 et dans la région RD207 [START_REF] Lam | Rv2820c of Beijing/W strains enhances Mycobacterium tuberculosis survival in human macrophages[END_REF]. A l'opposé M. bovis BCG est la souche la moins virulente parmi les MTC, son atténuation fait suite à la perte de la région RD1 dans son génome [START_REF] Lewis | Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Guérin Attenuation[END_REF].

INTERFACES ENVIRONNEMENT HOMME : PORTES D'ENTREE ET

MICROBIOTES

Depuis les travaux de Villemin et la mise en place de la culture de M. tuberculosis par Robert Koch de nombreux scientifiques ont essayé de déterminer comment la tuberculose est transmise à l'homme [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF][START_REF] Cambau | Steps towards the discovery of Mycobacterium tuberculosis by Robert Koch, 1882[END_REF]. Une grande partie des études sur la transmission de la tuberculose ont été réalisées à la fin du 19e / début du 20e siècle.

Ces études se sont concentrées sur la culture pour démontrer la viabilité, et l'inoculation à l'animal pour confirmer la pathologie [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF]. Ainsi, à l'époque plusieurs théories ont existé et Calmette et Guérin ont rapporté en 1905 l'origine intestinale de la tuberculose pulmonaire [START_REF] Calmette | Origine intestinale de la tuberculose pulmonaire[END_REF]. D'autres travaux ont rapporté la culture de M. tuberculosis dans l'environnement [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF]. Il faut prendre en considération qu'à l'époque les connaissances sur la diversité des mycobactéries (tuberculeuses et nontuberculeuses) étaient plus limitées de sorte que plusieurs travaux ont pu être réalisés avec mycobactéries environnementales, ou avec des MTC différentes de M. tuberculosis stricto sensu [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF]. Une partie importante de nos connaissances sur la transmission de la tuberculose provient de l'investigation des cas acquis au cours des soins. Il est bien admis depuis les années 50 que la voie respiratoire est la principale voie de transmission de M. tuberculosis, cependant, cela n'exclut pas d'autres voie de transmission [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF] (Figure 1).

Voie respiratoire.

Des études innovantes par Riley et Wells dans les années 50-60, ont montré que l'exposition de cobayes à des gouttelettes d'aérosols de patients atteints de tuberculose pulmonaire entraînait des taux substantiels de conversion de la tuberculine chez les cobayes [START_REF] Sultan | Tuberculosis Disseminators[END_REF][START_REF] Riley | Aerial Dissemination of Pulmonary Tuberculosis. A Two-Year Study of Contagion in a Tuberculosis Ward[END_REF] ; et ces travaux ont été reproduits récemment avec des résultats cohérents [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF]. Ces études ont clarifié les principes fondamentaux de la transmission aérienne de la tuberculose pulmonaire et sont actuellement largement acceptés comme la voie principale de la transmission [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF][START_REF] Yates | The transmission of Mycobacterium tuberculosis in high burden settings[END_REF].

Également, ont été rapportés des cas de transmission de M. tuberculosis par aérosols au cours d'autopsie [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF] et de procédures chirurgicales d'incision et l'irrigation d'abcès tuberculeux [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF]. La transmission respiratoire concerne aussi la tuberculose à M. bovis pour des personnes exposées aux aérosols provenant d'animaux infectés par M. bovis [START_REF] Couto | Zoonotic Tuberculosis in Humans: Control, Surveillance, and the One Health Approach[END_REF][START_REF] Olea-Popelka | Zoonotic tuberculosis in human beings caused by Mycobacterium bovis-a call for action[END_REF]; mais aussi en cas de transmission interhumaine de M. bovis [START_REF] Olea-Popelka | Zoonotic tuberculosis in human beings caused by Mycobacterium bovis-a call for action[END_REF][START_REF] Sunder | Humanto-Human Transmission of Tuberculosis Caused by Mycobacterium bovis in Immunocompetent Patients[END_REF][START_REF] Evans | Cluster of human tuberculosis caused by Mycobacterium bovis: evidence for person-toperson transmission in the UK[END_REF].

Chez la souris, l'infection par aérosols de M. tuberculosis entraîne une perte de la diversité de microbiote intestinal, 06 jours après infection [START_REF] Winglee | Aerosol Mycobacterium tuberculosis Infection Causes Rapid Loss of Diversity in Gut Microbiota[END_REF]. Ces changements rapides du microbiote sont attribués à la réponse immunitaire de l'hôte, toutes les souris ayant présenté un rétablissement de la diversité microbienne quelques jours plus tard [START_REF] Winglee | Aerosol Mycobacterium tuberculosis Infection Causes Rapid Loss of Diversity in Gut Microbiota[END_REF]. Le rôle du microbiote respiratoire dans l'apparition de la tuberculose reste incertain. De plus, les données montrent qu'il existe une diversité microbienne unique entre les patients tuberculeux et les témoins sains, mais les résultats sont jusqu'à présent controversés [START_REF] Eshetie | The respiratory microbiota: new insights into pulmonary tuberculosis[END_REF]. Il est important de noter pour le diagnostic de la tuberculose par culture au laboratoire, les protocoles comprennent une étape de décontamination chimique (soude) des crachats dans le but d'éliminer les microorganismes à croissance rapide afin de faciliter la croissance des mycobactéries [START_REF] Asmar | Rapid culture-based diagnosis of pulmonary tuberculosis in developed and developing countries[END_REF]. Cette décontamination a peut-être favorisé la non-observation par culture de certaines communautés bactériennes qui pourraient coexister avec M. tuberculosis dans les poumons et dans les voies respiratoires ; ouvrant des perspectives de travaux de microbiologie clinique.

Voie digestive

Historiquement, la voie digestive a été la première voie d'administration du vaccin BCG, illustrant la possibilité du passage systémique des MTC après ingestion [START_REF] Negre | Vaccination in Practice * Indian Medical Gazette[END_REF][START_REF] Calmette | Demonstration experimentale du passage des bacille-vaccins BCG a travers la muquese de l'intestin chez l'enfant et chez le singe[END_REF]. Ce mode d'administration a été abandonné dans la plupart des pays à la suite du « désastre de Lubeck », et en 1976, le Brésil a été le dernier pays à abandonner ce mode d'administration, du fait de la mauvaise réponse au test cutané et aussi pour des raisons économiques et opérationnelles [START_REF] Mortatti | Absorption of Mycobacterium bovis BCG administered by the oral route[END_REF][START_REF] Benévolo-De-Andrade | BCG Moreau Rio de Janeiro: an oral vaccine against tuberculosis -review[END_REF]. Actuellement, la possibilité d'une porte d'entrée digestive des MTC est négligée malgré les données publiées, illustrées par le « Désastre de Lübeck» en Allemagne en 1929-1933 [START_REF] Fox | Tuberculosis in Newborns: The Lessons of the "Lübeck Disaster" (1929-1933)[END_REF].

Au cours de cet épisode, l'administration orale du vaccin BCG contaminé accidentellement par M. tuberculosis à 251 nouveau-nés a provoqué une tuberculose chez 228 enfants qui ont tous présenté des atteintes ganglionnaires, 197 enfants ont présenté des formes abdominales, tandis qu'une forme pulmonaire a été rapportée chez 30 enfants ; avec 71 (28.3%) décès par tuberculose [START_REF] Fox | Tuberculosis in Newborns: The Lessons of the "Lübeck Disaster" (1929-1933)[END_REF]. Cet accident éclaire la transmissibilité de M. tuberculosis par voie digestive, et dans cet accident la forme de tuberculose à M. tuberculosis est semblable aux formes de tuberculose causée par M. bovis chez l'homme affectant le plus souvent les ganglions lymphatiques, après entrée de l'agent pathogène par voie digestive [START_REF] Fox | Tuberculosis in Newborns: The Lessons of the "Lübeck Disaster" (1929-1933)[END_REF]. Concernant M. canettii, les données cliniques et expérimentales [START_REF] Osman | Smooth Tubercle Bacilli: Neglected Opportunistic Tropical Pathogens[END_REF][START_REF] Bouzid | Ready Experimental Translocation of Mycobacterium canettii Yields Pulmonary Tuberculosis[END_REF] actuelles suggèrent l'existence d'un réservoir environnemental inconnu, et une transmission par voie digestive via des aliments avec une réplication locale dans l'oropharynx et les ganglions lymphatiques cervicaux et une dissémination accrue dans les voies respiratoires et digestives [START_REF] Osman | Smooth Tubercle Bacilli: Neglected Opportunistic Tropical Pathogens[END_REF].

La transmission digestive de la tuberculose est reconnue particulièrement pour la tuberculose zoonotique causé principalement par M. bovis [START_REF] Couto | Zoonotic Tuberculosis in Humans: Control, Surveillance, and the One Health Approach[END_REF]. La tuberculose zoonotique est essentiellement une maladie d'origine alimentaire qui fait suite à la consommation de lait ou de produits laitiers non pasteurisés [START_REF] Couto | Zoonotic Tuberculosis in Humans: Control, Surveillance, and the One Health Approach[END_REF]. La viande provenant d'animaux atteints de tuberculose n'est pas reconnue comme un véhicule de transmission de M. bovis, du moins lorsqu'elle est cuite, de plus M. bovis est rarement trouvée dans les muscles [START_REF] Couto | Zoonotic Tuberculosis in Humans: Control, Surveillance, and the One Health Approach[END_REF]. L'OMS a estimé que, en 2018 la tuberculose zoonotique à M. bovis a causé 143.000 nouveaux cas et une mortalité de 12.300 personnes [START_REF]Global tuberculosis report 2019[END_REF].

La tuberculose abdominale représente 12% des cas de tuberculose extrapulmonaire et 1 à 3% du total des cas de tuberculose [START_REF] Cho | Clinical features and outcomes of abdominal tuberculosis in southeastern Korea: 12 years of experience[END_REF]. 15% à 25% des cas de tuberculose abdominale ont une tuberculose pulmonaire concomitante [START_REF] Debi | Abdominal tuberculosis of the gastrointestinal tract: Revisited[END_REF]. Les modes d'infection par les MTC dans les cas de tuberculose abdominale comprennent la déglutition des expectorations infectées, l'ingestion d'aliments infectés, la propagation lymphatique à partir d'un foyer extra-abdominale, et par propagation contiguë à partir d'organes uro-génitaux [START_REF] Cho | Clinical features and outcomes of abdominal tuberculosis in southeastern Korea: 12 years of experience[END_REF][START_REF] Abu-Zidan | Diagnosis of abdominal tuberculosis: lessons learned over 30 years: pectoral assay[END_REF]. La tuberculose abdominale peut être classée en 4 formes: luminale (iléo-caecale), péritonéale, nodale et viscérale impliquant les organes solides intra-abdominaux [START_REF] Cho | Clinical features and outcomes of abdominal tuberculosis in southeastern Korea: 12 years of experience[END_REF]. Plusieurs séries de cas de tuberculose abdominale ont été rapportées, dans certains travaux cette localisation est majoritairement attribuée à M. bovis puis à M. tuberculosis [START_REF] Veeragandham | Abdominal tuberculosis in children: Review of 26 cases[END_REF][START_REF] Probert | Epidemiological study of abdominal tuberculosis among Indian migrants and the indigenous population of Leicester, 1972-1989[END_REF][START_REF] Torres-Gonzalez | Human tuberculosis caused by Mycobacterium bovis: a retrospective comparison with Mycobacterium tuberculosis in a Mexican tertiary care centre, 2000-2015[END_REF] Il a été rapporté dans chez des modèles murins que l'altération du microbiote intestinal après administration d'antibiotiques provoquait une sensibilité à la tuberculose. En effet les souris avec dysbiose du microbiote ont montré une augmentation significative de M. tuberculosis dans les poumons et une dissémination dans la rate et le foie [START_REF] Khan | Alteration in the Gut Microbiota Provokes Susceptibility to Tuberculosis[END_REF] ; des résultats similaires à ceux observés à l'induction de la dysbiose par l'infection par Helicobacter hepaticus [START_REF] Majlessi | Colonization with Helicobacter is concomitant with modified gut microbiota and drastic failure of the immune control of Mycobacterium tuberculosis[END_REF].

Tuberculose d'inoculation.

L'inoculation cutanée est aussi une voie de transmission, rapportée dans certains cas accidentels chez les personnels soignants [START_REF] Kramer | Primary cutaneous tuberculosis after a needlestick injury from a patient with AIDS and undiagnosed tuberculosis[END_REF][START_REF] Huang | Primary inoculation tuberculosis after an accidental scalpel injury[END_REF], ou chez des patients à la suite de l'injection de corticoïdes ou de traumatismes cutanés [START_REF] Jong | Non-respiratory tuberculosis with Mycobacterium tuberculosis after penetrating lesions of the skin: five case histories[END_REF] et de tatouage [START_REF] Horney | Cutaneous inoculation tuberculosis secondary to "jailhouse tattooing[END_REF]. Cette voie d'inoculation donne une tuberculose cutanée primaire. D'autre part, la tuberculose cutanée faciale, la tuberculose cutanée péri-oculaire ont été décrites et les auteurs ont émis l'hypothèse que cette présentation inhabituelle pourrait être due à un traumatisme mineur suivi d'une inoculation [START_REF] Mm | Facial cutaneous tuberculosis: an unusual presentation[END_REF]. Annobil et al. ont décrit une tuberculose primaire du pénis avec hypertrophie bilatérale des ganglions lymphatiques inguinaux chez un bébé de 4 mois circoncis à l'âge de 6 semaines ; peut-être liée au fait que le barbier-opérateur avait humecté par expectoration, son rasoir avant de l'aiguiser [START_REF] Annobil | Primary tuberculosis of the penis in an infant[END_REF]. Également, un rapport anecdotique de transmission de M. tuberculosis par voie sexuelle a été décrit, comportant une tuberculose cutanée pénienne suivie d'une tuberculose endométriale chez la partenaire du patient ; les deux souches de M. tuberculosis étant identiques par typage moléculaire [START_REF] Angus | Cutaneous tuberculosis of the penis and sexual transmission of tuberculosis confirmed by molecular typing[END_REF]. Dans de rares cas, la tuberculose peut être contractée après une transplantation d'organe solide, à partir d'un donneur d'organe infecté, et en raison de l'immunosuppression, les receveurs de greffe développent fréquemment une TB extra-pulmonaire ou disséminée, cependant la tuberculose après une transplantation d'organe solide est le plus souvent causée par une infection primaire ou la réactivation d'une infection latente [START_REF] Abad | Mycobacterium tuberculosis after solid organ transplantation: A review of more than 2000 cases[END_REF].

DEVENIR DE M. TUBERCULOSIS CHEZ L'HOMME

Une fois inhalé, M. tuberculosis transite de la trachée aux poumons où il est phagocyté par les macrophages alvéolaires dans lesquels il est internalisé dans les phagosomes, puis dans les phagolysosomes [START_REF] Pieters | Mycobacterium tuberculosis and the Macrophage: Maintaining a Balance[END_REF][START_REF] Cambier | Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids[END_REF]. Cependant, M. tuberculosis peut bloquer l'acidification et la maturation des phagosomes pour survivre dans les macrophages alvéolaires de l'hôte [START_REF] Ehrt | Mycobacterial survival strategies in the phagosome: defence against host stresses[END_REF][START_REF] Houben | ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria[END_REF]. Macrophages et autres cellules immunitaires s'organisent en agrégat pour former le granulome [START_REF] Pagán | The Formation and Function of Granulomas[END_REF] dans lequel les mycobactéries sont à la fois intracellulaires à l'intérieur des macrophages et extracellulaires [START_REF] Getahun | Latent Mycobacterium tuberculosis infection[END_REF]. Ce granulome est formé principalement par des macrophages infectés et non infectés par les bacilles M. tuberculosis entourés de cellules immunitaires comprenant des granulocytes, des cellules dendritiques (CD), des cellules tueuses naturelles (NK) et des lymphocytes T et B [START_REF] Pagán | The Formation and Function of Granulomas[END_REF] (Figure 3). Dans ces conditions, les défenses immunitaires innées et adaptatives sont impliquées dans la maîtrise de M. tuberculosis [START_REF] Dutta | Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms[END_REF] limitant leur réplication et aboutissant à un état d'équilibre entre l'organisme hôte et l'agent pathogène. Dans le granulome, les bacilles M. tuberculosis se trouvent exposés à diverses conditions de stress en particulier l'hypoxie favorisant l'entrée en dormance des mycobactéries [START_REF] Dutta | Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms[END_REF]. Cet état de dormance chez M. tuberculosis se traduit par une capacité à persister dans les tissus hôtes sans se répliquer pendant des mois voire des années, sans causer de tuberculose maladie et entraînant une infection chronique asymptomatique chez près de 90% des personnes infectées connue sous le nom d'infection tuberculeuse latente [START_REF] Bhavanam | Understanding the pathophysiology of the human TB lung granuloma using in vitro granuloma models[END_REF][START_REF] Wayne | Dormancy of Mycobacterium tuberculosis and latency of disease[END_REF][START_REF] Cardona | On the nature of Mycobacterium tuberculosislatent bacilli[END_REF][START_REF] Rittershaus | The Normalcy of Dormancy: Common Themes in Microbial Quiescence[END_REF]. En revanche, 5% des personnes infectées vont développer la tuberculose active [START_REF] Koul | The challenge of new drug discovery for tuberculosis[END_REF] alors que le reste des personnes seraient compétentes pour éliminer l'agent pathogène. Les mycobactéries dormantes peuvent résider dans les lésions pulmonaires granulomateuses anciennes (dans le macrophage et/ou caséum), les ganglions lymphatiques pulmonaires [START_REF] Parrish | Mechanisms of latency in Mycobacterium tuberculosis[END_REF] ou encore le tissu adipeux qui est décrit comme un vaste réservoir abritant les mycobactéries dormantes les préservant des agents antimicrobiens et du système immunitaire hôte [START_REF] Neyrolles | Is Adipose Tissue a Place for Mycobacterium tuberculosis Persistence?[END_REF]. Lorsque l'immunité de l'hôte est compromise et les conditions environnementales autour de M. tuberculosis deviennent favorables à sa réactivation, ces bacilles accélèrent leur réplication, ce qui conduit à la nécrose des macrophages infectés et à la libération des mycobactéries intracellulaires, qui pourraient infecter de nouvelles cellules et se propager à d'autres tissus [START_REF] Woolhiser | In vivo adaptation of the Wayne model of latent tuberculosis[END_REF][START_REF] Dietrich | Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology[END_REF]. Outre leur capacité à se réactiver, le rôle important de la dormance dans l'histoire naturelle de la tuberculose réside dans le fait que les mycobactéries dormantes sont potentiellement infectieuses. En effet, ceci a été démontré expérimentalement [START_REF] Woolhiser | In vivo adaptation of the Wayne model of latent tuberculosis[END_REF][START_REF] Dietrich | Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology[END_REF] mais aussi cliniquement où il a été constaté que les mycobactéries dormantes peuvent être encore plus infectieuses que les mycobactéries métaboliquement actives dans l'expectoration des patients atteints de tuberculose pulmonaire [START_REF] Datta | Sputum Microscopy With Fluorescein Diacetate Predicts Tuberculosis Infectiousness[END_REF].

TUBERCULOSE CHEZ LES ANIMAUX.

Les données de la littérature indiquent qu'à l'exception du M. bovis BCG qui est une souche vaccinale et de M. canettii, toutes les autres MTC (11/13 espèces) peuvent infecter une ou plusieurs espèces animales, y compris l'homme (Figure 2).

Les espèces MTC sont génétiquement très apparentées (> 99.9 % d'identité nucléotidique) et certains auteurs les classent en souches adaptées à l'hôte humain, telles que M. tuberculosis et M. africanum, et celles qui possèdent le pouvoir de se propager et de se transmettre dans une grande variété d'hôtes d'animaux sauvages et domestiques [START_REF] Brites | A New Phylogenetic Framework for the Animal-Adapted Mycobacterium tuberculosis Complex[END_REF]. Cependant, il est à noter que la barrière espèce n'est pas stricte et dans de rares situations M. tuberculosis peut infecter des animaux domestiques, ou sauvages en captivité. Plus de 15 espèces animales peuvent être infectées par M. tuberculosis y compris le perroquet [START_REF] Ghodbane | Non-human sources of Mycobacterium tuberculosis[END_REF][START_REF] Mukherjee | Isolation of Mycobacterium tuberculosis from Antelope cervicapra and Gazelle bennettii in India and confirmation by molecular tests[END_REF][START_REF] Adeogun | Mycobacterium tuberculosis and Dual M. tuberculosis/M. bovis Infection as the Cause of Tuberculosis in a Gorilla and a Lioness, Respectively, in Ibadan Zoo, Nigeria[END_REF] ; de même que M. africanum a été sporadiquement isolé chez des animaux, étant cultivée à partir de singes d'Afrique atteints de tuberculose active, et chez des vaches [START_REF] Yeboah-Manu | The Biology and Epidemiology of Mycobacterium africanum[END_REF][START_REF] Jong | Mycobacterium africanum-Review of an Important Cause of Human Tuberculosis in West Africa[END_REF]. Cinq MTC ont déjà été rapportés comme des zoonoses, causant la tuberculose chez les animaux et transmissibles à l'homme, incluant M. bovis [START_REF] Couto | Zoonotic Tuberculosis in Humans: Control, Surveillance, and the One Health Approach[END_REF][START_REF] Palmer | Mycobacterium bovis: Characteristics of Wildlife Reservoir Hosts[END_REF][START_REF] Broughan | Mycobacterium bovis infections in domesticated non-bovine mammalian species. Part 1: Review of epidemiology and laboratory submissions in Great Britain 2004-2010[END_REF][START_REF] Michelet | Mycobacterium bovis Infection of Red Fox, France[END_REF], M. caprae [START_REF] Prodinger | Mycobacterium caprae infection in humans[END_REF][START_REF] Krajewska | Transmission of Mycobacterium caprae in a herd of European bison in the Bieszczady Mountains, Southern Poland[END_REF], M. microti [START_REF] Panteix | Pulmonary tuberculosis due to Mycobacterium microti: a study of six recent cases in France[END_REF][START_REF] Michelet | Infection with Mycobacterium microti in Animals in France[END_REF][START_REF] Zanolari | Tuberculosis Caused by Mycobacterium microti in South American Camelids[END_REF], M. orygis [START_REF] Dawson | Transmission of Mycobacterium orygis (M. tuberculosis Complex Species) from a Tuberculosis Patient to a Dairy Cow in New Zealand[END_REF][START_REF] Marcos | Mycobacterium orygis Lymphadenitis in New York, USA[END_REF] et M. pinnipedii [START_REF] Zmak | From Peruvian mummies to living humans: first case of pulmonary tuberculosis caused by Mycobacterium pinnipedii[END_REF][START_REF] Rodriguez-Campos | Overview and phylogeny of Mycobacterium tuberculosis complex organisms: Implications for diagnostics and legislation of bovine tuberculosis[END_REF]. Cependant, l'agent principal de tuberculose chez les animaux est M. bovis qui peut infecter une large gamme de mammifères, cette mycobactérie a une répartition mondiale chez les bovins [START_REF] Palmer | Mycobacterium bovis: Characteristics of Wildlife Reservoir Hosts[END_REF][START_REF] Michel | Mycobacterium bovis at the animal-human interface: A problem, or not?[END_REF]. D'autres MTC identifiées chez des animaux n'ont jamais été rapportées chez l'homme, incluant M. mungi, M. suricattae,, Dassie bacillus et Chimpanzee bacillus [START_REF] Coscolla | Novel Mycobacterium tuberculosis Complex Isolate from a Wild Chimpanzee[END_REF][START_REF] Parsons | Novel Cause of Tuberculosis in Meerkats, South Africa[END_REF][START_REF] Rodriguez-Campos | Overview and phylogeny of Mycobacterium tuberculosis complex organisms: Implications for diagnostics and legislation of bovine tuberculosis[END_REF][START_REF] Alexander | Novel Mycobacterium tuberculosis Complex Pathogen, M. mungi[END_REF]. Ces données suggèrent qu'il n'y a pas de spécificité entre les espèces MTC et les espèces mammifères infectées, et que la répartition actuelle des espèces MTC parmi les espèces mammifères résulte de probabilité de contacts.

TUBERCULOSE DANS L'ENVIRONNEMENT (Figure 1).

Les données actuelles de la littérature autorisent un scénario dans lequel les MTC sont initialement des mycobactéries de l'environnement qui ont évolué à partir de pathogènes opportunistes d'eucaryotes unicellulaires (amibes) vers des pathogènes opportunistes des mammifères puis des pathogènes contagieux chez l'Homme. Quelques travaux ont montré la persistance de M. tuberculosis et M. bovis dans le sol inoculé de façon expérimentale au laboratoire, dans des conditions maîtrisées de température et d'hygrométrie, pour une durée de 12 mois [START_REF] Ghodbane | Long-term survival of tuberculosis complex mycobacteria in soil[END_REF]. Cette observation expérimentale a été relayée par des observations de terrain réalisées à Téhéran en Iran où 1% des prélèvements de sol et 10% des prélèvements d'eau ont cultivé M. tuberculosis, re-cultivée 09 mois après le prélèvement ; et dont les génotypes déterminés par spoligotyping correspondaient en partie à ceux des patients tuberculeux diagnostiqués à Téhéran [START_REF] Velayati | Identification and Genotyping of Mycobacterium tuberculosis Isolated From Water and Soil Samples of a Metropolitan City[END_REF]. Un travail récent a revu l'ensemble des données expérimentales et d'observations de terrain, permettant de confirmer la possibilité de conservation prolongée des MTC dans le sol [START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF]. Les patients atteints de tuberculose pulmonaire éliminent M. tuberculosis dans les selles, qui sont un prélèvement alternatif aux expectorations pour le diagnostic de la tuberculose par culture [START_REF] Asmar | A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens[END_REF] et par biologie moléculaire [START_REF] El Khéchine | Detection of Mycobacterium tuberculosis complex organisms in the stools of patients with pulmonary tuberculosis[END_REF]. Ainsi, les patients infectés par la tuberculose peuvent contaminer l'environnement [START_REF] Velayati | Identification and Genotyping of Mycobacterium tuberculosis Isolated From Water and Soil Samples of a Metropolitan City[END_REF]. De plus, il a été rapporté un cas de transmission de M. tuberculosis à un embaumeur à partir du cadavre d'un patient décédé de tuberculose pulmonaire [START_REF] Sterling | Transmission of Mycobacterium tuberculosis from a Cadaver to an Embalmer[END_REF] ; illustrant que les cadavres de patients tuberculeux pourraient être une source d'infection du sol [START_REF] Ghodbane | Non-human sources of Mycobacterium tuberculosis[END_REF].

2 RÉSUMÉ

 2 Plusieurs pièces du puzzle de l'histoire naturelle de la tuberculose sont assemblées dans cette revue afin d'illustrer les réservoirs et sources potentiels des mycobactéries du complexe Mycobacterium tuberculosis (MTC), leur transmission aux animaux et à l'Homme et leur devenir dans les populations, dans une perspective co-évolutive. Compagnes millénaires des populations mammifères et humaines, MTC sont détectées dans le sol dans lequel elles infectent et survivent dans les amibes végétatives et les kystes, à l'exception de Mycobacterium canettii. Jamais détectées dans la sphère des plantes, elles sont transmissibles par voie transcutanée, par voie digestive et par voie respiratoire et provoquent une infection du système lymphatique avec une dissémination secondaire dans la plupart des tissus dans lesquels elles déterminent une réaction inflammatoire granulomateuse spécifique et non pathognomonique. MTC survit sous forme dormante dans le granulome (intra/extracellulaire) et aussi dans le tissu adipeux et cela quel que soit l'espèce MTC et l'espèce mammifère ; indiquant que l'épidémiologie actuelle dans les populations mammifères est essentiellement gouvernée par les probabilités de contacts entre les espèces mammifères et les espèces MTBC. Les variabilités individuelles de l'expression clinique de la tuberculose sont liées à l'espèce MTC, la souche et l'inoculum ; les facteurs génétiques de l'hôte ; les modulations acquises de la réponse inflammatoire ; et probablement les microbiotes. Cette revue des données de la littérature suggère une histoire naturelle évolutive de mycobactéries environnementales telluriques, satellites d'eucaryotes unicellulaires, transmissibles aux mammifères principalement par voie respiratoire et par voie digestive, chez lesquelles elles déterminent une infection contagieuse mortelle primitivement lymphatique et une quiescence mimant l'enkystement. Cette revue ouvre des

  Par ailleurs, quelques observations expérimentales indiquent la survie des MTC à l'intérieur des formes végétative des amibes libres du genre Acanthamoeba[START_REF] Medie | Mycobacterium tuberculosis Complex Mycobacteria as Amoeba-Resistant Organisms[END_REF]. En particulier, il a été montré expérimentalement que les cinq amibes libres Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba lenticulata, Vermamoeba vermiformis et Dictyostellium discoideum sont infectables par M. bovis; que M. bovis est enkysté par chacune de ces cinq amibes; que M. bovis persiste au moins 60 jours à l'intérieur des kystes; et que l'inhalation expérimentale des amibes végétatives et des kystes infectés par M. bovis provoque une tuberculose pulmonaire chez la souris Balb/c[START_REF] Sanchez-Hidalgo | Mycobacterium bovis hosted by free-living-amoebae permits their long-term persistence survival outside of host mammalian cells and remain capable of transmitting disease to mice[END_REF]. Cette vie intra amibienne a été l'occasion d'échanges génétiques entre l'amibe hôte et les mycobactéries[START_REF] Lamrabet | The Genealogic Tree of Mycobacteria Reveals a Long-Standing Sympatric Life into Free-Living Protozoa[END_REF]. De façon intéressante, seule M. canettii n'est pas enkystée au moment de la formation du kyste d'amibe contrairement à M. tuberculosis qui probablement peut survivre pour des périodes étendues de temps à l'intérieur du kyste : il a été montré que les kystes l'amibe Acanthamoeba (ne contenant pas M. tuberculosis) pouvaient survivre pour 50 décades[START_REF] Appelt | Polyphasic Analysis of a Middle Ages Coprolite Microbiota, Belgium[END_REF]. Le mécanisme de sortie précoce de M. canettii n'est pas connu même si cette MTC code pour une cellulase active qui pourrait cliver la paroi cellulosique du kyste en cours de formation[START_REF] Medie | Mycobacterium tuberculosis Complex Mycobacteria as Amoeba-Resistant Organisms[END_REF]. Également, la modalité de survie des MTC à l'intérieur du kyste d'amibe est inconnue même s'il pourrait s'agir de mycobactéries dormantes[START_REF] Salah | Free-living amoebae, a training field for macrophage resistance of mycobacteria[END_REF]. L'ensemble de ces observations suggèrent la possibilité d'un cycle environnemental indépendant des hôtes habituels, dont la possibilité n'est pas documentée. Cependant, le rôle du sol comme source de contamination de certains mammifères, ne semble pas déraisonnable. Alors que le rôle des plantes dans l'histoire naturelle des MTC reste inconnu, on ignore si les MTC pourraient infecter des végétaux et si les MTC dans le sol pourraient passer dans les racines et puis infecter les parties aériennes des plantes.CONCLUSIONS ET PERSPECTIVESCette revue de la littérature issue de différentes sources géographiques et thématiques ouvre des scénarios de réservoirs, sources, modes de transmission et devenir des MTC dans les populations animales et humaines, plus larges que celles habituellement rapportées. En effet, la tuberculose peut être comprise comme une infection causée par les MTC affectant l'homme et de nombreux mammifères, transmises principalement par voie respiratoire et digestive. L'origine des MTC est initialement des bactéries telluriques pathogènes des eucaryotes unicellulaires, qui ont progressivement acquis la capacité à infecter les eucaryotes pluricellulaires puis à être directement transmissibles d'hôte à hôte conférant le caractère contagieux actuellement observé chez les populations humaines et animales.La focalisation des études sur les voies respiratoires restreint la connaissance de ces scénarios possibles, diminuant la vigilance sur les modalités alternatives actuelles ; et passées pour les populations préhistoriques.

Figure 01 .

 01 Figure 01. Réservoir des mycobactéries du complexe Mycobacterium tuberculosis et voies de transmission à l'homme.

Figure 02 .Figure 03 .

 0203 Figure 02. Répartition des espèces du complexe Mycobacterium tuberculosis chez les mammifères[START_REF] Ghodbane | Non-human sources of Mycobacterium tuberculosis[END_REF][START_REF] Martinez | Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications[END_REF][START_REF] Coscolla | Novel Mycobacterium tuberculosis Complex Isolate from a Wild Chimpanzee[END_REF][START_REF] Parsons | Novel Cause of Tuberculosis in Meerkats, South Africa[END_REF][START_REF] Couto | Zoonotic Tuberculosis in Humans: Control, Surveillance, and the One Health Approach[END_REF][START_REF] Mukherjee | Isolation of Mycobacterium tuberculosis from Antelope cervicapra and Gazelle bennettii in India and confirmation by molecular tests[END_REF][START_REF] Adeogun | Mycobacterium tuberculosis and Dual M. tuberculosis/M. bovis Infection as the Cause of Tuberculosis in a Gorilla and a Lioness, Respectively, in Ibadan Zoo, Nigeria[END_REF][START_REF] Jong | Mycobacterium africanum-Review of an Important Cause of Human Tuberculosis in West Africa[END_REF][START_REF] Palmer | Mycobacterium bovis: Characteristics of Wildlife Reservoir Hosts[END_REF][START_REF] Broughan | Mycobacterium bovis infections in domesticated non-bovine mammalian species. Part 1: Review of epidemiology and laboratory submissions in Great Britain 2004-2010[END_REF][START_REF] Michelet | Mycobacterium bovis Infection of Red Fox, France[END_REF][START_REF] Prodinger | Mycobacterium caprae infection in humans[END_REF][START_REF] Krajewska | Transmission of Mycobacterium caprae in a herd of European bison in the Bieszczady Mountains, Southern Poland[END_REF][START_REF] Panteix | Pulmonary tuberculosis due to Mycobacterium microti: a study of six recent cases in France[END_REF][START_REF] Michelet | Infection with Mycobacterium microti in Animals in France[END_REF][START_REF] Zanolari | Tuberculosis Caused by Mycobacterium microti in South American Camelids[END_REF][START_REF] Dawson | Transmission of Mycobacterium orygis (M. tuberculosis Complex Species) from a Tuberculosis Patient to a Dairy Cow in New Zealand[END_REF][START_REF] Marcos | Mycobacterium orygis Lymphadenitis in New York, USA[END_REF][START_REF] Zmak | From Peruvian mummies to living humans: first case of pulmonary tuberculosis caused by Mycobacterium pinnipedii[END_REF][START_REF] Rodriguez-Campos | Overview and phylogeny of Mycobacterium tuberculosis complex organisms: Implications for diagnostics and legislation of bovine tuberculosis[END_REF][START_REF] Michel | Mycobacterium bovis at the animal-human interface: A problem, or not?[END_REF][START_REF] Alexander | Novel Mycobacterium tuberculosis Complex Pathogen, M. mungi[END_REF]


Sébastien Albert

Olivier Flores

Cláudia Baider

F B Vincent Florens

Dominique Strasberg

With or without frugivores? Flying foxes sustain woody plant regenerationDiffering severity of frugivore loss contrasts the fate of native forests on the land of the Dodo (Mascarene archipelago)

Keywords: Frugivore extinction, island biology, Janzen-Connell effect, plant diversity loss, protected tropical forest

Frugivore loss has the potential to alter the regeneration of tropical hyperdiverse forests. However, no study has investigated tree diversity maintenance within old-growth protected forests on tropical oceanic islands where conservation stakes are considerable. In the Mascarenes, the largest frugivores including the Dodo went extinct after human colonization in the 17 th century. Mauritius, today devoid of almost all its native habitats, retains an important population of flying foxes, while Réunion, largely covered by native habitats, has lost all but one of its native frugivores, a bulbul. Using census data of 130 woody species from 3-ha permanent plots from Mauritius (MAU-BF) and Réunion (REU-ML), we show that most fleshy-fruited species regenerate poorly at REU-ML, in contrast to MAU-BF, although large-seeded fleshy-fruited species regenerate poorly at both sites. The difference in recruitment is all the more striking because local woody plant diversity at MAU-BF is double that at REU-ML. Changes observed in the understory will probably lead to a strongly impoverished canopy at REU-ML, and to canopy shift composition at MAU-BF. Hence, the protected but highly fragmented forests on Mauritius are likely more resilient than those on Réunion usually considered best preserved within the ecoregion. Although processes other than seed dispersal might also be at work, flying foxes probably ensure effective dispersal of numerous native fleshy-fruited plants on Mauritius, contrary to only bulbuls on Réunion. To efficiently protect tropical rainforests on islands and continents alike, there is an urgent need to protect extant frugivores populations and reinstate seed dispersal function wherever necessary.

INTRODUCTION

Vertebrates play an important role as mutualistic pollinators or seed dispersers in most tropical forests worldwide [START_REF] Dirzo | Defaunation in the Anthropocene[END_REF][START_REF] Turner | The ecology of trees in the tropical rain forest[END_REF]. Recently, it has become clear that their loss has the potential to deeply alter tropical forest composition (Albert et al., 2020b;[START_REF] Dirzo | Defaunation in the Anthropocene[END_REF][START_REF] Effiom | Bushmeat hunting changes regeneration of African rainforests[END_REF][START_REF] Terborgh | Tree recruitment in an empty forest[END_REF][START_REF] Trolliet | How complementary are large frugivores for tree seedling recruitment? A case study in the Congo Basin[END_REF][START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF]. Most conservation strategies have for decades involved integrating forest habitats into protected areas. However, preserving forests in which mutualistic vertebrates are absent or at low density might be ineffective when their functioning highly depends on plant-animals interactions [START_REF] Laurance | Averting biodiversity collapse in tropical forest protected areas[END_REF].

The various life-history strategies of trees complicate the assessment of the impact of frugivore extirpation, but studies at community level indicate an alteration of the recruitment for numerous fleshyfruited plants in defaunated protected forests of the Neotropics [START_REF] Terborgh | Tree recruitment in an empty forest[END_REF], Central Africa [START_REF] Effiom | Bushmeat hunting changes regeneration of African rainforests[END_REF][START_REF] Trolliet | How complementary are large frugivores for tree seedling recruitment? A case study in the Congo Basin[END_REF] and Borneo [START_REF] Harrison | Consequences of defaunation for a tropical tree community[END_REF]. Changes in recruitment were detected just a few decades after frugivore decline or loss, being mainly the consequence of the negative distance-and/or density-dependent effects on germination or seedling survival close to maternal trees [START_REF] Comita | Testing predictions of the Janzen-Connell hypothesis: a meta-analysis of experimental evidence for distance-and density-dependent seed and seedling survival[END_REF][START_REF] Connell | On the role of natural enemies in preventing competitive exclusion in some marine animals and in rain forest trees[END_REF][START_REF] Janzen | Herbivores and the number of tree species in tropical forests[END_REF]. Other works have also shown how fruit consumption by frugivores may enhance the recruitment of fleshy-fruited plants by removing potential physical/chemical dormancy [START_REF] Soltani | A meta-analysis of the effects of frugivory (endozoochory) on seed germination: role of seed size and kind of dormancy[END_REF][START_REF] Traveset | Seed trait changes in dispersers' gut and consequences for germination and seedling growth[END_REF] or more simply, by cleaning the seed of flesh that could attract pathogens and seed predators causing propagule mortality [START_REF] Levi | Dispersal vacuum in the seedling recruitment of a primate-dispersed Amazonian tree[END_REF]. Hence, frugivores play a central role in closing the loop of dispersal in habitats largely dominated by fleshy-fruited plants [START_REF] Wang | Closing the seed dispersal loop[END_REF].

In theory, the decline and extirpation of frugivores threaten tropical forests not only on continents, where they can be hyperdiverse [START_REF] Terborgh | Tree recruitment in an empty forest[END_REF], but also on oceanic islands [START_REF] Hansen | Seed dispersal and establishment of endangered plants on oceanic islands: The Janzen-Connell model, and the use of ecological analogues[END_REF]. Despite their isolation and small areas, oceanic islands can indeed host substantial richness in fleshy-fruited species, e.g. one forest patch on Mauritius harbours more than 60 tree species per hectare of which 89% are fleshy-fruited (Florens et al., 2017a[START_REF] Florens | Surviving 370 years of human impact: what remains of tree diversity and structure of the lowland wet forests of oceanic island Mauritius?[END_REF]. This relative diversity is comparable to some African continental tropical rainforests [START_REF] Turner | The ecology of trees in the tropical rain forest[END_REF]. Hence, one may expect a higher negative impact of frugivore loss on diversity maintenance on tropical oceanic islands since numerous frugivores went extinct after human colonization [START_REF] Hansen | The forgotten megafauna[END_REF][START_REF] Heinen | Extinction-driven changes in frugivore communities on oceanic islands[END_REF], and having no options of alternative seed dispersal systems [START_REF] Guimarães | Seed dispersal anachronisms: rethinking the fruits extinct megafauna ate[END_REF].

Fleshy-fruited plant species vary in their vulnerability following the extinction of frugivores [START_REF] Guimarães | Seed dispersal anachronisms: rethinking the fruits extinct megafauna ate[END_REF]. Plants with large fruits and/or large seeds are particularly at risk because large vertebrates often constitute the first guild to be extirpated [START_REF] Dirzo | Defaunation in the Anthropocene[END_REF][START_REF] Hansen | The forgotten megafauna[END_REF][START_REF] Heinen | Extinction-driven changes in frugivore communities on oceanic islands[END_REF]. Several studies have shown that insular large-seeded tree species experience recruitment failure at plant population level due to disperser loss [START_REF] Carpenter | Introduction of mammalian seed predators and the loss of an endemic flightless bird impair seed dispersal of the New Zealand tree Elaeocarpus dentatus[END_REF][START_REF] Hansen | Seed dispersal and establishment of endangered plants on oceanic islands: The Janzen-Connell model, and the use of ecological analogues[END_REF][START_REF] Wotton | Frugivore loss limits recruitment of large-seeded trees[END_REF]. Although large-seeded species are reputedly well adapted to the strong competition encountered in tropical understory [START_REF] Leishman | The evolutionary ecology of seed size[END_REF], their seedlings/saplings can be strongly depleted at community level in defaunated sites [START_REF] Effiom | Bushmeat hunting changes regeneration of African rainforests[END_REF][START_REF] Harrison | Consequences of defaunation for a tropical tree community[END_REF][START_REF] Terborgh | Tree recruitment in an empty forest[END_REF][START_REF] Trolliet | How complementary are large frugivores for tree seedling recruitment? A case study in the Congo Basin[END_REF]. To our knowledge however, the relationship between recruitment patterns inferred from size-distributions and defaunation has never been carried out before on islands, where one may expect a strong negative impact of large-bodied frugivore extinction on large-seeded plant recruitment.

This study aims to assess the impact of frugivore extinction on forest regeneration by comparing diameter cohorts of native woody species within two islands sharing a close biogeographic history, but with different trajectories since human colonization (Fig. 1 ). Mauritius and Réunion had lowland species-rich forests dominated by fleshy-fruited species [START_REF] Albert | Why are woody plants fleshy-fruited at low elevations? Evidence from a high-elevation oceanic island[END_REF]Florens et al., 2017a;[START_REF] Strasberg | Diversity, size composition and spatial aggregation among trees on a 1-ha rain forest plot at La Réunion[END_REF]. On Réunion, the extirpation of large frugivores from Réunion dates back two centuries (Fig. 1). Consequently, primary succession dynamics have been severely altered after frugivore extinctions and great uncertainty remains about the capacity of established forests to maintain their diversity in the near future (Albert et al., 2020b). On Mauritius by contrast, Pteropus niger still exists in large populations and is a keystone species which feeds fruits of at least 53% of woody plants (Florens et al., 2017a;Fig.1). With a mean body mass of 450 g, this flying-fox is about nine times larger than the bulbul Hypsipetes borbonicus, the largest extant frugivorous bird on Réunion [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF]. This critical difference in the defaunation history between the islands provides a relevant opportunity to use these two main islands of the Mascarenes as pseudo-replicates, and to isolate the effects of seed dispersal from other factors on plant recruitment. Although this biogeographical setting is relevant to address the effects of defaunation on forest regeneration, we note that Mauritius cannot be considered as a strict control in terms of frugivory interaction. Indeed, Mauritius also lost its largest frugivore species that were able to swallow large fruits (Fig. 1) and their extirpation has probably not been fully compensated for by the surviving fauna [START_REF] Howe | Making dispersal syndromes and networks useful in tropical conservation and restoration[END_REF][START_REF] Meehan | Potential disruptions to seed dispersal mutualisms in Tonga, Western Polynesia[END_REF][START_REF] Trolliet | How complementary are large frugivores for tree seedling recruitment? A case study in the Congo Basin[END_REF].

Using census data from permanent plots at Brise Fer on Mauritius (MAU-BF) and Mare Longue on Réunion (REU-ML), we address three complementary hypotheses in response to differential alteration of seed dispersal networks. First, we assume that plant diversity is better maintained at MAU-BF than at REU-ML. In particular, at community level, we expect that the diversity among juveniles should be similar or greater than that of adults at MAU-BF, whereas it should be lower at REU-ML. Second, within species, we assume a higher number of juveniles relative to adults at MAU-BF than at REU-ML. Indeed, few juveniles in proportion at a given time may indicate a declining population [START_REF] Condit | Predicting population trends from size distributions: a direct test in a tropical tree community[END_REF]. Third, we hypothesize seed size to have a strong influence on plant diversity and juveniles ratio. The extinction of all Mascarene frugivores able to swallow fruit >13 mm in diameter probably led to recruitment failure of large-seeded fleshy-fruited plants on both islands, although the extant flying foxes might have, to some extent, carried out the function of these extinct large frugivores on Mauritius.

METHODS

Study sites

Réunion (2512 km², 2-3 million years) and Mauritius (1865 km², 7.8 million years), the two largest islands of the Mascarene archipelago in the tropical Indian Ocean, are respectively 750 and 900 km east of Madagascar (Fig. 1). They are part of the biodiversity hotspot of South-West Indian Ocean islands [START_REF] Mittermeier | Hotspots revisited. Earth's biologically richest and most endangered terrestrial ecoregions[END_REF]. The Mascarene islands share most of their animal and plant lineages, with a great number of endemic species given their relatively small terrestrial area [START_REF] Kier | A global assessment of endemism and species richness across island and mainland regions[END_REF]. Mauritius and Réunion host 691 native flowering plant species (273 single island endemic and 150 Mascarene endemic) and 550 species (165 single-island endemic and 140 Mascarene endemic), respectively [START_REF] Baider | Status of plant conservation in oceanic islands of the Western Indian Ocean[END_REF]. Extinct and threatened species make 10.9% and 81.7% on Mauritius, while 1.2% and 50.9% on Réunion [START_REF] Baider | Status of plant conservation in oceanic islands of the Western Indian Ocean[END_REF]. Lower rates of plant extinction and threat on Réunion is attributed to its higher native cover (30%) than on Mauritius (4.4%), although lowland habitats have been almost completely transformed on Réunion [START_REF] Florens | Surviving 370 years of human impact: what remains of tree diversity and structure of the lowland wet forests of oceanic island Mauritius?[END_REF][START_REF] Strasberg | An assessment of habitat diversity and transformation on La Réunion Island (Mascarene Islands, Indian Ocean) as a basis for identifying broad-scale conservation priorities[END_REF]. In that context, permanent plots at Brise Fer on Mauritius (MAU-BF) and Mare Longue on Réunion (REU-ML) are used for conservation purposes to monitor the remnants of evergreen wet tropical rainforest in the Mascarenes [START_REF] Cadet | La végétation de l'île de la Réunion: étude phytoécologique et phytosociologique[END_REF][START_REF] Florens | Surviving 370 years of human impact: what remains of tree diversity and structure of the lowland wet forests of oceanic island Mauritius?[END_REF][START_REF] Safford | A survey of the occurrence of native vegetation remnants on Mauritius in 1993[END_REF][START_REF] Strasberg | An assessment of habitat diversity and transformation on La Réunion Island (Mascarene Islands, Indian Ocean) as a basis for identifying broad-scale conservation priorities[END_REF].

Mare Longue (Réunion)

The three 1-ha plots at REU-ML (centred around 21°21'13'' S, 55°44'40'' E) were in reserve (68 hectares), now included in the National park of Réunion (Fig. 1). This site is at the lower reaches of the last continuous corridor of vegetation in the Mascarenes running from 100 m asl to the volcano caldera at 2400 m. This forest corridor is surrounded by secondary vegetation and sugar cane fields at lower elevations. The studied plots are situated between 130 and 340 m asl and receive ca 4000 mm of mean annual rainfall [START_REF] Réchou | Spatio temporal variability of rainfall in a high tropical island: Patterns and large scale drivers in Réunion Island[END_REF]Tab.1). The forest at REU-ML is classified as a lowland tropical rainforest with no deciduous species [START_REF] Cadet | La végétation de l'île de la Réunion: étude phytoécologique et phytosociologique[END_REF] and is little invaded by alien plants . It is set on pahoehoe basaltic lava flow estimated at ca 430 years old (Albert et al., 2020a) and not yet significantly altered. Emergent trees can reach 25 m high and canopy is about 15-20 m, with an average of ca 900 stems >10 cm dbh per hectare and basal area over 70 m².ha -1 (Tab.1).

Brise Fer (Mauritius)

The three 1-ha plots at MAU-BF (centred around 20°22'20'' S, 57°26'10'' E) are near the western edge of Mauritius central plateau, in the Black River Gorges National Park (Fig. 1). This National Park has the largest remnants of native forests on Mauritius [START_REF] Florens | Conservation in Mauritius and Rodrigues: challenges and achievements from two ecologically devastated oceanic islands[END_REF]. The studied plots are located at 560-600 m asl and receive ca 2400 mm of mean annual rainfall (Tab.1). The forest at MAU-BF is classified as a tropical rainforest [START_REF] Lorence | Exotic species invasion into Mauritius wet forest remnants[END_REF]. Trees can reach 18 m high and canopy is about 12-15 m. MAU-BF has 1000-1600 stems >10 cm dbh per hectare but lower basal area than REU-ML (Tab.1). The area has been weeded of alien plant species since 1996 [START_REF] Baider | Control of invasive alien weeds averts imminent plant extinction[END_REF] .

Sampling

Monitoring of permanent plots began in the 1990s (at REU-ML), but we used the data sampled between 2010 and 2014 to avoid temporal bias. We used saplings reaching 1 cm diameter at breast high (dbh, measured at 1.3 m), as this was the threshold for measurements at REU-ML. We calculated an equivalent dbh for plants that had multiple stems.

Growth forms

We categorized the growth form of each woody species as either tree, shrub or climber according to the description in the Flore des Mascareignes (Bosser et al., 1976-ongoing) or newer publication, and expert knowledge of the local floras. Shrubs could reach up to 15 cm dbh, while trees reached a maximum of 94 cm at MAU-BF and 148.8 cm dbh at REU-ML.

Diameter cohorts

We excluded climbers and species without secondary growth (e.g. Monocots, fern trees) from the analyses and defined "young", "intermediate" and "adult" cohorts as stems with dbh of [1; 3[ cm, [3; 10] cm and >10 cm, respectively for trees, and [1; 1.5[ cm, [1.5; 3] cm and >3 cm, respectively for shrubs (Fig. 2). For each site, we extracted the number of young, intermediate and adult cohorts for each species.

Dispersal traits

We categorized the fruit type of each woody species as either fleshy (FF) or dry (DF), using the Flore des Mascareignes (Bosser et al., 1976-ongoing), and expert knowledge of the local floras when information was missing or incomplete. Fruits were classified as fleshy if they had noticeable fleshy pericarps or fleshy appendages when mature. In the Mascarenes, most DF plants rely on abiotic agents for their dispersal.

We used seed length as a proxy for seed size as this trait was known for all species, based on the Flore des Mascareignes (Bosser et al., 1976-onwards) and authors' field measurements. At MAU-BF and REU-ML, seed length of woody species ranged from 0.06 cm (Weinmannia tinctoria, Cunoniaceae) to 5.3 cm (Elaeocarpus integrifolius, Elaeocarpaceae), which represented an order of magnitude of 10 6 in seed mass. We assigned seed length to three size classes (Appendix Fig. A1): small-seeded plants [0.06; 0.6] cm, medium-seeded plants [0.6; 1.5] cm and large-seeded plants [1.5; 5.3] cm (following [START_REF] Corlett | Tropical rainforests and the need for cross-continental comparisons[END_REF][START_REF] Costa | Reduced availability of large seeds constrains Atlantic forest regeneration[END_REF]. Large seeds cannot be swallowed by the extant frugivores in the Mascarenes, but can be dispersed by flying foxes over short distances at MAU-BF (Florens et al., 2017a). Because there were no large-seeded and few medium-seeded DF species, we combined both classes to create a "dispersal trait" factor with four classes: (1) all DF species; and FF species that were (2) small-seeded, (3) medium-seeded, and (4) large-seeded.

Analyses

Dominance-diversity across diameter cohorts

To analyze changes in woody species dominance-diversity across diameter cohorts (i.e. from adults to youngs), we used a rank-abundance distribution approach. Rank-abundance distributions display logtransformed species abundance ranked in declining order of species abundance. This approach, recommended by [START_REF] Matthews | REVIEW: On the species abundance distribution in applied ecology and biodiversity management[END_REF], allows to describe the full distribution of commonness and rarity for the whole woody community and according dispersal traits at each site.

Diversity indices across diameter cohorts

We first used the Shannon diversity index (Ishannon) to synthetically characterize changes in diversity highlighted with rank-log abundance plots. We then assessed changes in phylogenetic diversity using the function pIa (R package 'adiv') that generalizes PD index of [START_REF] Faith | Conservation evaluation and phylogenetic diversity[END_REF] to account for species abundance [START_REF] Pavoine | adiv: An r package to analyse biodiversity in ecology[END_REF]. In this respect, we constructed phylogenetic trees for woody plant communities based on the phylogeny from [START_REF] Zanne | Three keys to the radiation of angiosperms into freezing environments[END_REF] (R package 'brranching').

Phylogenetic decoupling can show whether changes in diversity affect particular lineages. Phylogenetic decoupling was calculated as (pIa=1, adult / pIa=1, young) / (Ishannon, adult / Ishannon, young) and (pIa=1, adult / pIa=1, int.) / (Ishannon, adult / Ishannon, int.) for "adult vs young cohorts" and "adult vs intermediate cohorts" respectively. We assume values >1.1 and <0.9 would reveal stronger and weaker changes in phylogenetic diversity than in Shannon diversity, respectively.

Demographic status assessment

The demographic status of a tree population can be assessed via the ratio of juveniles to adults [START_REF] Condit | Predicting population trends from size distributions: a direct test in a tropical tree community[END_REF], hereafter called recruitment index. We therefore calculated the recruitment index of each woody species: nyoung / (nyoung + nadult) for youngs and nint / (nint + nint) for intermediate cohorts, including rare species, e.g. a single canopy tree without any conspecific individual in the understory. Finally, we estimated, at each site, the distribution of the recruitment index within classes of dispersal traits and growth forms (shrubs vs. trees). Analyses were done in R ver. 4.0.2 (R Core Team).

RESULTS

Total woody species richness was higher at Brise Fer (MAU-BF, 100 species: 65 trees, 35 shrubs) than at Mare Longue (REU-ML, 51 species: 42 trees, 9 shrubs). Woody plants were overwhelmingly fleshyfruited (FF) (91% at MAU-BF; 88% at REU-ML). MAU-BF and REU-ML shared only 20 of the 130 species studied in total, but shared 35 genera (51% of those at MAU-BF and 81% at REU-ML; Tab.A2). Eight other genera present at REU-ML, but absent at MAU-BF do exist on Mauritius. Among the 31 genera present at MAU-BF but absent at REU-ML, 19 were present on Réunion (often in subhumid habitats, e.g. Pleurostylia, Pyrostria, Securinega, Scutia) and 12 only occurred on Mauritius (Tab.A2). MAU-BF showed 1.47 species per genus on average against 1.16 species per genus at REU-ML, as a result of the existence of various radiated plant species at MAU-BF.

Dominance-diversity across diameter cohorts

Across diameter cohorts (from adults, to intermediate cohorts and youngs), woody species richness strongly decreased from 47, to 40 and 35 species at REU-ML, whereas it slightly decreased at MAU-BF from 87, to 84 and 80 species (Fig. 3). We detected a strong increase in the steepness of the dominance-diversity curves of woody species overall at REU-ML. Some species excessively dominated young cohorts, and rare species were more numerous among intermediate and young cohorts despite depauperate richness. On the contrary, we detected no increase in steepness in the dominancediversity curves at MAU-BF, but a tailing-off of rare species (Fig. 3).

Considering dispersal traits (DF plants, FF small-, medium-and large-seeded plants), all groups were negatively impacted at REU-ML, seen by a noticeable decline in species richness across diameter cohorts (Fig. 3). The increase in steepness curves of FF plants was related to one or several species becoming more dominant, e.g. the shrub Chassalia corallioides (Rubiaceae) and the tree Labourdonnaisia calophylloides (Sapotaceae) among medium-seeded and large-seeded plants, respectively. Actually, most ranks among young cohorts at REU-ML showed a strong deficit of abundance in comparison to adults, whatever dispersal traits (Fig. 3).

At MAU-BF, FF small-and medium-seeded plants showed stability and a slight increase of species richness across diameter cohorts, respectively. Both groups had higher juvenile abundance on most ranks (Fig. 3). Conversely, the strong increase in the steepness of dominance-diversity curve of FF large-seeded plants was accompanied by an important decline in species richness (-8 species; Fig. 3). Large-seeded species were diverse in the canopy of MAU-BF with five species exceeding 100 stems (surprisingly, only one species at REU-ML), but their stem proportion strongly decreased across diameter cohorts from 0.37 to 0.21. Hence, the tailing-off of the overall dominance-diversity curve at MAU-BF was mainly due to diversity loss of large-seeded plants.

Dry-fruited plants showed low species richness at both REU-ML and MAU-BF, but they were nevertheless fairly well represented in the canopies of both forests (Fig. 3). While dominance-diversity curves were relatively similar across diameter cohorts at MAU-BF, dry-fruited plants were rare among juveniles at REU-ML. This shows that these plants could not be involved in any replacement in the understory at both sites and, therefore, were not studied further below.

Diversity indices across diameter cohorts of fleshy-fruited plants

Similarly to dominance-diversity curves, Shannon indices of small and medium-seeded plants strongly decreased across diameter cohorts at REU-ML, while they increased at MAU-BF (Fig. 4). The Shannon index of large-seeded plants, which was already low for adults at REU-ML, declined further, and steadily decreased at MAU-BF. Phylogenetic diversity showed broadly similar trends across diameter cohorts for most dispersal traits at both sites. However, phylogenetic decoupling, i.e. values > 1.1 and < 0.9 (Fig. 4), was observed for medium-seeded plants at REU-ML and large-seeded plants at MAU-BF. At MAU-BF, the lower decrease in phylogenetic diversity of large-seeded plants was actually associated with the decline among youngs of several radiated large-seeded species within Eugenia / Syzygium (Myrtaceae), Labourdonnaisia (Sapotaceae) and Diospyros (Ebenaceae; Fig. A2). Conversely, the higher decrease in phylogenetic diversity of medium-seeded plants at REU-ML came from the high dominance of few medium-seeded Rubiaceae among youngs and from species of Ocotea (Lauraceae), Xylopia (Annonaceae) and Tambourissa (Monimiaceae) showing very few individuals, leading to low occurrence of Magnoliidae in the understory.

Demographic status assessment of fleshy-fruited plants

The median recruitment index of small-and medium-seeded FF trees was between three and five times higher at MAU-BF (between 0.7 & 0.8, Fig. 5) than at REU-ML (between 0.15 & 0.26). In other words, half of small-and medium-seeded tree species had at least three times as many adults as juveniles at REU-ML and at least three juveniles for every adult at MAU-BF. For large-seeded FF trees, the median recruitment index was low at both sites (between 0.11 & 0.43), and the situation was paradoxically better at REU-ML than at MAU-BF for youngs. If this result has to be tempered by the low diversity of large-seeded trees at REU-ML, the collapse of the recruitment index observed in this group at MAU-BF was particularly striking.

The median recruitment index of small-and medium-seeded FF shrubs was relatively similar at MAU-BF and REU-ML (between 0.57 & 0.69 and between 0.45 & 0.58 for youngs and intermediate cohorts, respectively, Fig. 5). The median recruitment index of shrubs at REU-ML was thus four times higher than that of trees with the same dispersal traits, despite the low number of shrub species. Conversely, although not low at MAU-BF, the median recruitment index of small-and medium-seeded shrubs was slightly lower than for juvenile trees. The few large-seeded shrubs in this study were only encountered at MAU-BF and showed a particularly low median recruitment index (< 0.09). For details of the results for each woody species, please report to the dataset (available at http://dx.doi.org/10.17632/r3x9hxt98s.1#file-2e500bfd-9b67-4bee-adac-e21e2d55f06c).

DISCUSSION

Comparing three one-hectare plots of Mare Longue on Réunion (REU-ML) and Brise Fer on Mauritius (MAU-BF), famous for being the land of the Dodo, is a unique opportunity to explore the consequences of frugivores extinctions on the maintenance of diversity within tropical forests, as initiated by [START_REF] Terborgh | Tree recruitment in an empty forest[END_REF] in Peru. On Réunion, all large-bodied native frugivores went extinct 200 years ago and the largest extant frugivore, the bulbul Hypsipetes borbonicus, weighs just over 50 g [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF]. On Mauritius, all frugivores above 600 g (including the Dodo), are also extinct, but MAU-BF still hosts a sister species of bulbul, but also a parakeet and more importantly a population of flying foxes (Pteropus niger) which weighs up to 540 g (Fig. 1 & Tab.A1). Our results support three assumed differences between REU-ML considered as treatment (defaunated forest) and MAU-BF as pseudo-control: (1) diversity is better preserved at MAU-BF than at REU-ML; (2) number of juveniles relative to adults is larger at MAU-BF than at REU-ML, indicating stronger forest regeneration at MAU-BF;

(3) large-seeded plants have difficulties in regenerating in the understory at both sites. Our results therefore indicate that the loss of frugivores likely has a severe impact on the regeneration of lowland tropical forests in the Mascarenes. Hence, even protected forests on oceanic islands would be just as much at risk as tropical forests in Neotropics [START_REF] Terborgh | Tree recruitment in an empty forest[END_REF], in Central Africa [START_REF] Effiom | Bushmeat hunting changes regeneration of African rainforests[END_REF][START_REF] Trolliet | How complementary are large frugivores for tree seedling recruitment? A case study in the Congo Basin[END_REF] and in Borneo [START_REF] Harrison | Consequences of defaunation for a tropical tree community[END_REF].

Concern about strong diversity loss at REU-ML, encouraging situation at MAU-BF

Overall woody species richness is about twice as high at MAU-BF than at REU-ML, and if the associated factors are beyond the scope of this study, the maintenance of diversity across diameter cohorts (from adult to young) at MAU-BF compared to REU-ML is noteworthy. Over a third (ca 35%) of woody species do not have even a single sapling in the understory at REU-ML, compared to less than a fifth (ca 18%) at MAU-BF, especially large-seeded FF plants. In accordance, the recruitment is notably higher at MAU-BF than at REU-ML, except for large-seeded FF species, for which it is low at both sites. Although REU-ML forest is considered as the best preserved tropical rainforest in Mascarene islands [START_REF] Strasberg | Diversity, size composition and spatial aggregation among trees on a 1-ha rain forest plot at La Réunion[END_REF], the extent of diversity loss in the understory and the poor recruitment of saplings for most species, questions the long-term sustainability of native forests on Réunion (Albert et al., 2020b). On the contrary, the important regeneration of numerous species at MAU-BF shows that weeding of invasive alien plants since the mid-1990s [START_REF] Lorence | Exotic species invasion into Mauritius wet forest remnants[END_REF][START_REF] Monty | Invasive alien plants elicit reduced production of flowers and fruits in various native forest species on the tropical island of Mauritius (Mascarenes, Indian Ocean)[END_REF], has allowed this today promising recruitment (Fig. A3). Moreover, these observed differences between sites are remarkable as invasive alien mammals as pigs, deer or macaques are present at MAU-BF and absent at REU-ML [START_REF] Florens | Conservation in Mauritius and Rodrigues: challenges and achievements from two ecologically devastated oceanic islands[END_REF]. However, while the situation is encouraging at MAU-BF, our results indicate that when immediate threats are removed, there are still ongoing problems, as most largeseeded plants cannot recover despite conservation actions [START_REF] Krivek | Invasive alien plant control improves foraging habitat quality of a threatened island flying fox[END_REF]. Therefore, it is likely that MAU-BF forest will probably experience a shift in composition in the future with considerably less large-seeded plants present in the canopy, as predicted elsewhere [START_REF] Effiom | Bushmeat hunting changes regeneration of African rainforests[END_REF][START_REF] Harrison | Consequences of defaunation for a tropical tree community[END_REF][START_REF] Terborgh | Tree recruitment in an empty forest[END_REF]. This would be all the more problematic as this loss would affect structural tree species derived from the largest radiations, e.g. within Eugenia, Syzygium or Diospyros genera.

One might argue that we did not study community dynamics, and that caution must be taken about the demographic status of plants, as few juveniles at a given time would not necessarily translate in a population decline [START_REF] Condit | Predicting population trends from size distributions: a direct test in a tropical tree community[END_REF]. Indeed, the non left-skewed diametric distribution might reflect the normal depletion in the understory of a fast-growing species sampled at an unfavourable time window. However, fast-growing species are very rare in the Mascarene flora [START_REF] Cadet | La végétation de l'île de la Réunion: étude phytoécologique et phytosociologique[END_REF]Florens et al., 2017b), and the lack of recruitment of numerous large-seeded plants that are theoretically adapted to deeply shaded understory with strong competition [START_REF] Leishman | The evolutionary ecology of seed size[END_REF] suggests that anomalies in static diameter distribution truly derive from failure in forest regeneration because of strong dispersal limitation. Taking into account all dbh (and not only >1 cm) at MAU-BF shows, for example, that the recruitment of large-seeded plants remains very low, contrary to small-and mediumseeded FF plants which show a (very) large number of saplings with dbh < 1cm (Fig. A4). The difficulty now lies in disentangling the mechanisms underlying this pattern of diversity, particularly since factors other than the availability in frugivores, such as vegetation structure or other post-dispersal limitations, fluctuate between the two sites.

The likely crucial role of flying foxes in woody plant dispersal on Mauritius

Diversity loss at both sites primarily involves FF plants that make the bulk of woody plants at both sites. The disparities observed in the results between REU-ML and MAU-BF may be strongly influenced by a more or less significant alteration of mutualism networks at archipelago scale [START_REF] Schleuning | At a loss for birds: insularity increases asymmetry in seed-dispersal networks[END_REF]. Such a discrepancy in diversity maintenance and sapling recruitment of FF plants, except for large-seeded ones, may be mainly due to the persistence of an important population of flying foxes on Mauritius. Indeed, this bat is today the most important keystone species at MAU-BF due to the extinction of all other large-bodied frugivores. Flying foxes are known to feed upon numerous native species on Mauritius (Florens et al., 2017a;[START_REF] Nyhagen | An investigation into the role of the Mauritian flying fox, Pteropus niger, in forest regeneration[END_REF] and the role of Pteropodidae as effective seed dispersers of small-and medium-seeded plants is well-known throughout the Paleotropics [START_REF] Bollen | Feeding ecology of Pteropus rufus (Pteropodidae) in the littoral forest of Sainte Luce, SE Madagascar[END_REF][START_REF] Mcconkey | Flying foxes cease to function as seed dispersers long before they become rare[END_REF][START_REF] Muscarella | The role of frugivorous bats in tropical forest succession[END_REF][START_REF] Oleksy | Flying foxes create extensive seed shadows and enhance germination success of pioneer plant species in deforested Madagascan landscapes[END_REF][START_REF] Van Toor | Linking colony size with quantitative estimates of ecosystem services of African fruit bats[END_REF]. Flying foxes probably ensure island-scale mobility for numerous small-seeded native species that are sometimes absent from MAU-BF canopies [START_REF] Oleksy | The movement ecology of the Mauritian flying fox (Pteropus niger): a long-term study using solar-powered GSM/GPS tags[END_REF], ultimately allowing maintenance of local diversity. At REU-ML, the largest frugivore is the Réunion bulbul, which occurs at a relative small density since the beginning of the 20th century [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF], and might not ensure effective dispersal of many plant species even though it can theoretically swallow their fruits [START_REF] Mcconkey | Loss of seed dispersal before the loss of seed dispersers[END_REF]. In fact, the good recruitment of FF shrubs that the Réunion bulbul preferentially disperse [START_REF] Albert | Rupture des interactions mutualistes plantes à fruits charnus-vertébrés frugivores, et 10 conséquences sur la régénération des forêts tropicales dans les Mascareignes[END_REF] suggests that it still plays an important role in the understory of old-growth forests. Similarly, the high increase in density of several FF shrubs after weeding at MAU-BF [START_REF] Baider | Control of invasive alien weeds averts imminent plant extinction[END_REF]) might be explained by consumption of their fruits by the Mauritius bulbul. The population levels of this bird nevertheless remain relatively low (< 600 individuals, [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF].

Unlike other FF woody plants, large-seeded plants do not regenerate any better at MAU-BF than they do at REU-ML on average. The weak increase of large-seeded tree recruitment at REU-ML relative to smaller-seeded plants might be counterintuitive because of the extinction of all large native frugivores since 1800, but it might also be an artifact. Indeed, several endangered large-fruited trees might have already died out within permanent plots, e.g. Sideroxylon majus (Sapotaceae), Hernandia mascarense (Hernandiaceae), Scolopia heterophylla (Salicaceae). This would explain why there is so low diversity among adults, and this would also lead to the overestimation of median recruitment of extant species. Isolated large-seeded trees that still survive near the plots, hardly regenerate and might go locally extinct, thus further impoverishing local diversity, and at the same time, increasing beta diversity at larger spatial scale [START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF]. Exceptions exist, as the large-seeded Labourdonnaisia calophylloides that dominates plots at REU-ML and regenerates well, because invasive rats may act as secondary seed dispersers [START_REF] Shiels | Are introduced rats (Rattus rattus) both seed predators and dispersers in Hawaii?[END_REF]. However, while rats could occasionally move large fruits, they are poor effective dispersers because their potential dispersal distance is limited, but more importantly, they do destroy most seeds, and those hoarded are usually clumped inside trees. Another possible contribution to this species regeneration are bird poachers that, for centuries, have made glue from the fruit flesh [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF], reproducing to some extent the behavior of extinct frugivores. Indeed, flesh persistence has a strong negative impact on the regeneration of this species [START_REF] Albert | Rupture des interactions mutualistes plantes à fruits charnus-vertébrés frugivores, et 10 conséquences sur la régénération des forêts tropicales dans les Mascareignes[END_REF], as already demonstrated elsewhere in the Sapotaceae family [START_REF] Levi | Dispersal vacuum in the seedling recruitment of a primate-dispersed Amazonian tree[END_REF].

At MAU-BF, flying foxes feed upon large-seeded plants (Florens et al., 2017a), but fail to recruit in the understory. Despite their relative large body mass allowing them to move large fruits, flying foxes cannot swallow large seeds. This incapacity has two major implications as already demonstrated at MAU-BF for the endangered endemic shrub Syzygium mamillatum (Myrtaceae): (1) seeds are dispersed close from parents trees resulting in increased Janzen-Connell effects on seedlings and saplings; (2) seeds are not cleaned properly, leading to the persistence of potential dormancy-inducing or pathogen-attracting compounds, or increased attraction of introduced mammals [START_REF] Hansen | Seed dispersal and establishment of endangered plants on oceanic islands: The Janzen-Connell model, and the use of ecological analogues[END_REF]. Anyhow, the effectiveness of large-seeded plant dispersal by extinct large-bodied frugivores was probably much higher in the past. Entire lineages that had likely complementary functional roles in frugivory went extinct at both sites: fruit pigeons including the dodo [START_REF] Meehan | Potential disruptions to seed dispersal mutualisms in Tonga, Western Polynesia[END_REF][START_REF] Whittaker | The role of frugivorous bats and birds in the rebuilding of a tropical forest ecosystem, Krakatau, Indonesia[END_REF]Wotton andKelly, 2011, 2012), parrots with the exception of the extant endangered parakeet [START_REF] Sebastián-González | The extent, frequency and ecological functions of food wasting by parrots[END_REF][START_REF] Tella | Conserving the diversity of ecological interactions: The role of two threatened macaw species as legitimate dispersers of "megafaunal" fruits[END_REF], skinks [START_REF] Zuël | Ingestion by an endemic frugivore enhances seed germination of endemic plant species but decreases seedling survival of exotics[END_REF] or giant tortoises [START_REF] Hansen | Seed dispersal and establishment of endangered plants on oceanic islands: The Janzen-Connell model, and the use of ecological analogues[END_REF]. Their absence probably plays a major role in recruitment failure of numerous large-seeded plants at both sites.

Potential post-dispersal factors: seed predation, herbivory and light availability

Recruitment failure in some large-seeded species may also be due to seed predation, such as in the large tree Canarium paniculatum (Burseraceae) of which seeds are often predated by introduced rats at MAU-BF [START_REF] Auchoybur | Threats to the maintenance of Canarium paniculatum (Burseraceae) a Mauritian endemic canopy tree[END_REF]. However, seed predation by invasive mammals may be a secondary cause of recruitment failure as already shown at MAU-BF [START_REF] Hansen | Seed dispersal and establishment of endangered plants on oceanic islands: The Janzen-Connell model, and the use of ecological analogues[END_REF], on Réunion [START_REF] Albert | Rupture des interactions mutualistes plantes à fruits charnus-vertébrés frugivores, et 10 conséquences sur la régénération des forêts tropicales dans les Mascareignes[END_REF] or in New Zealand [START_REF] Wotton | Frugivore loss limits recruitment of large-seeded trees[END_REF]. Furthermore, seedlings and saplings are impacted by invasive herbivores, such as macaques or deers, as already reported by Thompson in the late 19th century [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF]. Large-seeded plants such as Sapotaceae or Diospyros spp. may have specific vegetative traits which would make them more palatable than other plants [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF]. Again, herbivory might be a secondary limitation on large-seeded plants that would primarily have difficulty establishing because of strong dispersal limitation.

One may argue that some species are shade-intolerant and have major difficulties in regenerating under closed canopy where competition for light is strong. This is especially true for most DF trees that include the smaller-seeded woody plants in the Mascarenes, as elsewhere [START_REF] Leishman | The evolutionary ecology of seed size[END_REF]. The fact that saplings of anemochorous long-lived pioneer species such as Agarista salicifolia (Ericaceae), Homalium paniculatum (Salicaceae) or Nuxia verticillata (Stilbaceae) are absent or very rare at REU-ML reinforces this hypothesis [START_REF] Cadet | La végétation de l'île de la Réunion: étude phytoécologique et phytosociologique[END_REF][START_REF] Strasberg | Diversity, size composition and spatial aggregation among trees on a 1-ha rain forest plot at La Réunion[END_REF], and contributes to the observed decline in diversity. This influence of light availability is shown at MAU-BF where DF plants such as Homalium integrifolium or N. verticillata, only regenerate well where canopy cover is the lowest (Fig. A5). Canopy tree species with large, wind-dispersed seeds are absent in the Mascarenes, a common feature of oceanic islands [START_REF] Whittaker | The rebuilding of an isolated rain forest assemblage: how disharmonic is the flora of Krakatau?[END_REF]. Therefore, a gradual replacement of largeseeded FF plants in the understory, as shown in continental tropical forests [START_REF] Harrison | Consequences of defaunation for a tropical tree community[END_REF][START_REF] Terborgh | Tree recruitment in an empty forest[END_REF], is not possible.

Finally, native large-fruited light-demanding trees are usually absent in gaps at REU-ML because they are not dispersed to such areas [START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF] and most native tree species do not have a seedling/sapling bank prior to disturbance. These gaps are often occupied by alien FF smallseeded plants that benefit from high levels of dispersal by small frugivores [START_REF] Albert | Rupture des interactions mutualistes plantes à fruits charnus-vertébrés frugivores, et 10 conséquences sur la régénération des forêts tropicales dans les Mascareignes[END_REF][START_REF] Baret | Effects of canopy gap size on recruitment and invasion of the non-indigenous Rubus alceifolius in lowland tropical rain forest on Réunion[END_REF][START_REF] Mandon-Dalger | Relationships between alien plants and an alien bird species on Reunion Island[END_REF], indicating the importance of seed dispersal in forest dynamics.

RECOMMENDATIONS FOR CONSERVATION

The protected tropical rainforest of REU-ML shows insufficient regeneration of most of its species because of large-bodied frugivore loss, while that of MAU-BF shows an important recruitment of FF plants, indicating their capacity of being effectively dispersed. There is consequently an urgent need to protect flying foxes that face repeated mass culling on Mauritius [START_REF] Florens | Mass-culling of a threatened island flying fox species failed to increase fruit growers' profits and revealed gaps to be addressed for effective conservation[END_REF] and to promote the recolonization of larger forested areas by this species, which has now only a small population on Réunion, after over 200 years of absence. While the ideal would be to rewild ecosystems with large-gape frugivores as already undertaken in several South-West Indian Ocean islands [START_REF] Griffiths | Resurrecting extinct interactions with extant substitutes[END_REF][START_REF] Pedrono | Using a surviving lineage of Madagascar's vanished megafauna for ecological restoration[END_REF], direct sowing actions of large-seeded species should nevertheless be considered to support their recruitment. Ultimately, we provide additional evidence of a bleak future for diverse tropical rainforest if seed dispersal networks are not restored wherever necessary. [1; 1.5[ cm and [1; 3[ cm (blue), respectively. Intermediate cohorts of shrubs and trees have a dbh of [1.5; 3[ cm and [3; 10[ cm (green), respectively. Adult cohorts of shrubs and trees have a dbh>3 cm and >10 cm (red), respectively. Two species with a left-skewed diametric distribution illustrate the assignment. Fig. 3 Rank-abundance diagrams at MAU-BF (black) and REU-ML (grey) for woody species overall and according dispersal traits: dry-fruited plants that were mainly anemochorous at both sites and fleshy-fruited (FF) small-seeded, medium-seeded and large-seeded plants of which mean seed length was [0.06; 0.6[, [0.6; 1.5[ and [1.5; 5.3]

Fig. 2

 2 Fig.2 Definition of diameter cohorts according to growth forms. Young cohorts of shrubs and trees show a diameter at breast height (dbh) of[1; 1.5[ cm and [1; 3[ cm (blue), respectively. Intermediate cohorts of

  cm, respectively. Solid, dashed-dotted and dotted lines respectively displayed adult, intermediate and young cohorts. Note the different scale for the x-axis.

Fig. 4

 4 Fig.4 Changes in diversity indices of fleshy-fruited plant assemblages across diameter cohorts at MAU-BF (black) and REU-ML (grey). Left, Shannon diversity index; Center, phylogenetic diversity index pIa=1 of Pavoine (2020); Right, phylogenetic decoupling, e.g. for "adult vs young" (pIa=1, adult / pIa=1, young) / (Ishannon, adult / Ishannon, young). Values of phylogenetic decoupling > 1 and < 1 indicated stronger and lower changes in phylogenetic diversity than in Shannon diversity across diameter cohorts, respectively. Circles, squares and triangles displayed large-, medium-and small-seeded fleshy-fruited plants, respectively. Note the different scale for the y-axis.

Fig. 5

 5 Fig.5 Violin plots of recruitment index of fleshy-fruited plants calculated as nyoung/(nyoung+nadult) and nint./(nint +nadult) per species at MAU-BF (black) and REU-ML (grey) in relation to dispersal traits and growth forms. A value of 0.5 and 0.25 for young cohorts means that a species has as many youngs as adults and three times as many adults as youngs, respectively. Black dots display the median of observed distributions. Note that areas of violins are scaled proportionally to the number of species.

ACKNOWLEDGEMENTS

We would like to thank all those who participated in the data collection within Mascarene permanent plots and relevant agencies (National Parks and Conservation Service, for Mauritius) for the permits, and reviewers for constructive comments.

DATA ACCESSIBILITY

Data associated with this study will be archived in the Mendeley Data. DOI: http://dx.doi.org/10.17632/r3x9hxt98s.1#file-2e500bfd-9b67-4bee-adac-e21e2d55f06c

FIGURES & TABLE

dispersible by small frugivores. Actually, the Mascarenes initially showed highly asymmetric dispersal networks with relatively few species of frugivores compared to the diversity of plants (see [START_REF] Schleuning | At a loss for birds: insularity increases asymmetry in seed-dispersal networks[END_REF]. Illustrations: giant tortoises, dodo, flying foxes, parrots, fruit pigeons, starling by J. Hume; white-eyes by J.G. Keulemans; skinks by J. Noseworthy; bulbuls by D. Strasberg. For more details, see Appendix Tab.A1.

Tab.1 Descriptors of permanent plots at MAU-BF and REU-ML. Main environmental factors are shown:

elevation ("asl": above sea level) and annual precipitation ("Precip.")