Sébastien Albert

Olivier Flores

Dominique Strasberg

Collapse of dispersal trait diversity across a long-term chronosequence reveals a strong negative impact of frugivore extinctions on forest resilience

Keywords: Anemochory, biodiversity loss, defaunation, island biology, plant-animal mutualistic interactions, seed dispersal disruption, succession, zoochory anno Domini, ADF: anemochorous dry-fruited, asl: above sea level, LFF

Understanding how seed dispersal disruption may alter plant community diversity and dynamics over a large temporal scale remains a challenge. Here, we use a longterm chronosequence to compare changes in the richness and composition of different dispersal trait assemblages in communities established before and after human colonisation in the Mascarene archipelago.

2.

Our study was located on Réunion on the slopes of the Piton de la Fournaise, one of the most active volcanoes worldwide. We analysed 151 vegetation surveys on lava flows dated between 1401-AD and 1956-AD and in tropical rainforests established on older substrata. We defined five classes of substratum age, according to the wellknown chronology of native frugivore defaunation: "old substrata" and [1401, 1665[ when frugivores were abundant and diverse before permanent human settlement; [1665, 1800[ when large-bodied frugivore populations were strongly reduced;

[1800, 1900[ when large-bodied frugivores went extinct and small-bodied frugivores were still abundant; [1900, 1956] decline in the populations of small-bodied frugivores.

https://doi. org/10.1111/1365-2745.13359 Based on dispersal traits, we categorised 146 native woody species as anemochorous, small fleshy-fruited or large fleshy-fruited, i.e. plant of which fruit could not theoretically be dispersed by extant frugivores.

3.

Changes in dispersal trait diversity strongly correlated with the chronology of defaunation. Species-rich communities settled before human colonisation were strongly dominated by fleshy-fruited species. Large fleshy-fruited plants in the oldest communities settled after human colonisation declined markedly and almost disappeared after 1800. The richness of small fleshy-fruited plants decreased less rapidly across the chronosequence, with medium levels on [1665, 1800[ and [1800, 1900[ lava flows and low levels on [1900, 1956] lava flows. Conversely, the richness of anemochorous plants remained unchanged. Communities settled before human colonisation had a similar composition. Fleshy-fruited assemblages showed strong species loss across the chronosequence, while anemochorous assemblages showed strong species turnover, which was probably due to lower dispersal limitation.

Synthesis.

Our results provide the first insights into the tremendous impact that frugivore extinction has on plant colonisation dynamics over 300 years. The dramatic loss of fleshy-fruited plant diversity on historical lava flows highlights the irreplaceable dispersal role played by frugivores, especially large-bodied species. The conservation of plant-animal mutualistic interactions is invaluable and refaunation efforts need to be undertaken in areas where frugivores have been extirpated.

Accepted Manuscript https://doi.org/10.1111/1365-2745.13359 Résumé 1. Comprendre comment la rupture des interactions de frugivorie peut altérer la diversité et la dynamique des communautés végétales sur une grande échelle temporelle demeure un défi. Ici, nous utilisons une chronoséquence de plus de six siècles pour comparer les changements dans la richesse et la composition de différents assemblages basés sur les traits de dispersion au sein de communautés établies avant et après la colonisation humaine de l'archipel des Mascareignes. [1800, 1900[ quand les grands frugivores étaient éteints et les petits frugivores encore abondants; [1900,1956] déclin des populations de petits frugivores. Nous caractérisons 146 espèces ligneuses indigènes selon leurs traits de dispersion : anémochores, à petits fruits charnus ou à gros fruits charnus, i.e. les fruits ne peuvent être théoriquement dispersés par les frugivores relictuels.

3.

Les changements dans la diversité des traits de dispersion sont fortement corrélés à la chronolgie de la défaunation. Les communautés avec une grande richesse spécifique établies avant la colonisation humaine sont largement dominées par les espèces à fruits charnus. Les plantes à gros fruits charnus dans les communautés éta-https://doi.org/10.1111/1365-2745.13359 blies peu après le début de la colonisation humaine déclinent très fortement et disparaissent quasiment après 1800. La richesse des plantes à petits fruits charnus diminue moins vite le long de la chronoséquence, avec des niveaux moyens sur les coulées de lave de [1665, 1800[ et [1800, 1900[, puis

Synthèse.

Nos résultats montrent pour la première fois l'impact considérable de l'extinction des frugivores sur la dynamique de colonisation des plantes sur plus de 300 ans. L'effondrement de la diversité des plantes à fruits charnus sur les coulées de lave historiques souligne le rôle irremplaçable joué par les frugivores, en particulier les espèces de grande taille. La conservation des interactions mutualistes plantesanimaux est primordiale, et des efforts de refaunation doivent être entrepris dans les zones où les frugivores ont disparu.

Introduction

The decline in large vertebrates is a global phenomenon [START_REF] Blackburn | Avian Extinction and Mammalian Introductions on Oceanic Islands[END_REF][START_REF] Dirzo | Defaunation in the Anthropocene[END_REF][START_REF] Hansen | The Forgotten Megafauna[END_REF][START_REF] Steadman | Prehistoric extinctions of Pacific island birds: biodiversity meets zooarchaeology[END_REF][START_REF] Young | Patterns, Causes, and Accepted Manuscript[END_REF]. For decades it has been raising serious concern with regard to the consequences for plant-animal mutualistic interactions [START_REF] Bond | Do mutualisms matter? Assessing the impact of pollinator and disperser disruption on plant extinction[END_REF][START_REF] Emmons | Tropical rain forests: why they have so many species and how we may lose this biodiversity without cutting a single tree[END_REF][START_REF] Janzen | Deflowering of Central America[END_REF]. Notably, frugivore vertebrates (hereafter referred to as "frugivores") play a prominent role and their extirpation alters seed dispersal process [START_REF] Babweteera | Can remnant frugivore species effectively disperse tree seeds in secondary tropical rain forests?[END_REF]2016;[START_REF] Jordano | Differential contribution of frugivores to complex seed dispersal patterns[END_REF][START_REF] Mcconkey | Flying foxes cease to function as seed dispersers long before they become rare[END_REF][START_REF] Meehan | Potential disruptions to seed dispersal mutualisms in Tonga, Western Polynesia[END_REF][START_REF] Riera | Breakage of mutualisms by exotic species: the case of Cneorum tricoccon L. in the Balearic Islands (Western Mediterranean Sea)[END_REF][START_REF] Wotton | Do larger frugivores move seeds further? Body size, seed dispersal distance, and a case study of a large, sedentary pigeon[END_REF][START_REF] Young | Patterns, Causes, and Accepted Manuscript[END_REF]. Ecological and evolutionary consequences at plant population level include altered regeneration [START_REF] Levi | Dispersal vacuum in the seedling recruitment of a primate-dispersed Amazonian tree[END_REF][START_REF] Wotton | Frugivore loss limits recruitment of large-seeded trees[END_REF][START_REF] Zeng | Cascading effects of forested area and isolation on seed dispersal effectiveness of rodents on subtropical islands[END_REF], reduced connectivity (Pérez-Méndez, [START_REF] Pérez-Méndez | Persisting in defaunated landscapes: Reduced plant population connectivity after seed dispersal collapse[END_REF], downsized dispersal traits [START_REF] Galetti | Functional extinction of birds drives rapid evolutionary changes in seed size[END_REF] and altered genetic diversity (Carvalho, Galetti, Colevatti, & Jordano, 2016). Recent studies reported the transforming effects of frugivore loss on plant communities, by comparing seedling recruitment between intact and defaunated sites [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] Vanthomme | Bushmeat Hunting Alters Recruitment of Large-seeded Plant Species in Central Africa[END_REF][START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF][START_REF] Wright | The Plight of Large Animals in Tropical Forests and the Consequences for Plant Regeneration[END_REF]. However, to our knowledge, no study has yet examined how seed dispersal disruption may jeopardise whole tree communities over a period exceeding thirty years. Our research uses a long-term chronosequence of lava flows that spans a frugivore defaunation time range of 300 years on Réunion, an oceanic island that belongs to the Mascarenes. It has been one of the latest tropical archipelagos to be colonised by humans in the 17 th century, which makes it a unique study system, particularly since: (1) the kinetics of frugivore extinction is well documented by subfossil records [START_REF] Hume | A synopsis of the pre-human avifauna of the Mascarene Islands[END_REF]Mourer-https://doi.org/10.1111/1365-2745.13359 Chauvire, Bour, Ribes, & Moutou, 1999) and historical archives [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF][START_REF] Lougnon | Sous le signe de la tortue: voyages anciens à l'Île Bourbon, 1611-1725[END_REF]; and (2) high volcanic activity has produced numerous lava flows, which provide an ideal natural experiment to date and explain patterns of plant community changes (Fig. 1).

Seed dispersal is a key process for ecosystem recovery after disturbance, such as lava flows. Dispersal is the only possible pathway by which plant colonise disturbed areas where the living biomass, including soil seed banks, has been totally destroyed [START_REF] Walker | Primary succession and ecosystem rehabilitation[END_REF]. In relation to seed dispersal strategies, fruit traits are important for understanding plant colonisation mechanisms: plants with large or small fleshy fruits mainly depend on frugivores for their dispersal, whereas most plants with dry fruits depend on abiotic dispersal vectors, e.g. wind [START_REF] Howe | Ecology of seed dispersal[END_REF][START_REF] Van Der Pijl | Principles of Dispersal in Higher Plants (Third Revised and Expanded Edition)[END_REF]. In most tropical rainforests, frugivorous vertebrates are key components of forest succession, since they play a far greater role than wind in dispersing woody plants [START_REF] González-Castro | How does avian seed dispersal shape the structure of early successional tropical forests?[END_REF][START_REF] Ingle | Seed dispersal by wind, birds, and bats between Philippine montane rainforest and successional vegetation[END_REF][START_REF] Whittaker | Plant Recolonization and Vegetation Succession on the Krakatau Islands, Indonesia[END_REF]. Hence, past and present frugivore defaunation probably has severe consequences on ecosystem recovery and forest dynamics after disturbance. This is especially the case on tropical oceanic islands, where a mass extinction of frugivore vertebrates has frequently followed human colonisation [START_REF] Blackburn | Avian Extinction and Mammalian Introductions on Oceanic Islands[END_REF][START_REF] Heinen | Extinctiondriven changes in frugivore communities on oceanic islands[END_REF][START_REF] Steadman | Prehistoric extinctions of Pacific island birds: biodiversity meets zooarchaeology[END_REF]. Therefore, we would expect drastic changes in the diversity and composition of fleshy-fruited plant assemblages on lava flows, before and after human colonisation, compared to anemochorous dry-fruited (ADF) plant assemblages, which remain unaffected.

The fruit of fleshy-fruited plants varies considerably in size, typically ranging from a few millimetres to over 50 cm [START_REF] Guimarães | Seed Dispersal Anachronisms: Rethinking the Fruits Extinct Megafauna Ate[END_REF]. This suggests that different sized frugivores are required for effective dispersal (Hansen & https://doi.org/10.1111/1365-2745.13359 Galetti, 2009;[START_REF] Naniwadekar | Large frugivores matter: insights from network and seed dispersal effectiveness approaches[END_REF]. Critical fruit size thresholds exist among plant communities: for example, the early extinction of megafauna limited the dispersal of many large-seeded plants [START_REF] Federman | Implications of lemuriform extinctions for the Malagasy flora[END_REF][START_REF] Guimarães | Seed Dispersal Anachronisms: Rethinking the Fruits Extinct Megafauna Ate[END_REF]. Fruit size of ca 13 mm is documented as a critical threshold, both on the mainland [START_REF] Dowsett-Lemaire | Fruit choice and seed dissemination by birds and mammals in the evergreen forests of upland Malawi[END_REF][START_REF] Galetti | Functional extinction of birds drives rapid evolutionary changes in seed size[END_REF] and on oceanic islands [START_REF] Mcconkey | Low redundancy in seed dispersal within an Accepted Manuscript[END_REF]. As defaunation primarily extirpates large vertebrates [START_REF] Dirzo | Defaunation in the Anthropocene[END_REF], most oceanic islands are strongly depauperate in largebodied frugivores [START_REF] Mcconkey | Low redundancy in seed dispersal within an Accepted Manuscript[END_REF][START_REF] Schleuning | At a loss for birds: insularity increases asymmetry in seed-dispersal networks[END_REF]. In some places, including Réunion, they have been totally extirpated and therefore absent for several centuries [START_REF] Heinen | Extinctiondriven changes in frugivore communities on oceanic islands[END_REF]. In addition to the contrast between ADF vs fleshy-fruited species, we would also expect to see dramatic changes in diversity and composition within fleshy-fruited species given that large fleshy-fruited (LFF) plant assemblages are likely to have been impacted for a longer period (since human colonisation) than small fleshy-fruited (SFF) plant assemblages.

In this paper, we use a comparative approach to investigate whether dispersal failure caused by defaunation leads to severe diversity loss in plant communities studied across a long-term chronosequence of lava flows. We focus on changes in the richness and composition of ADF, SFF and LFF plant assemblages settled on substrata belonging to five age classes, which we consider as a proxy of dispersal agent availability (Fig. 1). We compare plant communities settled before permanent human settlement in 1665-AD, when frugivores were abundant and diverse, to more recent communities established after the extinction of large-bodied frugivores and the decline in the population of small-bodied frugivores began. First, we test whether changes in richness relate to the availability of frugivores. We expect the richness of LFF and SFF plant to be high on ancient lava flows that were colonised when frugivores were still https://doi.org/10.1111/1365-2745.13359 abundant. In theory, LFF plant richness should decrease faster across the chronosequence (from oldest to youngest lava flows) than SFF plant richness because largebodied frugivores became extinct sooner than small frugivores. On the contrary, dispersal limitation for ADF plants is low, therefore, variation in ADF plant richness should be independent of the defaunation history. It may even increase across the chronosequence because early primary successional stages are often dominated by ADF species [START_REF] Walker | Primary succession and ecosystem rehabilitation[END_REF]. Second, we analyse β-diversity and its two components, i.e. nestedness and turnover, which respectively result from the loss (or gain) of taxa and from the replacement of taxa [START_REF] Baselga | Partitioning the turnover and nestedness components of beta diversity[END_REF], to detect variation in plant community composition across the chronosequence. We expect strong dispersal limitation in fleshy-fruited species to lead to a significant increase of nestedness across the chronosequence, especially for LFF plants. On the contrary, if ADF species suffer no major dispersal limitation and can all reach lava flows, we should detect higher species replacement or species gain on recent lava flows [START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF].

Material and methods

Study area

Réunion (2512 km²) is the largest island in the Mascarene archipelago. It is located in the biodiversity hotspot that characterises the South-West Indian Ocean islands [START_REF] Mittermeier | Hotspots Revisited. Earth's Biologically Richest and Most Endangered Terrestrial Ecoregions[END_REF]) (Fig. 2). The study site is located in the South-East of Réunion on the slopes of the Piton de la Fournaise. This shield volcano is one of the most active in the world. More than 238 eruptions have been recorded since the second half of the 17th century [START_REF] Morandi | Pre-historic (< 5 kiloyear) Explosive Activity at Piton de la Fournaise Volcano[END_REF]. For 5000 years, most lava flows have occurred in the most recent caldera of the Piton de la Fournaise. Eruptions have also occurred https://doi.org/10.1111/1365-2745.13359 outside the caldera along three rift zones [START_REF] Morandi | Pre-historic (< 5 kiloyear) Explosive Activity at Piton de la Fournaise Volcano[END_REF]. The Piton de la Fournaise area has a humid tropical climate throughout the year below 750 m asl, i.e. annual mean temperature ranges from 19 to 25°C and precipitation in the driest month ranges from 75 to 350 mm, with large inter-and intra-annual variations [START_REF] Jumeaux | Atlas climatique de la Réunion. Sainte-Clotilde (Réunion)[END_REF]. This area shelters almost all that remains of the tropical rainforest on Réunion and represents the last sanctuary of its kind in the Mascarene archipelago [START_REF] Strasberg | An assessment of habitat diversity and transformation on La Réunion Accepted Manuscript[END_REF].

Data collecting

Vegetation sampling and frugivore guild characterisation

We used a historical dataset sampled in the 1970s by the French botanist [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF]. It is invaluable because it includes forests that have since been destroyed (Appendix Fig. S1). We retained 151 vegetation plots sampled in pristine or relatively undisturbed habitats, where alien plants were rare or had little overall cover (Fig. 2).

Twenty-eight plots were settled on well-developed soils as reported by [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF] in his technical notes. We did not have proper dating for these substrata, but based on the very likely slow pedogenesis, we assumed that they were at least 1000 years old [START_REF] Meunier | The output and bio-cycling of Si in a tropical rain forest developed on young basalt flows (La Reunion Island)[END_REF]. One-hundred and twenty-three plots were located on 27 lava flows dated between 1401 AD and 1956 AD: (i) three lava flows were dated by 14 C [START_REF] Morandi | Pre-historic (< 5 kiloyear) Explosive Activity at Piton de la Fournaise Volcano[END_REF], (ii) sixteen lava flows were known from historical sources [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF][START_REF] Stieltjes | Coulées historiques de Piton de La Fournaise [Carte géologique 1/25.000e[END_REF] and (iii) eight lava flows were dated using an indirect approach based on the size distribution of long-lived ADF pioneer trees found on lava flows [START_REF] Strasberg | Evaluation des aléas "coulée de lave" et "gaz volcaniques[END_REF]. Because 14 C dating could not be used https://doi.org/10.1111/ 1365-2745.13359 in several areas such as the caldera that, however, hosted isolated old remnant forests, the tree size method allowed to expand the chronosequence and to add some heterogeneity in the sampled ecological situations.

We assigned the 151 vegetation plots to five substrata age classes, defined according to the availability of dispersal agents, as documented by subfossils records [START_REF] Hume | A synopsis of the pre-human avifauna of the Mascarene Islands[END_REF][START_REF] Mourer-Chauvire | The avifauna of Re-Accepted Manuscript[END_REF] and historical archives [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF]) (Fig. 1). Namely, the first class was "old substrata" on which plant communities assembled when frugivores were numerous and where pedological conditions were supposed to be more favorable. This class therefore included our reference control plots (n = 28 plots); the second class was [1401, 1665[, when frugivores were still numerous and diverse (n = 36 plots); the third class was [1665, 1800[, when largebodied frugivore populations were severely reduced, while small-bodied frugivore populations were less impacted (n = 23 plots); the fourth class was [1800, 1900[, when large-bodied frugivores were extinct and small-bodied frugivores, especially the native bulbul (Hypsipetes borbonicus) and the hoopoe starling (Fregilupus varius), were still abundant (n = 37 plots); the fifth and most recent class was [1900,1956], when populations of extant small-bodied frugivores further declined to extremely low densities (n = 27 plots) (Fig. 1).

Spatial distribution of study plots was not random given the volcanic eruption pattern, as well as difficulties in accessing some remote locations (Fig. 2 & Appendix Fig. S2). All plots on "old substrata" were outside the caldera; plots on [1401, 1665[ lava flows were all located in the southern half of the Fournaise area, whereas those on [1665, 1800[ lava flows showed a wider distribution; plots located on [1800, 1900[ and [1900, 1956] lava flows were respectively located in south-central Fournaise area and in the caldera. In the latter area, old growth native forests were already rare https://doi.org/10.1111/ 1365-2745.13359 and very fragmented in 1975[START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF] also used different sampling areas ranging from 500 to 1000 m² on pre-defaunation lava flows and from 100 to 1000 m² on historical lava flows (Fig. 2).

For each plot, [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF] recorded the cover of tree, shrub and herbaceous strata and maximum canopy height. For each stratum, he recorded the list of tracheophyta at species level, including seedlings with the Braun-Blanquet semi-quantitative method. These data allowed us to quantify species richness, composition and vegetation structure. We focused on woody species, i.e. woody angiosperms and arborescent ferns because they were taxonomically well known in the 1970s, unlike herbaceous species. In addition, they contribute significantly to the structure of plant communities from the early stages of vegetation development on [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF].

Dispersal traits of woody species

Woody species were assigned to three functional groups based on dispersal traits.

Fleshy-fruited plants that were originally dispersed by a rich native frugivore fauna (Fig. 1 & Appendix Tab. S1) were assigned to two dispersal trait groups based on fruit size [START_REF] Izhaki | Fruit Traits and Fruit Removal in East Mediterranea[END_REF][START_REF] Mcconkey | Low redundancy in seed dispersal within an Accepted Manuscript[END_REF]: small fleshy-fruited (SFF) and large fleshy-fruited (LFF) species. These two groups were defined according to the mean value for fruit (or arillate seed) diameter, i.e. above or below 13 mm, which is the maximum gape width of Hypsipetes borbonicus, the largest extant frugivore on Réunion (body mass = 55 g) [START_REF] Cheke | An ecological history of the Mascarene Islands, with particular reference to extinctions and introductions of land vertebrates[END_REF]. Plants that use wind dispersal mechanisms and do not depend on animal dispersal were categorised as anemochorous dry-fruited (ADF). Dry-fruited plant species whose dispersal strategy remained unclear were included in the overall analysis of diversity, but not assigned to a specific dispersal trait group. These species are not common in lowland forests on Réunion. https://doi.org/10.1111/1365-2745.13359

Statistical analyses

We constructed a presence-absence matrix for each plot and each dispersal trait group based on species presence at any growth stage, i.e. in any vegetation stratum. Species could thus be present at seedling or sapling stage only.

Plant species richness across the chronosequence

We modeled plant species richness across the chronosequence using a multiple regression approach to take into account the relative influence of plot area, elevation, precipitation of driest month and isolation that substantially varied across study plots (Appendix Fig. S2). To assess plot isolation, we used a habitat map to construct a binary raster of native closed-canopy forests at 100 m resolution and calculated the proportion of native forests in the 100 hectares surrounding each plot. The five variables used for analyses showed moderate collinearity with maximum variance inflation factors < 4.

The relationship between count data responses (species richness of all woody plants, ADF, SFF, LFF and not-assigned plants) and explanatory variables including the five variables and four interaction terms, i.e. age of lava flow x (isolation + elevation + precipitation + area), were analysed using generalized linear models (GLMs) with a Poisson distribution [START_REF] O'hara | Do not log-transform count data[END_REF]. Using a type-II analysis of deviance, we dropped the non-significant terms for each GLM. We used the Moran eigenvector filtering function (R package "spdep") to account for possible effects of spatial autocorrelation on parameter estimates [START_REF] Bivand | Package 'spdep[END_REF]. Pseudo-R ² of GLMs were calculated by the relationship "1 -residual deviance/null deviance". We finally estimated the standardised values of species richness (Sst) across the chronosequence by fixing https://doi.org/10.1111/1365-2745.13359 the four other variables to their mean value (sampling area = 591 m², elevation = 354 m asl, precipitation of driest month = 226 mm, isolation = 46 % of native forests).

Community composition across the chronosequence

We performed pairwise dissimilarity analyses between vegetation plots and used the beta.pair function (R package betapart) to decompose total β-diversity (measured by Sorensen dissimilarity) into its two components, nestedness and turnover (measured by Simpson dissimilarity) [START_REF] Baselga | betapart : an R package for the study of beta diversity: Betapart package[END_REF]. We then constructed six distance matrices by calculating the absolute values of the difference between each pair of plots for age of lava flow, sampling area, elevation, precipitation of driest month and isolation. Because age of lava flow was an ordinal variable, we converted the five age classes to numerical values from 1 (old substrata) to 5 ([1900; 1956]). We also calculated the Euclidean geographical distance for each plot pair. To take into account the influence of sampling area, elevation, precipitation of driest month, isolation and Euclidean distance on dissimilarities across the chronosequence, we could not use a multiple regression as we did for species richness because of obvious violation of the assumption on independence [START_REF] Anderson | Navigating the multiple meanings of β diversity: a roadmap for the practicing ecologist: Roadmap for beta diversity[END_REF]. Consequently, we performed partial Mantel tests with 1000 permutations (R package ecodist) to examine the influence of age of lava flow on each dissimilarity while controlling for other variables, and vice versa [START_REF] Goslee | The ecodist package for dissimilarity-based analysis of ecological data[END_REF].

Results

Species richness across the chronosequence

The age of lava flow was by far the best predictor of species richness for woody plants overall (n = 151, p = 1.7e-27; pseudo-R² = 0,87), small fleshy-fruited (SFF) plants (p = https://doi.org/10.1111/1365-2745.13359 4.7e-27; pseudo-R² = 0,84) and large fleshy-fruited LFF plants (p = 6.2e-41; pseudo-R² = 0,87) (Appendix Tab. S2 & Fig. S3). Conversely, elevation was the best predictor for anemochorous dry-fruited (ADF) plant richness (p = 3.7e-16; pseudo-R² = 0,70).

Isolation also had a strong influence on the richness of the four groups, but only on post-defaunation lava flows for all woody plants, SFF and LFF plants.

The estimated standardised species richness (Sst) for woody plants overall decreased considerably across the chronosequence, i.e. with decreasing substrata age (Fig. 3): Sst was similar on pre-defaunation substrata, i.e. 32.9 and 35.4 in "old substrata" and [1401; 1665] respectively, then declined markedly to 17.6 in [1665; 1800[ and [1800; 1900[, and dropped down to 11.5 species in [1900; 1956]. This general downward trend across the chronosequence concealed very different changes that were evidenced when dispersal trait groups were considered separately (Fig. 3).

Sst for ADF plants ranged from 4.8 to 6.2 species and showed no significant trend across the chronosequence. Changes in species richness of fleshy-fruited plants drove the general trend of woody plant species. Sst for SFF plants was similar on predefaunation substrata, i.e. 20.9 and 22.5 in "old substrata" and [1401;1665], respectively, then strongly decreased in [1665;1800[ to 10.8 species. It was similar in [1800;;1900[ with 11.3 species anddropped down to 5.4 species in [1900;1956]. Sst for LFF plants followed a similar pattern to SFF plants, although their decline was more abrupt:

LFF plant richness was similar on pre-defaunation substrata, i.e. 6. 1 and 7.3 in "old substrata" and [1401; 1665], respectively. It showed a strong decrease to 1.1 species in [1665; 1800[, fell to 0.4 species in [1800; 1900[ and 0 in [1900; 1956]. The independently standardized estimated richness across dispersal trait groups overall summed to the overall richness of woody species with a low average relative difference of 0.5%, which suggested a good accuracy of the separate estimates (Appendix Accepted Manuscript https://doi.org/10.1111/1365-2745.13359 Fig. S4). Lastly, we also performed statistical analyses without taking into account lava flows whose dating was based on pioneer trees development and obtained similar conclusions (Appendix Fig. S5).

Community composition across the chronosequence

The total β-diversity measured by Sorensen dissimilarity with control plots as reference ("old substrata") showed a highly significant increase across the chronosequence for woody plants overall (from 0.35 to 0.90), as well as within each dispersal trait group (ADF, from 0.35 to 0.78; SFF, from 0.35 to 0.89; LFF, from 0.33 to 0.62) when other variables were controlled (partial Mantel test, p< 0.001) (Fig. 4 & Appendix Fig. S6).

For woody plants overall, turnover and nestedness increased significantly across the chronosequence (partial Mantel tests, p<0.001 & p<0.001). However, turnover was the main driver of composition changes, in relation with fleshy-fruited (both LFF and SFF) and ADF species dominating assemblages on old and recent lava flows, respectively (Fig. 3). Within dispersal trait groups, the general increase in Sorensen dissimilarity concealed very different situations evidenced when the two components of βdiversity were considered separately.

For ADF assemblages, the turnover strongly increased across the chronosequence from 0.26 to 0.64 (partial Mantel test, p<0.001), whereas nestedness was low and stable (partial Mantel test, NS) and mostly influenced by elevation and isolation (Fig. 4 & Appendix Fig. S6). For SFF assemblages, the turnover was mostly influenced by elevation and showed a weak increase from 0.26 to 0.32 across the chronosequence (partial Mantel test, p = 0.04), whereas nestedness strongly increased from 0.09 to 0.57 (partial Mantel test, p<0.001). For LFF assemblages, dissimilarity analyses involve a shorter chronosequence, given that LFF plants are totally absent on https://doi. org/10.1111/1365-2745.13359 [1900; 1956] lava flows, and showed similar trends as SFF assemblages. The turnover was mostly influenced by elevation and remained stable between 0.19 and 0.29 (partial Mantel test, NS), whereas nestedness strongly increased from 0.12 to 0.37 (partial Mantel test, p<0.001). Overall, species replacement was the main phenomenon driving species composition of ADF assemblages across the chronosequence, while SFF and LFF plant assemblages that remained on recent lava flows constituted a subset of the communities found on "old substrata".

Discussion

Frugivory disruption as a major cause of fleshy-fruited plant diversity loss

Plant dispersal trait diversity is strongly related to the availability of dispersal agents across a long-term chronosequence on the Piton de la Fournaise volcano. This suggests that the extinction of large-bodied frugivores has severely limited dispersal for many plant species since the early 18 th century, i.e. less than a century after the first human settlements. It appears to be a major cause of the fleshy-fruited plant diversity loss on historical lava flows. The negative impact of frugivore defaunation on tree recruitment has already been shown at community level [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] Vanthomme | Bushmeat Hunting Alters Recruitment of Large-seeded Plant Species in Central Africa[END_REF][START_REF] Wright | The Plight of Large Animals in Tropical Forests and the Consequences for Plant Regeneration[END_REF]. Nevertheless, to our knowledge, our study is the first to reveal such a dramatic collapse of dispersal trait diversity of the temporal scale of several centuries. Our research was made possible because frugivore defaunation in the Mascarene archipelago has been well documented [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF][START_REF] Lougnon | Sous le signe de la tortue: voyages anciens à l'Île Bourbon, 1611-1725[END_REF] and because the high volcanic activity on Réunion provides numerous lava flows that constitute a natural experiment acting as a "time machine". https://doi.org/10.1111/1365-2745.13359

Forests on old substrata and lava flows that occurred before permanent human settlements are widely 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]. They include LFF plants, that are usually big trees [START_REF] Diaz | The plant traits that drive ecosystems: evidence from three continents[END_REF][START_REF] Thompson | Do big plants have big seeds?[END_REF] with an important structural role in native forests (Appendix Fig. S7 & Fig. S8). LFF plants are far less present on lava flows that occurred while the large-bodied frugivores were strongly decreasing in number.

Hence, it is likely that effective dispersal at ecosystem level was disrupted before the dispersers of most LFF species actually went extinct, as shown elsewhere [START_REF] Mcconkey | Loss of seed dispersal before the loss of seed dispersers[END_REF]. LFF plants, including seedlings, are very scarce on lava flows that occurred after the extinction of all large-bodied frugivores in 1800. This shows that the latter play an extremely important role because they can swallow large fruits and are more likely to deposit seeds away from the parent tree [START_REF] Naniwadekar | Large frugivores matter: insights from network and seed dispersal effectiveness approaches[END_REF][START_REF] Wotton | Do larger frugivores move seeds further? Body size, seed dispersal distance, and a case study of a large, sedentary pigeon[END_REF]. This also suggests that potential dispersal alternatives are very unreliable, e.g. secondary dispersal by introduced vertebrates or cyclone-mediated seed dispersal [START_REF] Babweteera | Can remnant frugivore species effectively disperse tree seeds in secondary tropical rain forests?[END_REF][START_REF] Federman | Implications of lemuriform extinctions for the Malagasy flora[END_REF]. The richness of SFF plants also decreases on lava flows that occurred after 1665, despite the abundance of several important frugivores for this species group, the native bulbul and the hoopoe starling. Large-bodied frugivores do not just eat LFF plants and also play an important role in SFF plant dispersal [START_REF] Bollen | Feeding Ecology of Pteropus rufus (Pteropodidae) in the Littoral Forest of Sainte Luce, SE Madagascar[END_REF][START_REF] Florens | Disproportionately large ecological role of a recently mass-culled flying fox in native forests of an oceanic island[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]. In fact, their extinction has probably led to a decrease in the dispersal efficiency of most fleshy-fruited plants [START_REF] Babweteera | Can remnant frugivore species effectively disperse tree seeds in secondary tropical rain forests?[END_REF]Carvalho et al., 2016) and to the inability of many SFF plants to reach isolated post-defaunation lava flows [START_REF] Jordano | Differential contribution of frugivores to complex seed dispersal patterns[END_REF][START_REF] Wotton | Do larger frugivores move seeds further? Body size, seed dispersal distance, and a case study of a large, sedentary pigeon[END_REF]. We show that SFF richness eventually collapses on lava flows from the 20 th century, as already suggested [START_REF] Chevennement | La colonisation végétale d'un champ de lave de la Réunion[END_REF][START_REF] Strasberg | Dynamique des forêts tropicales de l'île de La Réunion: processus d'invasions et de régénération sur les coulées volcaniques[END_REF][START_REF] Thébaud | Plant dispersal in fragmented landscapes: A field study of woody colonization in rainforest remnants of the Mascarene Archipelago[END_REF] Accepted Manuscript https://doi.org/10.1111/1365-2745.13359 [START_REF] Thébaud | Plant dispersal in fragmented landscapes: A field study of woody colonization in rainforest remnants of the Mascarene Archipelago[END_REF]. We also reveal that the few fleshy-fruited species that colonise these areas are usually present on older substrata. Conversely, ADF plant richness remains unchanged across the chronosequence, despite strong species replacement. This finding suggests that successional changes occur within ADF assemblages across the chronosequence, but only rarely in fleshy-fruited assemblages [START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF].

Potential post-dispersal limitations

The pattern that we detected could partly be explained by post-dispersal factors, such as environmental filtering, e.g. light requirements, depth soil, or biotic interactions, e.g. seed predation [START_REF] Wang | Closing the seed dispersal loop[END_REF].

We have consistent evidence that the total absence of many fleshy-fruited plant species, even at early life stages, on lava flows dating from [1665; 1800[ and [1800; 1900[ is not a consequence of environmental filtering. Although shade tolerance or light requirements were not considered in our analyses, it is likely that some SFF understorey shrubs have difficulty germinating in open canopy vegetation, especially on the most recent lava flows. However, the proportions of LFF and SFF plants do not depend on canopy cover (Appendix Fig. S9). Many vegetation plots on historical lava flows show a well-developed canopy, but totally lack a LFF tree that is very common on pre-defaunation lava flows (Labourdonnaisia calophylloides, Sapotaceae, Appendix Fig. S10). Moreover, the presence of long-lived pioneer trees across the chronosequence provides relevant insights: a SFF pioneer tree (Sideroxylon borbonicum, Sapotaceae) is common on lava flows that occurred when small frugivores were abundant, but is much less prevalent on [1900; 1956] lava flows, despite favorable light conditions. On the contrary, an ADF pioneer tree (Agarista salicifolia, Ericaceae) is present across the entire chronosequence (Appendix Fig. S10). Finally, soil type is https://doi.org/10.1111/1365-2745.13359 another factor that might limit recruitment, but our analyses show a standardised richness as high on old substrata as on [1401, 1665[ lava flows and a low β-diversity between these categories. Hence, most big trees found on well-developed soils are able to establish and develop on [1401, 1665[ lava flows that usually display a very thin soil on continuous rock [START_REF] Meunier | The output and bio-cycling of Si in a tropical rain forest developed on young basalt flows (La Reunion Island)[END_REF] (Appendix Fig. S11).

Seed predation may also be an important mechanism that limits plant recruitment [START_REF] Forget | Seed fate: predation, dispersal, and seedling establishment[END_REF][START_REF] Hubbell | Seed Predation and the Coexistence of Tree Species in Tropical Forests[END_REF][START_REF] Janzen | Herbivores and the number of tree species in tropical forests[END_REF]. Rats are usually the main seed predators on oceanic islands [START_REF] Shiels | Barriers to seed and seedling survival of oncecommon Hawaiian palms: the role of invasive rats and ungulates[END_REF] and probably reached high densities in the late 17 th century, after their introduction on Réunion [START_REF] Cheke | Lost land of the dodo: an ecological history of Mauritius, Réunion & Rodrigues[END_REF]. Since then, rat populations may have remained at high levels. It is however unlikely that seed predation by rats could explain the temporal shift observed between LFF and SFF plants across the chronosequence. A few LFF plants are common at all growth stages in extant mature rainforest on pre-defaunation lava flows, despite the abundance of rats [START_REF] Strasberg | Diversity, size composition and spatial aggregation among trees on a 1-ha rain forest plot at La Réunion[END_REF], whereas they are totally absent from post-1800 lava flows. Hence, seed predation may play a secondary role in limiting recruitment, as shown by [START_REF] Wotton | Frugivore loss limits recruitment of large-seeded trees[END_REF] in New Zealand.

Primary succession disruption and implications for conservation

Our analyses show that, in less than five centuries, primary succession on lava flows has led to plant assemblages of woody plants similar in richness and composition to those found on old substrata. The fact that LFF plants probably lost their dispersal capacity after 1800 shows that they were already present on historical lava flows in 1800. Hence, well under 200 years might have been enough for the establishment of most shade-tolerant tree species. This temporal scale is consistent with Thornton https://doi.org/10.1111/ 1365-2745.13359 (1997), who documents the assembly of the Krakatau Islands ecosystem after its destruction in 1886, i.e. three years after the islands' sterilisation: despite the sea barrier (12 km from the closest source and 40 km from mainland Java and Sumatra), six zoochorous plants were recorded as soon as 1896, 23 in 1905, 35 in 1920, 68 in 1934 and 124 species in 1992. In the case of the Krakatau Islands, [START_REF] Whittaker | The Role of Frugivorous Bats and Birds in the Rebuilding of a Tropical Forest Ecosystem, Krakatau, Indonesia[END_REF] emphasise the crucial role played by fruit pigeons able to transport large-seeded plants that became established in the decades following the initial disturbance.

The absence of many native species on historical lava flows has probably had severe consequences on succession processes. For instance, the absence of LFF tree species may have caused competition release [START_REF] Hurtt | The consequences of recruitment limitation: reconciling chance, history and competitive differences between plants[END_REF], possibly benefiting alien plants with low dispersal limitation. Recent surveys on post-1800 lava flows reveal the presence of the same native plant communities as those surveyed by [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF], as well as the generally low or absent regeneration of native species [START_REF] Albert | Depauperate seed rain but effective recruitment after reinstated dispersal evidence strong impacts of frugivore extinctions on native forest regeneration after natural disturbance[END_REF], suggesting arrested succession [START_REF] Cohen | Releasing Rain Forest Succession: A Case Study in the Dicranopteris linearis Fernlands of Sri Lanka[END_REF]. Together, these findings demonstrate that plant communities of different ages do not follow the same trajectory and that the vegetation stages originally described by [START_REF] Cadet | La Végétation de l'Ile de La Réunion: Etude Phytoécologique et Phytosociologique[END_REF] do not represent successional seres [START_REF] Walker | The use of chronosequences in studies of ecological succession and soil development[END_REF]. Therefore, it is unlikely that the structure and diversity of native plant communities on historical lava flows will ever be as rich as the tropical rainforests on pre-defaunation substrata.

The persistently high LFF plant richness on pre-defaunation lava flows supports the fact that, despite the loss of all large-bodied frugivores since 1800, plant communities can display a high level of resilience in situ, but a very low level of resilience by migration [START_REF] Grubb | Some generalizing ideas about colonization and succession in green plants and fungi[END_REF]. The plant communities on pre-defaunation lava flows are https://doi.org/10.1111/1365-2745.13359 among the most important and well-preserved testimony of Mascarenes' lowland ecosystems [START_REF] Strasberg | Diversity, size composition and spatial aggregation among trees on a 1-ha rain forest plot at La Réunion[END_REF], and their protection is considered crucial [START_REF] Mittermeier | Hotspots Revisited. Earth's Biologically Richest and Most Endangered Terrestrial Ecoregions[END_REF]. Our results confirm that their loss would be particularly dramatic because these ecosystems have lost their resilience. Although most of the remaining lowland ecosystems are protected as part of the Réunion National Park, the high frequency of lava flows in this area of Réunion may lead to their gradual destruction. In 2007, for example, the last fragment of tropical rainforest at sea level was destroyed (Appendix Fig. S1). Moreover, the apparent maintenance of diversity on pre-defaunation lava flows is probably due to the fact that our study uses presence/absence data and does not take into account a potential failure of local recruitment for many species of the canopy. Several shade-tolerant long-lived trees hardly regenerate [START_REF] Strasberg | Diversity, size composition and spatial aggregation among trees on a 1-ha rain forest plot at La Réunion[END_REF] and long-lived pioneer trees are often unable to reach gaps nearby, which makes them particularly sensitive to local extinction in the near future [START_REF] Wandrag | Seed dispersal increases local species richness and reduces spatial turnover of tropical tree seedlings[END_REF].

Today, frugivory disruption combined with alien plant invasion (see [START_REF] Potgieter | Casuarina Invasion Alters Primary Succession on Lava Flows on La Réunion Island[END_REF] leads to the development of novel ecosystems with extremely low taxonomic and functional diversity. Given the dramatic loss of native fleshy-fruited plant diversity on recent lava flows, conservation strategies should include the lack of native vegetation recovery as a matter of urgency. Restoring the seed dispersal function will be necessary in defaunated ecosystems [START_REF] Correia | Refaunation and the reinstatement of the seed-dispersal function in Gorongosa National Park: Refaunation and Seed-Dispersal Function[END_REF], as has already been done on several islands in the Indo-Pacific region [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]. The fact that there will be no altitudinal escape for most fleshy-fruited plants in the context of climate change makes the situation even more urgent [START_REF] Mcconkey | Seed dispersal in changing landscapes[END_REF].

Accepted Manuscript https://doi.org/10.1111/1365-2745.13359

Conclusion

Defaunation can cause negative impacts on ecosystem functioning comparable with those from major drivers of global change [START_REF] Young | Patterns, Causes, and Accepted Manuscript[END_REF]. However, its long term impact on dynamics of very long-lived trees is not sufficiently documented. Our study brings a first evidence of frugivore defaunation effect at community level over a temporal scale of 300 years. The studied chronosequence of lava flows acting as a "time machine" and harboring plant communities settled before and after human colonisation provides a natural experiment on one of the last tropical islands colonised by humans. To our knowledge, this is the first time that such a collapse of dispersal trait diversity has been shown. It strongly argues in favour of restoration programs targeting the re-establishment of seed dispersal interactions by promoting native frugivore populations and rewilding projects focusing on large vertebrates.

2.

  Notre étude est située à La Réunion sur les pentes du Piton de la Fournaise, l'un des volcans les plus actifs au monde. Nous analysons 151 relevés de végétation sur des coulées de lave datées entre 1401-AD et 1956-AD, et en forêt tropicale humide sur des substrats plus anciens. Nous définissons cinq classes d'âge de substrat, selon la chronologie bien connue de la défaunation des frugivores indigènes : "vieux subtrats" et [1401, 1665[ quand les frugivores étaient abondants et diversifiés avant la colonisation humaine permanente; [1665, 1800[ quand les populations des grands frugivores étaient fortement réduites;

  faibles sur les coulées de lave du 20 ème siècle. En revanche, la richesse des plantes anémochores ne change pas de manière significative. Les communautés établies avant la colonisation humaine ont une composition comparable. Les assemblages de plantes à fruits charnus montrent une forte perte d'espèces le long de la chronoséquence tandis que les assemblages anemochores montrent un fort remplacement, probablement en raison d'une faible barrière à la dispersion.

Figures 533 Fig. 1 .

 5331 Figures

Acknowledgements

This study would not have been possible without the remarkable field and taxonomic work performed by Thérésien Cadet, who died in 1987. We would like to thank Frédéric Chiroleu, Anna Doizy and Sohan Sauroy-Toucouère for their help with the statistical and spatial analyses, Laurent Michon for his expertise in lava flow dating and Julian Hume for his unique illustrations. Christophe Thébaud and Daniel Simberloff helped to collect and measure the fleshy fruits. We would also like to thank the institutions that funded the research conducted by SA DS OF or provided facilities for the study (FEDER Région Réunion "DIVINES", OSU-Réunion & Marelongue Research Station, Université de la Réunion and CIRAD, Parc National de La Réunion, ONF Réunion).

We are grateful to two anonymous reviewers from which suggestions greatly improved the manuscript.

Accepted Manuscript https://doi. org/10.1111/1365-2745.13359

Authors' contributions

Sébastien Albert, Olivier Flores and Dominique Strasberg developed the ideas for the research and collected data. Sébastien Albert and Olivier Flores analysed the data.

All authors contributed critically to the drafts of the paper and have given their approval for publication.

Supporting information

Appendix. This appendix includes