Sam Bruun-Lund

Brecht Verstraete

Finn Kjellberg

Nina Rønsted

Rush hour at the Museum -Diversification patterns provide new clues for the success of figs (Ficus L., Moraceae)

Keywords: Diversification rates, Ficus, Key-innovations, Rainforest diversity, Hemi-epiphytes

Tropical rainforests harbour much of the earth's plant diversity but little is still known about how it evolved and why a small number of plant genera account for the majority. Whether this success is due to rapid turnover or constant evolution for these hyper-diverse plant genera is here tested for the species-rich genus Ficus L. (figs). The pan-tropical distribution of figs makes it an ideal study group to investigate rainforest hyper-diversification patterns. Using a recently published, dated and comprehensive phylogenetic hypothesis, we infer that figs are an old lineage that gradually accumulated species and exhibits very low extinction rates, which corresponds to the 'museum model' of evolution. Overall, no major significant shifts in evolutionary dynamics are detected, yet two shifts with lower probability are found. Hemiepiphytism, monoecy, and active pollination are traits that possibly are associated with the hyper-diversity found in figs, making it possible for the plants to occupy new niches followed by extensive radiation over evolutionary time scales. Figs possess unique diversification patterns compared to other typical rainforest genera.

Introduction

Rainforests harbor the majority of biodiversity on Earth and are viewed as old ecosystems that have been prevalent throughout much of the Cenozoic (65-0 Ma). However, rainforest hyper-diversity is not well understood and it has variously been attributed to be a museum of diversity, showing constant speciation and low extinction [START_REF] Wallace | Tropical Nature, and Other Essays[END_REF][START_REF] Stebbins | Flowering Plants: Evolution above the Species Level[END_REF], or a cradle of diversity, referring to more recent and rapid speciation [START_REF] Pennington | The origins of tropical rainforest hyperdiversity[END_REF][START_REF] Richardson | Rapid diversifcation of a species-rich genus of Neotropical rain forest trees[END_REF]. Previous phylogenetic studies of diversification patterns in typical rainforest plant families, such as Annonaceae, Arecaceae, and Menispermaceae, have suggested that they are old lineages having experienced a gradual and more or less constant lineage accumulation over time, supporting the museum model (Couvreur et al., 2011a(Couvreur et al., , 2011b;;[START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF]. In contrast, other studies have inferred more recent radiations of rainforest lineages at the genus level for Inga, Costus, Guatteria, and Renealmia, supporting the cradle model of recent diversification [START_REF] Erkens | A rapid diversification of rainforest trees (Guatteria; Annonaceae) following dispersal from Central into South America[END_REF][START_REF] Kay | Rapid speciation and the evolution of hummingbird pollination in neotropical Costus subgenus Costus (Costaceae): evidence from nrDNA ITS and ETS sequences[END_REF][START_REF] Richardson | Rapid diversifcation of a species-rich genus of Neotropical rain forest trees[END_REF][START_REF] Särkinen | Recent oceanic long-distance dispersal and divergence in the amphi-Atlantic rain forest genus Renealmia L.f. (Zingiberaceae)[END_REF]. Further complicating the picture, [START_REF] Koenen | Recently evolved diversity and convergent radiations of rainforest mahoganies (Meliaceae) shed new light on the origins of rainforest hyperdiversity[END_REF] recently documented that despite the Meliaceae family originated already in the Eocene (56-34 Ma), the majority of the rainforest species diversity is recent and associated with higher speciation rates compared to non-rainforest lineages.

In line with Couvreur et al. (2011b) who suggested a mixed model of steady processes and mixed diversification, [START_REF] Koenen | Recently evolved diversity and convergent radiations of rainforest mahoganies (Meliaceae) shed new light on the origins of rainforest hyperdiversity[END_REF] proposed a concept of highly dynamic diversification processes across ecosystems that are linked to environmental changes [START_REF] Xing | Fossils and a large molecular phylogeny show that the evolution of species richness, generic diversity, and turnover rates are disconnected[END_REF] rather than to climatic stability, as suggested by the museum model. This view is, for example, supported by the Miocene (23-5 Ma) radiation of succulents [START_REF] Arakaki | Contemporaneous and recent radiations of the world's major succulent plant lineages[END_REF] and grasses [START_REF] Edwards | The origins of C4 grasslands: integrating evolutionary and ecosystem science[END_REF], which was interpreted as an effect of global cooling and the subsequent spread of seasonally dry ecosystems. Furthermore, fossil evidence has shown that Eocene rainforests were more diverse than contemporary rainforests but experienced a period of decreasing diversity up to the Early Miocene, implying both ancient rapid diversification and high past extinction rates [START_REF] Jaramillo | Effects of rapid global warming at the paleocene-eocene boundary on neotropical vegetation[END_REF][START_REF] Wilf | High plant diversity in eocene south America: evidence from patagonia[END_REF]. More examples are needed to help clarify if there are general patterns explaining rainforest diversification or if individual plant groups exhibit specific patterns.

Figs (Ficus L., Moraceae) are an old Cretaceous (75-90 Ma; [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF][START_REF] Rønsted | 60 million years of co-divergence in the fig-wasp symbiosis[END_REF][START_REF] Zerega | Biogeography and divergence times in the mulberry family (Moraceae)[END_REF] but mega-diverse (?800 species; [START_REF] Berg | Moraceae (Ficus)[END_REF] and a significant component of tropical forests with many species having wide distribution ranges and ubiquitously high alpha-diversity in lowland tropical rainforests [START_REF] Berg | Moraceae (Ficus)[END_REF][START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]. [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF] argued that no diversity pattern of any other genus resembles that of figs: other diverse genera have either geographically restricted distributions or are especially diverse in only one region. This indicates that figs possess certain traits that make them exceptionally well adapted to a broad range of niches, which has led to their high diversity all over the tropics [START_REF] Corner | Botanical Society of Edinburgh[END_REF].

A range of different traits have been suggested as possible drivers for fig diversification [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]Jousselin et al., 2003a;[START_REF] Weiblen | Correlated evolution in fig pollination[END_REF]. They exhibit a variety of growth forms, including typical rainforest habits like hemi-epiphytes (stranglers and banyans), trees (with/ without buttress roots), and large woody climbers. Furthermore, many species are pioneers of forest succession and possess many pioneer traits, such as small seeds [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]. Furthermore, fig wasps have evolved a unique active pollination behaviour that is very uncommon in most other plants except a few known cases such as in Yucca. Fig wasps use coxal combs to collect pollen into pollen pockets and deposit the pollen onto receptive flowers before oviposition. Active pollination is found in two thirds of the species. For the other third of the genus, passive pollination happens when wasps carry pollen dispersed on their bodies without specialized behaviour [START_REF] Cook | Mutualists with attitude: coevolving fig wasps and figs[END_REF][START_REF] Kjellberg | Pollination mode in fig wasps: the predictive power of correlated traits[END_REF]. Due to their obligate pollination mutualism with short-lived wasps (Agaonidae), crops are available at population level year-round, providing an important food source for over 1200 mammal species globally [START_REF] Shanahan | Colonization of an island volcano, Long Island, Papua New Guinea, and an emergent island, Motmot, in its caldera lake. V. Colonization by figs (Ficus spp.), their dispersers and pollinators[END_REF].

In addition, quick responses to climatic changes and/or the presence of key innovations could promote high diversification [START_REF] Drummond | Multiple continental radiations and correlates of diversification in Lupinus (Leguminosae): testing for key innovation with incomplete taxon sampling[END_REF][START_REF] Heard | Key evolutionary innovations and their ecological mechanisms[END_REF][START_REF] Hodges | Spurring plant diversification: are floral nectar spurs a key innovation[END_REF][START_REF] Mayhew | Why are there so many insect species? Perspectives from fossils and phylogenies[END_REF][START_REF] Sanderson | Shifts in diversification rates with the origin of angiosperms[END_REF][START_REF] Silvestro | Disentangling the effects of key innovations on the diversification of Bromelioideae (Bromeliaceae)[END_REF][START_REF] Vamosi | Factors influencing diversification in angiosperms: at the crossroads of intrinsic and extrinsic traits[END_REF]. The idea of key innovations as a single causal explanation for the rapid diversification of a lineage has largely been abandoned in favour of more complex models developed across angiosperms [START_REF] Smith | Understanding angiosperm diversification using small and large phylogenetic trees[END_REF] and using more nuanced explanations involving multiple interacting traits assembled step-wise through evolution referred to as synnovation [START_REF] Donoghue | Confluence, synnovation, and depauperons in plant diversification[END_REF]. Therefore, traits that are shared among species from diverse genera could very well be responsible for the rise of high species richness in general [START_REF] Lovette | Clade-specific morphological diversification and adaptive radiation in Hawaiian songbirds[END_REF]. Certain comparative phylogenetic methods allow for testing and comparing the influence of certain traits on diversification patterns. Using those techniques to study the evolutionary history of a pan-tropical diverse genus such as the figs, and to identify diversification rates shifts and/or specific traits linked to higher diversification and survival might provide insights into the evolution of tropical biodiversity across ecosystems and continents in general.

Using a time-calibrated phylogenetic tree of figs, we address the following questions: (1) does the diversification pattern of figs support the museum model of ancient gradual diversification or the cradle model of recent rapid radiations? (2) Do significant diversification shifts within Ficus exist and if so, (3) are they correlated with specific traits representing one or more keyinnovations?

Materials and methods

Taxon sampling

As input for the different diversification rate analyses, the most recently published dated phylogenetic hypothesis of figs was used [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF]. This phylogenetic hypothesis is also the most comprehensive, including 200 species (> 25% of the diversity of the genus) and was constructed based on the 5 low or single copy nuclear markers ITS, ETS, G3pdh, ncpGS, and waxy. Sampling broadly covers the diversity of Ficus with section Oreosycea and subgenus Synoecia being the least inclusive with 12% of the species of these lineages included (Supplemetary data S1). Although support and resolution of certain infrageneric relationships within Ficus are still low, the phylogenetic tree of [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF] represents by far the best hypothesis available to date rooted with four taxa of Castilleae, the sister lineage of Ficus [START_REF] Clement | Morphological evolution in the mulberry family (Moraceae)[END_REF].

Diversification rate analyses

A lineage through time (LTT) plot for the entire genus was generated with the R package paleotree 2.7 [START_REF] Bapst | Paleotree: an R package for paleontological and phylogenetic analyses of evolution[END_REF]. One thousand random post-burnin trees from the BEAST inference analysis by [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF] were used as input. A median curve with the 95% confidence interval is displayed.

A Bayesian Analysis of Macro-evolutionary Mixtures (BAMM 2.5) [START_REF] Rabosky | Analysis and visualization of complex macroevolutionary dynamics: an example from Australian Scincid lizards[END_REF]) was used to infer shifts in speciation and extinction rates across the phylogenetic tree. To account for non-random incomplete taxon sampling, total diversity and sampling fractions for all clades (see Supplementary data file S1) within figs were obtained from the literature [START_REF] Berg | Moraceae (Ficus)[END_REF]Rønsted et al., 2008a). The MCMC analysis was run for 1 million generations with sampling every 1000 generations and, after checking for convergence with the R package coda 0.19-1 [START_REF] Plummer | CODA: convergence diagnosis and output analysis for MCMC[END_REF], the first 10% was discarded as burn-in. We used the R package BAMMtools 2.1.6 [START_REF] Rabosky | Analysis and visualization of complex macroevolutionary dynamics: an example from Australian Scincid lizards[END_REF] and ape 4.0 [START_REF] Paradis | APE: analyses of phylogenetics and evolution in R language[END_REF] to summarize rates over each branch of the phylogenetic tree (visualized in the so-called 'phylorate' plot), to plot the 95% credible shift set (CSS) with sampling frequencies of the different shift configurations, and to obtain the shift configuration with the maximum a posteriori (MAP) probability.

Problems with estimating extinction rates have been reported for earlier versions of BAMM [START_REF] Moore | Critically evaluating the theory and performance of bayesian analysis of macroevolutionary mixtures[END_REF] and although they have been accounted for in the latest version, we decided to include a MEDUSA analysis as a second method to infer diversification rate shifts [START_REF] Alfaro | Nine exceptional radiations plus high turnover explain species diversity in jawed vertebrates[END_REF]. MEDUSA requires species richness to be assigned to unresolved clades and we therefore pruned the original dated phylogenetic tree of [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF] to section level and assigned diversity to each terminal. The R package geiger 2.0.6 [START_REF] Alfaro | Nine exceptional radiations plus high turnover explain species diversity in jawed vertebrates[END_REF] was used to perform the MEDUSA analysis.

State-dependent diversification rate analyses

State-dependent diversification rate analyses were performed with the Binary-State Speciation and Extinction (BiSSE) model [START_REF] Maddison | Estimating a binary character's effect on speciation and extinction[END_REF] and the MultiState Speciation and Extinction (MuSSE) model, as implemented in the R package diversitree 0.9-9 [START_REF] Fitzjohn | Diversitree: comparative phylogenetic analyses of diversification in R[END_REF]. The influence of two traits on the diversification rates was analysed using the BiSSE model: 1) pollination mode with the states active versus passive pollination, and 2) monoecious versus (gyno)dioecious plants. The influence of habit was analysed using the MuSSE model and four different states were defined: shrub or small tree (< 30 m, understory), climber, large tree (?30 m, canopy), and hemiepiphyte. Information on the trait states for each taxon was extracted from the literature (Fig. 1; [START_REF] Berg | Taxonomic Studies on Ficus (Moraceae) in the West Indies, Extra-Amazonian Brazil and Bolivia, Ilicifolia[END_REF][START_REF] Berg | Moraceae[END_REF][START_REF] Berg | Moraceae (Ficus)[END_REF][START_REF] Berg | African fig trees and fig wasps[END_REF][START_REF] Burrows | Figs of southern & south-central africa[END_REF][START_REF] Corner | Ficus in the Solomon Islands and its bearing on the post-jurrasic history of Melanesia[END_REF]Zhengyi et al., 2003; supplemented by our own taxonomic knowledge). For each analysis, we corrected for state-specific incomplete taxon sampling. Likelihood ratio tests were performed to test for significant differences between the unconstrained and constrained rate models and these found the unconstrained model to be the best-fit model for all characters. The MCMC analysis was performed with a standard Cauchy distribution as hyper-prior to avoid zero rates [START_REF] Burin | Omnivory in birds is a macroevolutionary sink[END_REF] and was run for 10,000 generations with sampling every 1000 generations. Chain convergence and effective sampling size (ESS) parameters were inspected with the R package coda 0.19-1 [START_REF] Plummer | CODA: convergence diagnosis and output analysis for MCMC[END_REF]. The mean speciation, extinction, and net diversification rates and their respective 95% credibility intervals were calculated for each state of each character. A Cohen's d effect size test was performed to test for differences between these means. The results were visualized in posterior probability distribution plots. Fig. 1. Phylorate plot of Ficus with branches colored according to speciation rate (lineages/Ma), resulting from the Bayesian Analysis of Macro-evolutionary Mixtures (BAMM) analysis. For 70% of the samples in the posterior distribution no shift is observed. However, at a lower probability, a shift in diversification dynamics is observed at the lineage leading to section Americanae (20% of the samples in the posterior distribution) and at the lineage containing both section Americanae and section Galoglychia (3.6% of the samples in the posterior distribution), as indicated with red circles. The M is located on the branch where a rate shift occurs according to the MEDUSA analysis. The scored traits along with the species are indicated on the tips of the tree.

Results

Diversification shifts

The BAMM analysis revealed that 70% of the samples in the posterior distribution exhibit no shifts in speciation rates, thus a constant model of evolution is favoured with no sudden increase in diversification rates for any clades (Fig. 1). However, shifts in diversification rates are detected with lower probability at the branch leading to section Americanae (20% of the samples in the posterior distribution) and at the branch leading to the clade containing both section Americanae and section Galoglychia (3.6% of the samples in the posterior distribution) (Fig. 1; red circles & Supplementary data file S3). The detected shifts with a lower probability (20% and 3.6%) are at a lower marginal shift probability and therefore not as well supported and significant as a model of constant evolutionary rate. MEDUSA identified significant acceleration of net speciation rates at one point in the tree where a branch leading to the clade containing both section Americanae and section Galoglychia (Fig. 1; red circle with an M). Overall net diversification rates inferred by MEDUSA and BAMM are very similar, confirming the robustness of our result.

The LLT plot shows a gradual increase in lineage accumulation through time, meaning that no sudden radiation of lineages is detected at any point (Fig. 2). This demonstrates consistency between the patterns inferred by BAMM, where no strongly supported shifts in rate dynamics is observed either.

State-dependent diversification rates

Following the methodology of the Cohen d effect size test, the differences in diversification rates between traits found in the BiSSE analysis are defined as 'very small', 'small', 'large', 'very large', and 'huge' and this terminology is used in the description of the results. See supplementary data file S2 for all inferred rates by BiSSE and MuSSE analysis.

Pollination mode. The BiSSE analysis inferred a huge difference in net diversification rates between active and passive pollination: species with active pollination (r = 0.071) diversify faster than species with passive pollination (r = 0.052)see supplementary data file S2. There is a large difference in extinction and speciation rate between the two traits, with extinction rate being higher for passively pollinated fig species and speciation faster for actively pollinated figs, as also suggested in [START_REF] Kjellberg | Pollination mode in fig wasps: the predictive power of correlated traits[END_REF]. The transition rate from passive to active pollination was found to be larger than the reverse.

Monoecy and dioecy. When comparing monoecious and dioecious figs, the BiSSE method inferred a huge difference in net diversification rate between monoecious and dioecious fig species. We found that monoecious figs evolve faster (r = 0.076) than dioecious species (r = 0.055). Extinction rate is similar with a very small difference between the two trait states (thus, less different than for the mode of pollination). The speciation rate is hugely different between dioecious and monoecious species, with monoecious species evolving faster than the dioecious ones (see supplementary data file S2). The transition rate from dioecy to monoecy is also much higher than the reverse.

Life forms (habits) of Figs. In order to account for the various life forms of figs, a statedependent diversification analysis using the MuSSE model was performed and four trait states were defined: shrubs/ small trees, large trees, climbers, and hemi-epiphytes. Net diversification rate of hemi-epiphytes (r = 0.076) is much higher than net diversification for the three other life forms (shrubs/small trees, large trees, climbers) which also show very similar rates (r between 0.049 and 0.057). The speciation and extinction rates for all life forms are somewhat similar, however, the hemi-epiphytic figs show a slightly higher speciation rate and a slightly lower extinction rate, resulting in the generally higher net diversification rate.

Transition rates between life forms. The rate of transitions between life forms varies considerably (see Fig. 3). The highest transition rates are inferred for figs transitioning from large canopy trees (?30 m) to smaller understory trees (< 30 m) or shrubs (mean = 0.033), as well as for the opposite direction (mean = 0.029). The transition rate from either of these life forms as well as climbers to hemi-epiphytic figs is low (from 0.006 up to 0.035). However, the transition rates from hemiepiphytes to any of the other three life forms are much lowerall ranging between 0.001 and 0.003.

Discussion

Our results, which are based on the largest and most recent phylogenetic tree of figs, provide possible explanations for some of the diversification patterns that have been hypothesized up till now. Our aim was to (1) test if the diversification patterns of Ficus support a museum model of ancient gradual diversification or a cradle model of recent, rapid radiation. Also, (2) to see if any significant shifts in diversifications patterns occur within Ficus and (3) if they are linked with specific traits.

Diversification patterns

To investigate diversification patterns in figs, we constructed a lineage-through-time (LTT) plot and performed an analysis using both BAMM and MEDUSA to test how lineages evolve and diversify through time. Diversification of figs seems to correspond with a museum model of constant evolution and little extinction, leading to the gradual accumulation of lineages, possibly in response to long-lasting and stable tropical ecosystems (Figs. 1 and3). However, it is worth noting that other examples of rainforest lineages supporting the museum model of gradual diversification are detected for pan-tropical rainforest tree families (Couvreur et al., 2011a,b;[START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF], whereas generic level studies have primarily focused on the Neotropics and largely support the cradle model of recent diversification [START_REF] Richardson | Rapid diversifcation of a species-rich genus of Neotropical rain forest trees[END_REF][START_REF] Kay | Rapid speciation and the evolution of hummingbird pollination in neotropical Costus subgenus Costus (Costaceae): evidence from nrDNA ITS and ETS sequences[END_REF][START_REF] Erkens | A rapid diversification of rainforest trees (Guatteria; Annonaceae) following dispersal from Central into South America[END_REF][START_REF] Särkinen | Recent oceanic long-distance dispersal and divergence in the amphi-Atlantic rain forest genus Renealmia L.f. (Zingiberaceae)[END_REF]. These patterns could suggest that Neotropical rainforests may be cradles of recent diversity within a pantropical museum model.

Consequently, the diversification patterns we have found lend support to the statement by [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF] that no other genus compares to the diversity pattern of Ficus as other diverse genera have either geographically restricted distributions or are especially diverse in only one region. Furthermore, the overall net diversification rate for the genus (r = 0.1) is higher, largely explained by a lower extinction rate (? = 0.009), than the overall net diversification rate inferred across all angiosperms (r = 0.07-0.09) [START_REF] Magallon | Absolute diversification rates in angiosperm clades[END_REF].

Overall, the BAMM analysis inferred no significant rate shifts in most of the samples from the 95% credibility set, but at a lower marginal probability two shifts towards increased speciation rates are found (Fig. 1). The shift with the second highest probability is found at the lineage leading to section Americanae and the third rate shift is detected at the branch leading towards the clade containing both section Americanae and section Galoglychia (Fig. 1; red circles). The latter rate shift was also detected by the MEDUSA analysis on the branch leading towards a clade with both sections. Even though the analysis do detect a shift in rate diversification this is only at a very low probability and the most supported analysis detects no shifts in the genus. Fig. 2. Lineage-though-time plot depicted on the dated phylogenetic tree from [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF]. A gradual increase of lineages through time is observed and no sudden bursts of speciation are present within the genus.

State-dependent diversification patterns

The aim of the diversification analyses was to explore the potential link between traits or key innovations and varying diversification rates. Our separate results show higher diversification rates linked to figs that are actively pollinated, monoecious, and hemi-epiphytes compared to passively pollinated, dioecious figs exhibiting other life forms. Furthermore, these traits all occur in clades associated with higher diversification rates from BAMM and MEDUSA (sections Americanae and Galoglychia; see Fig. 1). It should also be that the traits were analysed separately and are perhaps not necessarily linked together.

Active pollination in fig species is positively linked with higher diversification rates, i.e. species that are actively pollinated evolve faster. It has previously been hypothesized that active pollination could be a driver of speciation, which is in line with our results (e.g. [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]Jousselin et al., 2003b;[START_REF] Kjellberg | Pollination mode in fig wasps: the predictive power of correlated traits[END_REF]. It has also been suggested that active pollination might have evolved as a way for wasps to ensure fertilization of the flowers in which they oviposit (Jousselin et al., 2003a). [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF] suggests that maintaining high levels of heterozygosity due to ensured outcrossing, together with a very high proportion of inflorescences pollinated could also be a driver of faster speciation in actively pollinated figs. Also, active pollination could be maintained over evolutionary time by actively pollinating species being less prone to extinction than passively pollinating ones [START_REF] Kjellberg | Pollination mode in fig wasps: the predictive power of correlated traits[END_REF], which is confirmed by results exhibiting a lower extinction rate.

The efficiency of active pollination has enabled figs to occupy rare (micro)-niches and it is therefore an important factor for the high alphadiversity in figs, since the ability to outcross at extremely low population densities may also have lowered extinction rates [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]. Our results support these hypotheses by inferring an extinction rate almost twice as low for actively pollinating species (0.007 vs. 0.016) compared to passively pollinated species.

Monoecy or (gyno)-dioecy. Being monoecious is correlated with increased rates of diversification in figs. In contrast to angiosperms in general [START_REF] Käfer | Dioecy is associated with higher diversification rates in flowering plants[END_REF], monoecious fig species have a higher diversification rate and this has also been shown elsewhere for other plant species [START_REF] Laenen | Increased diversification rates follow shifts to bisexuality in liverworts[END_REF], which has been explained by the longterm advantages of outcrossing. In figs, however, this does not seem to be the case. It could instead be explained by self-compatible bisexual species being more likely to establish new colonies following longdistance dispersal than unisexual or self-incompatible ones, potentially promoting allopatric speciation and finding new niches [START_REF] Heilbuth | Lower species richness in dioecious clades[END_REF][START_REF] Laenen | Increased diversification rates follow shifts to bisexuality in liverworts[END_REF]. Fig. 3. Transitions rates between life forms (habits). The circle size is proportional to the number of species with that trait state and the thickness of the arrows is proportional to the transition rates. Hardly any species evolve from a hemiepiphytic life form to any of the three other states. Life forms. Figs that are hemi-epiphytic have an increased diversification rate compared to other life forms, which all show a somewhat similar net diversification rate (see supplementary data file S2). Around half of the species are hemi-epiphytic, further suggesting that this life form is advantageous for figs [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF][START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]. They seem to possess some key innovations or a syndrome of advantageous traits explaining their diversification success. The ability to grow on rocky outcrops using aerial roots has probably been very important in the diversification and evolution of figs. Even though hemiepiphytic figs are present in low population densities, most hemi-epiphytic species have large ranges related to a more diffuse dispersal mechanism as opposed to terrestrial species, which tend to have more locally restricted dispersal abilities [START_REF] Harrison | Dispersal of fig pollinators in Asian tropical rain forests[END_REF]. Dispersal range also differs between dioecious figs, which may be more restricted compared to monoecious figs.

Whereas pollinators of monoecious figs disperse by ascending above the canopy and drifting in the wind until they reach the fume of receptive fig odour emitted by a tree, the pollinators of dieocious figs disperse more locally and do not use the wind for dispersal [START_REF] Harrison | Dispersal of fig pollinators in Asian tropical rain forests[END_REF].

The hemi-epiphytic life form allows for occupation of niches that most plants do not readily colonize. Hemi-epiphytes are adapted to grow in the canopy layer with low water availability and high light conditions. Adaptive root flexibility may also explain transitioning from forest habitats to similar dry environments expressed by lithophytes in Australia [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF][START_REF] Harrison | The diversity of hemi-epiphytic figs in a Bornean lowland rain forest[END_REF][START_REF] Harrison | Seventy-seven ways to be a fig: overview of a diverse plant assemblage[END_REF]Rønsted et al., 2008b).

Transitioning between life forms. The rate of which the traits tested evolve and transition into each other is an indication of the diversification dynamics of figs and may offer explanations as to how they became so successful. We scored large canopy trees and small understory trees/shrubs as separate life forms, but our results indicate that it is easy to transition between these two states, probably because it does not require large evolutionary sacrifices or changes.

The rate at which figs become hemi-epiphytic is not high but the rate of hemi-epiphytic figs transitioning back towards another life form is very low (Fig. 3). Consequently, the hemiepiphytic life-form appears to be a more stable state providing an adaptive advantage and explaining why such a high proportion of the figs are hemi-epiphytic [START_REF] Berg | Moraceae (Ficus)[END_REF].

What has made figs so successful?

The origin of figs has been dated to the late Cretaceous (75-90 Ma; [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF] which is before the K/Pg boundary (?65 Ma) where a lot of plant diversity is hypothesized to have gone extinct [START_REF] Wilf | Land plant extinction at the end of the Cretaceous: a quantitative analysis of the North Dakota megafloral record[END_REF]. For figs, this might have created opportunities to expand into new niches, as they are generally opportunistic species with low competitive abilities [START_REF] Berg | Moraceae (Ficus)[END_REF][START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF]. After having established in such new niches, figs would have kept evolving at a constant rate as competition is low in many (micro)-niches [START_REF] Frenzke | Evolution of epiphytism and fruit traits act unevenly on the diversification of the species-rich genus Peperomia (Piperaceae)[END_REF][START_REF] Gentry | Diversity and biogeography of neotropical vascular epiphytes[END_REF]. Figs do possess many traits typical of pioneer plantssmall seeds, high assimilation and growth rates, high fecundity, and flexible rooting habitsthat are advantageous in colonising new areas as also expressed by the genus Piper and epiphytic Orchids [START_REF] Harrison | Figs and the diversity of tropical rainforests[END_REF].

Furthermore, the renowned pollination system of figs with its unique fig-pollinating wasps ensuring high pollination success is beneficial when population densities are low. Even though fig species and their pollinators show high levels of co-diversification and specificity [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and figpollinating wasps[END_REF], multiple pollinators for a single fig species are frequently reported (Machado et al., 2005). Hybridization and introgression could also have contributed to the large genetic and species diversity in figs [START_REF] Bruun-Lund | First plastid phylogenomic study reveals potential cyto-nuclear discordance in the evolutionary history of Ficus L. (Moraceae)[END_REF].

Conclusion

Overall, the diversification rate analyses show that figs generally follow the museum model of evolution with a gradual accumulation of species over time and with very low extinction rates and no significant evolutionary shifts. However, the trait state-dependent analyses show that monoecy, active pollination, and having a flexible root habit (i.e. hemi-epiphytes) are linked with higher diversification rates and this is further underpinned by these character states being expressed by a subclade with higher diversification rate. These trait states are found throughout most of the phylogenetic tree suggesting that they represent key innovations or syndromes, possibly responsible for making the figs so successful in terms of species diversity and accumulating high species diversity over an evolutionary time scale.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.actao.2017.11.001.


Larissa Galante Elias

Finn Kjellberg

Fernando Henrique

Antoniolli Farache

Eduardo A B Almeida

Jean-Yves Rasplus

Astrid Cruaud

Yan-Qiong Peng

Da-Rong Yang

Rodrigo Augusto

Santinelo Pereira

Ovipositor morphology correlates with life history evolution in agaonid fig wasps

Keywords: Ficus, Functional morphology, Parasitic Hymenoptera, Parasitoid, Plant-insect interaction

The high adaptive success of parasitic Hymenoptera might be related to the use of different oviposition sites, allowing niche partitioning among co-occurring species resulting in life history specialization and diversification. In this scenario, evolutionary changes in life history and resources for oviposition can be associated with changes in ovipositor structure, allowing exploitation of different substrates for oviposition. We used a formal phylogenetic framework to investigate the evolution of ovipositor morphology and life history in agaonid wasps. We sampled 24 species with different life histories belonging to all main clades of Agaonidae including representatives of all described genera of non-pollinating fig wasps (NPFW). Our results show an overall correlation between ovipositor morphology and life history in agaonid fig wasps. Ovipositor morphologies seem to be related to constraints imposed by features of the oviposition sites since ovipositor morphology has experienced convergent evolution at least four times in Sycophaginae (Agaonidae) according to the resource used. Nongalling species have more distantly spaced teeth with uneven spacing, as opposed to the observed morphology of galling species. Our results suggest that the ancestral condition for ovipositor morphology was most likely the presence of one or two apical teeth. Regarding life history, ovary galling species that oviposit in receptive figs possibly represent the ancestral condition. Different ovipositor characteristics allow exploitation of new niches and may be related to resource partitioning and species co-existence in the fig-fig wasp system.

Introduction

Oviposition is a crucial step for the reproductive success of insects because finding and assessing suitable oviposition sites play a decisive role in the successful development of offspring. Within this context, oviposition behaviour and ovipositor structure have an important adaptive role, allowing exploitation of diverse oviposition sites [START_REF] Gauld | The Hymenoptera[END_REF]). Among Hymenoptera, there is a wide diversity of resources used for oviposition, which is related to the highly diversified life history strategies of the group, including galling, endo-and ectoparasitoid habits [START_REF] Sharkey | Phylogeny and classification of Hymenoptera[END_REF][START_REF] Heraty | Evolution of the hymenopteran megaradiation[END_REF]. The high adaptive success of parasitic Hymenoptera might be related to exploitation of different substrates for oviposition, allowing niche partitioning among co-occurring species and leading to specialization and diversification [START_REF] Pellmyr | Non mutualistic yucca moths and their evolutionary consequences[END_REF][START_REF] Ronquist | Phylogeny, classification and evolution of the Cynipoidea[END_REF][START_REF] Devictor | Defining and measuring ecological specialization[END_REF]. In this scenario, evolutionary changes in life history and resource use can be associated with changes in ovipositor structure, facilitating the exploitation of different substrates for oviposition [START_REF] Quicke | Ovipositor structure and relationships within the Hymenoptera, with special reference to the Ichneumonoidea[END_REF][START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF][START_REF] Le Ralec | Comparative morphology of the ovipositor of some parasitic Hymenoptera in relation to characteristics of their hosts[END_REF][START_REF] Brajković | Morphological adaptations of the ovipositor of braconid wasps (Braconidae: Hymenoptera) associated to biological characteristics of their hosts[END_REF][START_REF] Vilhelmsen | The ovipositor apparatus of basal Hymenoptera (Insecta): phylogenetic implications and functional morphology[END_REF][START_REF] Belshaw | Inferring life history from ovipositor morphology in parasitoid wasps using phylogenetic regression and discriminant analysis[END_REF][START_REF] Sharkey | Phylogeny and classification of Hymenoptera[END_REF][START_REF] Ghara | Nature's Swiss Army Knives: ovipositor structure mirrors ecology in a multitrophic fig wasp community[END_REF][START_REF] Kawakita | Revision of the Japanese species of Epicephala Meyrick with descriptions of seven new species (Lepidoptera, Gracillariidae)[END_REF].

Ovipositor penetration in the substrate involves sliding movements of the two pairs of gonapophyses derived from the 8th and 9th abdominal segments. These structures form the ovipositor and are linked by a tongue and groove mechanism called olistheter, which allows the parts to slide in relation to one another [START_REF] Quicke | Ovipositor steering mechanisms in Braconid wasps[END_REF][START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF]. Teeth and notches on the tip of gonapophyses grip the substrate, anchoring the ovipositor by its distal extremity. Then, the reciprocal movement allows ovipositor insertion into the substrate, as one of the halves grips the substrate while the other is pushed inside [START_REF] Quicke | Ovipositor steering mechanisms in Braconid wasps[END_REF][START_REF] Vincent | The mechanism of drilling by wood wasp ovipositors[END_REF]oring et al., 2009). The structural and mechanical complexity of ovipositors thus allows movement and precise egg deposition in different substrates [START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF]. However, the understanding of the paths leading to this functional diversity is not trivial, because different characters and strategies are highly conserved in some groups whereas they might have convergently evolved to a specific niche in others [START_REF] Le Ralec | Les hyménoptères parasitoïdes: adaptations de l'appareil reproducteur femelle[END_REF].

Agaonid wasps associated with Ficus Tourn ex Linn (i.e., fig wasps) represent a model group for investigating such functional diversity regarding ovipositors in an evolutionary framework. The family represents a monophyletic group of Chalcidoidea [START_REF] Heraty | A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera)[END_REF], which is strictly associated with Ficus plants, using Ficus inflorescences (figs) as their exclusive oviposition site. In the context of Apocrita phylogeny, Chalcidoidea is placed as sister group of Diaprioidea [START_REF] Peters | Evolutionary history of the Hymenoptera[END_REF]. Fig wasps are distributed worldwide and have a wide diversity of life histories and oviposition strategies [START_REF] Cook | Mutualists with attitude: coevolving fig wasps and figs[END_REF]Cruaud et al., 2011a;[START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF]. They usually lay their eggs in the ovaries of pistillate flowers or in the fig receptacle, inducing a gall [START_REF] Galil | On the pollination ecology of Ficus sycomorus in east Africa[END_REF] [START_REF] Weiblen | How to be a fig wasp[END_REF]. In this case, ovipositors must move inside the figs until they reach an adequate oviposition site [START_REF] Ghara | Nature's Swiss Army Knives: ovipositor structure mirrors ecology in a multitrophic fig wasp community[END_REF][START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF]. These movements involve steering and bending [START_REF] Kundanati | Biomechanics of substrate boring by fig wasps[END_REF], as described for braconid parasitoids of twig-boring larvae [START_REF] Quicke | Ovipositor steering mechanisms in Braconid wasps[END_REF][START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF]. Steering mechanisms allow these species to reach the host in a concealed environment and to deposit the egg in a precise location.

Physical characteristics vary along the ontogenetic development of the fig and represent different constraints for oviposition by wasps. At the beginning of development, figs are small and flowers are still immature within them. Some NPFW species deposit their eggs and induce galls in the fig receptacle [START_REF] Bronstein | Natural history of Anidarnes bicolor (Hymenoptera: Agaonidae), a galler of the Florida strangler fig (Ficus aurea)[END_REF]Ghara et al., 2014) (i.e., receptacle gallers), which mainly consists of parenchymatous cells [START_REF] Verkerke | Anatomy of Ficus ottoniifolia (Moraceae) syconia and its role in the figfig wasp symbiosis[END_REF][START_REF] Verkerke | Syconial anatomy of Ficus asperifolia (Moraceae), a gynodioecious tropical fig[END_REF]. Egg deposition by these species does not occur at a very precise location and consequently ovipositor manoeuvrability is possibly less important in such a homogeneous substrate. Therefore, it is expected that ovipositor teeth are not required for thorough manoeuvering.

Later during fig development, all pollinating fig wasps and some NPFW species use ovaries of pistillate flowers as their oviposition sites, inducing galls (i.e., ovary gallers). They usually insert their ovipositors through the flower stigma, as is the case for some NPFW that oviposit from outside [START_REF] Grandi | Studio morfologico e biologico della Blastophaga psenes[END_REF][START_REF] Galil | Further studies on the pollination ecology of Ficus sycomorus L. (Hymenoptera, Chalcidoidea, Agaonidae)[END_REF][START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF] and lay their eggs at a very precise location-between the nucellus and inner integument or inside the embryo sac, depending on the species [START_REF] Grandi | Istituzioni di Entomologia Generale[END_REF][START_REF] Galil | The effects of Sycophaga sycomori L. on the structure and development of the synconia in Ficus sycomorus L[END_REF][START_REF] Verkerke | Structure and function of the fig[END_REF][START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF][START_REF] Jansen-González | Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp[END_REF]. Pollinating wasps enter the fig and have direct access to the stigma surfaces, where the ovipositor is inserted. At least some ovary gallers that oviposit externally are subject to additional bending movements in order to insert their ovipositors through flower stigmas, with manoeuvrability and movement accuracy being even more crucial for these species [START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF]. Thus, ovipositor teeth are expected to play a role in the oviposition process of externally ovipositing gallers, ensuring accuracy of movement. As fig tissues are rather homogeneous in texture at the stage when the ovary gallers deposit their eggs, the morphology of ovipositor teeth is expected to be adapted to this type of substrate [START_REF] Ghara | Nature's Swiss Army Knives: ovipositor structure mirrors ecology in a multitrophic fig wasp community[END_REF].

After oviposition by galling species, fig flowers undergo abrupt modifications such as cell hypertrophy and hyperplasia, altering the environment inside the fig [START_REF] Jansen-González | Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp[END_REF], 2014). Cleptoparasite and parasitoid fig wasps oviposit in already induced galls, so that their larvae feed on vegetal tissue or directly on the galler larvae, respectively [START_REF] Joseph | Recherches sur les chalcidiens, Blastophaga psenes (L.) et Philotrypesis caricae (L.), du figuier (Ficus carica L.)[END_REF][START_REF] Tzeng | Confirmation of the parasitoid feeding habit in Sycoscapter, and their impact on pollinator abundance in Ficus formosana[END_REF]. At this stage, the internal structure of the fig is completely modified, with enlarged, thickwalled galls filling almost completely the fig interior. To deposit the eggs, the ovipositor has to thrust into tissues that are heterogeneous in texture, from swollen cells and meristematic tissue inside galled flower ovaries to the thickened gall walls, which pose different mechanic challenges regarding anchoring and penetration. Therefore, a variable ovipositor tooth system is expected, permitting appropriate grip in different-textured tissues.

Ovipositor tooth characters have been investigated in depth in parasitic wasps within a mechanistic framework (e.g., Belshaw et al., 2003 in Ichneumonoidea, andGhara et al., 2011 in fig wasps). [START_REF] Ghara | Nature's Swiss Army Knives: ovipositor structure mirrors ecology in a multitrophic fig wasp community[END_REF] demonstrated that the ovipositor microstructure of NPFW species associated with F. racemosa matches resource use, i.e. the ovipositor of parasitoid species has a larger number of sensilla and ovipositor teeth and the extent of sclerotisation of the ovipositor tip correlates with the force required to penetrate the fig wall. However, studies investigating changes in life history and oviposition sites in relation to changes in ovipositor morphology in an explicitly evolutionary framework are largely missing. Within this context, we studied 24 agaonid species with different life histories (e.g., galling and nongalling species), which, therefore, use different resources for oviposition. Study species represent all main clades of Agaonidae, including representatives of four pollinating species and all described genera of NPFW species. In addition to acquisition of data for various species, recent improved phylogenetic understanding of NPFW taxa (e.g., Cruaud et al., 2011aCruaud et al., , 2011b) ) has permitted an explicit comparative analysis. We used a formal phylogenetic framework 1) to investigate the correlation between the evolution of overall ovipositor morphology and life history in agaonid wasps, and 2) to identify functional traits related to ovipositor teeth and to estimate how these traits evolved in Agaonidae. We hypothesised that fig wasps with different life histories and, therefore, using different resources for oviposition, would exhibit differences regarding ovipositor structure. We adopted a comparative approach aimed at understanding the evolution of different strategies related to the solution of a common problem regarding resource use.

Methods

Study species

We sampled 24 species with different life histories and therefore ovipositing in different substrates throughout fig development in order to maximize the ecological and phylogenetic diversity under consideration. The present study species belong to all main clades of Agaonidae including representatives of all described genera of NPFW (according to the phylogenetic hypotheses of Cruaud et al., 2011aCruaud et al., , 2011b) ) in addition to four genera of pollinating wasps. All study species' life histories are known from the literature or from personal observation by the authors [START_REF] Galil | The effects of Sycophaga sycomori L. on the structure and development of the synconia in Ficus sycomorus L[END_REF][START_REF] Elias | Colonization sequence of nonpollinating fig wasps associated with Ficus citrifolia in Brazil[END_REF][START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF][START_REF] Peng | The population dynamics of a non-pollinating fig wasp on Ficus auriculata at Xishuangbanna, China[END_REF][START_REF] Wang | Structure of a fig wasp community: temporal segregation of oviposition and larval diets[END_REF]Cruaud et al., 2011a;[START_REF] Farache | Taxonomic revision and molecular phylogeny of the fig wasp genus Anidarnes (Hymenoptera: Sycophaginae) Bouček, 1993[END_REF]. We included 10 Neotropical species (associated with six Ficus hosts), 12 Indo-Australasian species (associated with 10 Ficus hosts), and two Afrotropical species (associated with two Ficus hosts) (Table 1). Vouchers are deposited at CBGP (INRA), Montferrier-sur-Lez, France.

Ovipositor characters

Observation and measurements were made from fresh material, as well as from 100% ethanol-preserved material and dry specimens. Body length was estimated by adding the measures of head length, mesosome length and metasome length. Measurements were made on 10 to 20 individuals of each species under a Leica MZ 16 stereomicroscope (40X magnification). Ovipositors from two to five individuals of each species were dissected, dehydrated in alcohol series when necessary, gold coated for 280 s and examined with a Zeiss EV050 Scanning Electron Microscope (SEM). Based on a functional approach, we used characters related to ovipositor teeth for their potential role in drilling and/or anchoring the ovipositor, allowing its movement through the substrate [START_REF] Le Ralec | Les hyménoptères parasitoïdes: adaptations de l'appareil reproducteur femelle[END_REF][START_REF] Le Ralec | Comparative morphology of the ovipositor of some parasitic Hymenoptera in relation to characteristics of their hosts[END_REF][START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF][START_REF] Belshaw | Inferring life history from ovipositor morphology in parasitoid wasps using phylogenetic regression and discriminant analysis[END_REF][START_REF] Vilhelmsen | Per arborem ad astra: morphological adaptations to exploiting the woody habitat in the early evolution of Hymenoptera[END_REF] (Table 2 andFigure S1).

We did not include ovipositor length or ovipositor diameter in the analysis since these characters are correlated with fig size [START_REF] Zhen | Ovipositor length of three Apocrypta species: effect on oviposition behaviour and correlation with syconial thickness[END_REF][START_REF] Tzeng | The effect of fig wall thickness in Ficus erecta var. beecheyana on parasitism[END_REF] rather than relative measurements because no correlation between ovipositor traits and wasp size was detected (Supplementary material -Figure S5).

DNA sequencing, sequence alignment and phylogenetic analyses

The molecular protocols were applied as described by Cruaud et al. (2011a) and specimen sampling locations, hosts, and GenBank sequences are presented in Supplementary material (Table S1).

In order to obtain a more robust phylogenetic inference and to avoid tree-search artifacts caused by subsampling and long-branch attraction, the species analysed in this study were combined with the ingroup species used in the most comprehensive dataset for Sycophaginae to date (Cruaud et al., 2011b). A time-calibrated tree was obtained using BEAST v 2.4.2 [START_REF] Drummond | BEAST: Bayesian evolutionary analysis by sampling trees[END_REF] (Figure S6) and subsequently pruned in order to keep only the studied taxa (Figure S7).

Correlation between ovipositor traits and life history

In order to determine which ovipositor traits are related to life history, the relationship between each morphological character (Table 2) and life history (0 = galler or 1 = non-galler) was analysed with a phylogenetic generalised linear model for binomial response [START_REF] Ives | Phylogenetic logistic regression for binary dependent variables[END_REF] using the "phylolm" package [START_REF] Ho | A Linear-time algorithm for Gaussian and non-Gaussian trait evolution models[END_REF], as implemented in R environment (R Development Core Team, 2015).

As recommended by [START_REF] Revell | Phylogenetic signal and linear regression on species data[END_REF], the phylogenetic signal is estimated simultaneously with the regression coefficients. This measure of phylogenetic signal is associated with α, that gives the change rates among trait values. A value of a (-log α) greater than one represents a strong phylogenetic signal, whereas values of -4.0 or less indicate a low phylogenetic signal [START_REF] Ives | Phylogenetic logistic regression for binary dependent variables[END_REF].

We used a model comparison approach [START_REF] Burnham | Model Selection and Multimodel Inference: a Practical Information e Theoretic Approach[END_REF] based on Akaike Information Criterion (AIC) to rank competing models. We calculated the difference between the AIC of each model and the lowest AIC value (ΔAICi = AICi -AICmin); therefore, the best model had a ΔAIC = 0. We estimated the relative weight of each model (wAIC), which represents the likelihood of a given model to be the best one among a set of concurrent models [START_REF] Johnson | Model selection in ecology and evolution[END_REF]. Models with ΔAIC ≤2 and wAIC ≥0.10 were considered equally plausible to explain the observed data [START_REF] Burnham | Model Selection and Multimodel Inference: a Practical Information e Theoretic Approach[END_REF][START_REF] Johnson | Model selection in ecology and evolution[END_REF]. As a baseline for comparison, we included a null model (i.e., dependent variable ∼ constant), which refers to the absence of effect.

Because morphological characters (Table 2) reflect ovipositor tooth morphology, they do not apply to species that lack ovipositor teeth. Therefore, only species that bear teeth in their ovipositors were included in this analysis (16 species).

Evolution of ovipositor traits

In order to better understand how morphological characters (Table 2) evolved in Agaonidae, we performed ancestral state Table 3 Ovipositor and life history traits coded into discrete (i.e., categorical) characters. NA: not applicable. reconstruction analysis. We used a likelihood framework implemented on the R package phytools [START_REF] Revell | Phytools: phylogenetic Tools for comparative biology (and other things)[END_REF]. For each character, values were colour-coded, so that the variation in colour seen in the reconstruction reflected variation in character values.

Furthermore, to investigate how both general ovipositor morphology and life history change throughout the evolutionary history of Agaonidae, ancestral states were also estimated. In this case, characters were coded as multistate to provide summarised information. Multistate codings for overall ovipositor morphology and life history are detailed in Table 3. Analyses were performed in R environment with the package phytools [START_REF] Revell | Phytools: phylogenetic Tools for comparative biology (and other things)[END_REF], using a stochastic reconstruction that allowed the calculation of a Bayesian posterior probability distribution. The results of 1000 stochastic character maps for each trait were used to estimate probabilities at each node [START_REF] Revell | Two new graphical methods for mapping trait evolution on phylogenies[END_REF]. States for each character were colour-coded and probabilities of each state at nodes were represented by pie charts.

Results

Morphological and phylogenetic data

Overall ovipositor morphology and tooth measurements, as well as the tree representing the phylogenetic relationships and divergence times among the 24 study species were used as the framework for all comparative studies and are shown in the Supplementary material (Figures S2-S4, Figure S7 and Table S2).

Eukoebelea was recovered as the sister group for all other Sycophaginae. Idarnes and Sycophaga were recovered as monophyletic groups and each Idarnes species group was also recovered as monophyletic, with the carme species group being sister to the flavicollis + incerta species groups. Pseudidarnes + Anidarnes + Conidarnes were also monophyletic, in agreement with Cruaud et al. (2011aCruaud et al. ( , 2011b)).

All receptacle-galling species lack teeth on their ovipositors, regardless of the clade they belong to (Idarnes sp.7 (incertus group) is sister to a clade of tooth-bearing Idarnes). Even though vestigial teeth are present in the females of this species (Figure S2), they are not functional. The ovipositors of Agaonidae pollinating species bear one or two apical teeth. On the other hand, ovary-galling NPFW have multiple teeth in their ovipositors. Idarnes galling species bear fewer and shorter teeth than Sycophaga galling species (5-6 vs. 7-11, respectively, Table S2). However, internally ovipositing Sycophaga (Sycophaga sycomori and Sycophaga silvestrii) seem to have reduced ovipositor teeth which were the shortest among the study species. The non-galling NPFW also have multiple teeth in their ovipositors, Idarnes and Eukoebelea species have irregular serrated teeth, while non-galling Sycophaga have uniform teeth similar to those of the non-pollinating galling species.

Correlation between ovipositor traits and life history

The coefficient of variation of distance between teeth and the mean distance between teeth were equally plausible to predict the life history of fig wasps (ΔAIC ≤ 0.5, ∑wAIC = 0.903), i.e. species with more widely spaced teeth and more uneven tooth spacing are more likely to be non-gallers. All other tested models contributed little to the explanation of fig wasp life history. Indeed, most of them were less plausible than the null model (Table 4). Most a values were higher than 1.0, indicating a phylogenetic signal in the response variable (life history). Our approach using a general phylogenetic linear model was therefore suitable.

Evolution of ovipositor traits

We presented ancestral character state reconstructions only for the ovipositor traits significantly correlated with life history (ΔAIC < 2 and wAIC > 0.1) (Fig. 2). Reconstructions for all study characters are presented in Supplementary material (Figures S8-S9).

In Sycophaga, both mean and coefficient of variation of distance between teeth are higher in the non-galling species (violet to black colours in Fig. 2). In Idarnes, more spaced and uneven spacing among teeth also occurred in non-galling species (dark blue to black colours in the nongalling clade Idarnes sp.1 + Idarnes sp.9 + Idarnes punctata, Fig. 2).

The ancestral condition of the overall ovipositor morphology of Agaonidae was probably uniform teeth (67% posterior probability) or one-two apical teeth (32% posterior probability). Regarding life history, pollinating or non-pollinating ovary gallers were probably the ancestral state (70% and 28%, respectively) (Fig. 1). Loss of ovipositor teeth occurred independently at least twice in Sycophaginae lineages in which wasps are receptacle gallers. However, this number may have been underestimated since some Sycophaga that gall fig receptacles (Cruaud et al., 2011a) were not included in our analyses. Non-galling life history evolved at least three times in Sycophaginae, i.e., Sycophaga spinitarsus + Sycophaga agraensis clade, Eukoebelea, and Idarnes carme species group clade (Fig. 1).

Shifts in life history and resource use were correlated with shifts in overall ovipositor structure in the Idarnes clade. Within Idarnes, all shifts in life history were associated with changes in overall ovipositor morphology. Ovipositors exhibit multiple uniform teeth in ovarygalling species, whereas non-galling species have multiple serrated teeth. Species that induce galls in the receptacle lack teeth on their ovipositors. On the other hand, within Sycophaga, the shift from ovary-galling to non-galling life history in the clade Sycophaga agraensis + Sycophaga spinitarsus was not followed by changes in overall ovipositor morphology, which maintained the ancestral condition of uniform teeth. Reconstructions were performed under a likelihood framework. Species in grey correspond to galling life history, while species in black are non-gallers. Character mapping schemes were mirrored to allow the simultaneous appreciation of a pair of characters being reconstructed.

Discussion

Our results showed that overall ovipositor morphology is generally correlated with life history in agaonid fig wasps. This correlation seems to be driven by the characteristics of the substrate used for oviposition [START_REF] Kawakita | Revision of the Japanese species of Epicephala Meyrick with descriptions of seven new species (Lepidoptera, Gracillariidae)[END_REF]. Ovipositor morphologies seem to be related to constraints imposed by features of the oviposition sites, since ovipositor morphology has experienced convergent evolution at least four times in Sycophaginae according to the resource used (i.e., receptacle, flower ovary, induced galls; Fig. 2). The use of the fig receptacle is associated with absence of ovipositor teeth. Among ovary gallers, pollinating wasps have one or two teeth in the distal extremity of their ovipositors and galling NPFW species have multiple homogeneously sized and spaced teeth. On the other hand, most non-galling fig wasps have multiple teeth with heterogeneous size and spacing.

Non-pollinating ovary gallers that enter the fig receptacle to lay eggs (i.e., Sycophaga sycomori and S. silvestrii), however, do not completely follow this pattern, but a more detailed analysis reveals morphological convergences that support the morphology-life history correlation. Both S. sycomori and S. silvestrii have multiple teeth like their congeneric species that oviposit externally. Nonetheless, the teeth of these species are less developed, being 2-3 times shorter than those of the externally ovipositing Sycophaga (Figure S3 and Table S2). These less developed teeth may suggest that manoeuvrability is not an issue for internal galling species. Indeed, pollinators (Agaonidae) oviposit internally and have one or two teeth in their ovipositors, as ovipositor insertion occurs along the vascular bundle of receptive flowers, which is used as a guide [START_REF] Verkerke | Syconial anatomy of Ficus asperifolia (Moraceae), a gynodioecious tropical fig[END_REF]. On the other hand, Sycophaga wasps evolved strategies differing from those of pollinators and have a different mode of ovipositor insertion in the flower and location of egg deposition [START_REF] Galil | The effects of Sycophaga sycomori L. on the structure and development of the synconia in Ficus sycomorus L[END_REF]; data on Sycophaga sycomori). In both cases, there seems to be limited pressure for selecting a complex tooth system in ovipositors, supporting the idea of a potentially simpler ovipositor in terms of capacity of movements and/or anchoring.

The scenario is different for externally ovipositing ovary gallers. Their ovipositors reach the interior of the fig and have to bridge gaps between flowers until they are inserted [START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF]data on Idarnes sp.3). Tooth morphology may be involved in ovipositor movement inside the fig, through flowers that are used as anchoring points until the exact oviposition site is reached [START_REF] Vincent | The mechanism of drilling by wood wasp ovipositors[END_REF]. These wasps also insert their ovipositors through the stigma of mature pistillate flowers, as pollinators do [START_REF] Elias | Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig-fig wasp interaction[END_REF]; data on Idarnes sp.3). Therefore, oviposition in young flower ovaries involves precise ovipositor insertion, which seems to be related to the uniform ovipositor structure found in galling Sycophaginae wasps.

The association between life history and ovipositor traits suggested by descriptive data was confirmed by phylogenetic regression analysis. Mean distance between teeth and the coefficient of variation of distance between teeth showed that non-galling species have more spaced teeth, with uneven spacing, as opposed to what was observed in galling species. The other variables analysed (tooth number, mean tooth height, coefficient of variation of tooth height, and relative ovipositor length that bears teeth) did not show a significant correlation, possibly because they are not related to life history or because they have no biological meaning alone. Our results suggest that maneuverability relies on tooth spacing and on the unevenness of tooth spacing. Teeth that are closer together possibly permit fine movement, while teeth that are further apart would permit the use of substrates with different textures and structures.

Regarding the evolution of life histories and ovipositor traits within Agaonidae, our results suggest that the ancestral condition for ovipositor morphology was most likely the presence of one to two apical teeth. Regarding life history, ovary galling species that oviposit in receptive figs possibly represent the ancestral state. It is important to note that the results of both independent analyses were biologically compatible, increasing our confidence in these data.

Receptacle galling life history arose at least two times (our sampling did not include receptacle gallers within Sycophaga) and was always correlated to the loss of ovipositor teeth. In the clade Pseudidarnes + Anidarnes + Conidarnes, ovipositor teeth were completely lost. However, Idarnes sp.7 (incertus group) shows some vestigial teeth that could be related to a recent loss, associated with a recent shift to receptacle galler habit. Loss of ovipositor teeth also occurred in other fig-related Chalcidoidea that are receptacle gallers (L.G. Elias, unpublished data on Epichrysomallinae and Otitesellinae). This relationship is also observed in other Parasitica species that oviposit in shallow substrates or in exposed hosts and have fewer or no teeth. This is the case for Braconidae wasps that parasitise exposed hosts or hosts that live close to the surface [START_REF] Brajković | Morphological adaptations of the ovipositor of braconid wasps (Braconidae: Hymenoptera) associated to biological characteristics of their hosts[END_REF], sawflies that glue their eggs to the leaf surface [START_REF] Weltz | The saws of sawflies: exploring the morphology of the ovipositor in Tenthredinoidea (Insecta: Hymenoptera), with emphasis on Nematinae[END_REF] and Platygaster diplosisae (Platygasteridae) that parasitises hosts with a very thin egg envelope [START_REF] Nacro | Comparative study of the morphology of the ovipositor of Platygaster diplosisae (Hymenoptera: Platygasteridae) and Aprostocetus procerae (Hymenoptera: Eulophidae) two parasitoids associated with the African rice gall midge, Orseolia oryzivora (Diptera: Cecidomyiidae)[END_REF]. Loss of ovipositor teeth in these groups supports the hypothesis that multiple teeth are related to drilling through the fig wall and to ovipositor movement inside figs.

Evolution of non-galling habit occurred three times independently in our reconstructions, but general ovipositor morphology did not undergo substantial modifications in Sycophaga agraensis + Sycophaga spinitarsus. These wasps have uniform teeth like other Sycophaginae, supporting our idea of versatility of this ovipositor morphology, which seems to be adapted to Moreover, oviposition in harder substrates is also usually correlated to serrated ovipositors in other parasitic Hymenoptera [START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF].

Our results provide new data that contribute to the understanding of the origin and maintenance of the fig-fig wasp mutualism. The ancestral Agaonidae probably used flower ovaries as oviposition sites due to the high nutritional value of these structures. Flower ovaries are expensive resources because they are directly linked to the reproductive success of the plant. Nonetheless, the exchange of such valuable resource was maintained throughout the evolutionary history of the mutualism. Indeed, the Ficus-fig wasp association is exceptional among nursery mutualisms because it represents a stable relationship, with very few reversals (but see [START_REF] Compton | Studies of Ceratosolen galili, a non-pollinating agaonid fig wasp[END_REF][START_REF] Peng | Co-ocurrence of two Eupristina species on Ficus altissima in Xishuangbann, SW China[END_REF]. The use of other resources for oviposition (fig receptacle or galls) emerged later in the evolution of the group in what seems to be a case of adaptive radiation related to the occupancy of new niches [START_REF] Schluter | The Ecology of Adaptive Radiation[END_REF]. Paradoxically, the greatest species diversification within Sycophaginae has occurred among non-galling wasps, probably due to their wider niche amplitudes [START_REF] Elias | Colonization sequence of nonpollinating fig wasps associated with Ficus citrifolia in Brazil[END_REF]. However, the evolution of this life history, as well as the receptacle galling life history, were conditional on the previous existence of mutualism, highlighting the role of mutualistic interactions as sources of biological diversification.

Our results show that, in most cases, ovipositor characteristics are related to the use of different resources as oviposition sites [START_REF] Kawakita | Revision of the Japanese species of Epicephala Meyrick with descriptions of seven new species (Lepidoptera, Gracillariidae)[END_REF]. Different ovipositor characteristics allow the exploitation of new niches and may be related to resource partitioning and species co-existence in the fig-fig wasp system. The evolutionary approach used in the present study unravelled how different life histories evolved in correlation with morphological traits in fig wasps and opens new perspectives for studies regarding other species involved in multitrophic interactions.

Fig. 1 .

 1 Fig. 1. Patterns of evolution of mean distance between teeth (left) and coefficient of variation of distance between teeth (right) in the ovipositors of fig wasp species (Hymenoptera: Agaonidae). Colour shifts reflect changes in these characters throughout evolution of the group. Scale bars at the bottom show correspondence of colours and values for each character.Reconstructions were performed under a likelihood framework. Species in grey correspond to galling life history, while species in black are non-gallers. Character mapping schemes were mirrored to allow the simultaneous appreciation of a pair of characters being reconstructed.

Fig. 2 .

 2 Fig. 2. Patterns of evolution of life history (left) and ovipositor morphology (right) in the studied fig wasp species (Hymenoptera: Agaonidae). Squares representing each terminal taxon are colour-coded according to life history on the left tree (pink = pollinating ovary gallers, blue = non-pollinating ovary gallers, black = receptacle gallers, and red = non-gallers) and according to overall ovipositor morphology on the right tree (pink = one or two apical teeth, blue = multiple uniform teeth, black = no teeth, and red = multiple serrated teeth). Pie charts at each node represent probabilities of ancestral states using Bayesian posterior probabilities. Absent taxa on the right tree indicate missing data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

  figs possibly represent the ancestral state. It is important to note that the results of both independent analyses were biologically compatible, increasing our confidence in these data.Receptacle galling life history arose at least two times (our sampling did not include receptacle gallers within Sycophaga) and was always correlated to the loss of ovipositor teeth. In the clade Pseudidarnes + Anidarnes + Conidarnes, ovipositor teeth were completely lost. However, Idarnes sp.7 (incertus group) shows some vestigial teeth that could be related to a recent loss, associated with a recent shift to receptacle galler habit. Loss of ovipositor teeth also occurred in other fig-related Chalcidoidea that are receptacle gallers (L.G. Elias, unpublished data on Epichrysomallinae and Otitesellinae). This relationship is also observed in other Parasitica species that oviposit in shallow substrates or in exposed hosts and have fewer or no teeth. This is the case for Braconidae wasps that parasitise exposed hosts or hosts that live close to the surface[START_REF] Brajković | Morphological adaptations of the ovipositor of braconid wasps (Braconidae: Hymenoptera) associated to biological characteristics of their hosts[END_REF], sawflies that glue their eggs to the leaf surface[START_REF] Weltz | The saws of sawflies: exploring the morphology of the ovipositor in Tenthredinoidea (Insecta: Hymenoptera), with emphasis on Nematinae[END_REF] and Platygaster diplosisae (Platygasteridae) that parasitises hosts with a very thin egg envelope[START_REF] Nacro | Comparative study of the morphology of the ovipositor of Platygaster diplosisae (Hymenoptera: Platygasteridae) and Aprostocetus procerae (Hymenoptera: Eulophidae) two parasitoids associated with the African rice gall midge, Orseolia oryzivora (Diptera: Cecidomyiidae)[END_REF]. Loss of ovipositor teeth in these groups supports the hypothesis that multiple teeth are related to drilling through the fig wall and to ovipositor movement inside figs.Evolution of non-galling habit occurred three times independently in our reconstructions, but general ovipositor morphology did not undergo substantial modifications in Sycophaga agraensis + Sycophaga spinitarsus. These wasps have uniform teeth like other Sycophaginae, supporting our idea of versatility of this ovipositor morphology, which seems to be adapted to oviposition in different substrates (Ficus flowers or developed galls). Alternatively, in the Idarnes clade and in Eukoebelea, a different, serrated ovipositor structure evolved in correlation with the shift in life history, demonstrating different solutions to explore analogous substrates among fig wasp clades. In fact, serrated ovipositors occur in the majority of non-galling fig wasps (L.G. Elias, unpublished data on Sycoryctinae and other fig-associated Chalcidoidea).Moreover, oviposition in harder substrates is also usually correlated to serrated ovipositors in other parasitic Hymenoptera[START_REF] Quicke | Ovipositor Structure and Function in the Parasitic Hymenoptera with an Exploration of New Hypothesis[END_REF].Our results provide new data that contribute to the understanding of the origin and maintenance of the fig-fig wasp mutualism. The ancestral Agaonidae probably used flower ovaries as oviposition sites due to the high nutritional value of these structures. Flower ovaries are expensive resources because they are directly linked to the reproductive success of the plant. Nonetheless, the exchange of such valuable resource was maintained throughout the evolutionary history of the mutualism. Indeed, the Ficus-fig wasp association is exceptional among nursery mutualisms because it represents a stable relationship, with very few reversals (but see[START_REF] Compton | Studies of Ceratosolen galili, a non-pollinating agaonid fig wasp[END_REF][START_REF] Peng | Co-ocurrence of two Eupristina species on Ficus altissima in Xishuangbann, SW China[END_REF]. The use of other resources for oviposition (fig receptacle or galls) emerged later in the evolution of the group in what seems to be a case of adaptive radiation related to the occupancy of new niches[START_REF] Schluter | The Ecology of Adaptive Radiation[END_REF]. Paradoxically, the greatest species diversification within Sycophaginae has occurred among non-galling wasps, probably due to their wider niche amplitudes[START_REF] Elias | Colonization sequence of nonpollinating fig wasps associated with Ficus citrifolia in Brazil[END_REF]. However, the evolution of this life history, as well as the receptacle galling life history, were conditional on the previous

  . Some of them also pollinate pistillate flowers, establishing an obligate mutualistic relationship with Ficus. Pollinating fig wasps (Agaonidae: Agaoninae, Kradibiinae, Tetrapusiinae) enter the inflorescence to oviposit and pollinate Ficus flowers. Other agaonid fig wasps (Agaonidae: Sycophaginae) generally do not enter figs and do not perform pollination, being called "nonpollinating fig wasps" (NPFW henceforth)

Table 1

 1 Study species, taxonomic and ecological information. NG = Non-galler; OG = Ovary galler; Poll = Pollinator; RG = Receptacle galler. * indicates non-pollinating species that enter syconia.

	Life	Fig wasp species	Fig wasp	Host Ficus species	Country	Specimen
	history		subfamily	(Ficus section)
	Poll	Ceratosolen sp	Kradibiinae	F. hispida (Sycocarpus) Laos	5372_0199
	Poll	Pegoscapus sp	Agaoninae	F. citrifolia	Brazil	2672_0195
				(Americanae)
	Poll	Platyscapa sp	Agaoninae	F. concinna	China	1660_0101
				(Urostigma)
	Poll	Tetrapus sp	Tetrapusinae	F. obtusiuscula	Brazil	2685_0101
				(Pharmacosycea)
	OG	Idarnes sp.3	Sycophaginae	F. eximia (Americanae) Brazil	2565_0295
		(flavicollis group)
	OG	Idarnes flavicollis	Sycophaginae	F. obtusifolia	Brazil	2579_0295
				(Americanae)

Table 2

 2 Ovipositor traits coded into continuous characters for this study.

	Character	Formula
	1. Number of teeth on
	gonapophysis IX	-
	2. Mean tooth height	Ʃ tooth height/tooth
	(mm)	number
	3. Coefficient of variation	SD of tooth
	of tooth height	height/mean tooth
		height
	4. Mean distance between	Ʃ distance between
	teeth (mm)	teeth/(tooth number)-
		1
	5. Coefficient of variation	SD of distance
	of distance between	between teeth/mean
	teeth	distance between
		teeth
	6. Relative ovipositor	Portion bearing
	length bearing teeth	teeth/total ovipositor
	(mm)	length
	Phylogenetic analyses and molecular dating are described in detail in the Supplementary
	material.

Table 4

 4 Models tested to predict fig wasp life history (0-galler, 1-non-galler). Models with ΔAIC < 2 and wAIC > 0.1 were considered equally plausible. wAIC = model weight, a = phylogenetic signal estimated for the regression, na = not applicable.

	Model	Slope AIC ΔAIC wAIC a
	Predictor
	Coefficient of
	variation of
	distance	+	15.52 0	0.508 0.70
	between
	teeth
	Mean distance	+	16.02 0.5	0.395 1.10
	between teeth
	Null model	na	22.33 6.81 0.017 1.40
	Number of	+	23.98 8.46 0.007 1.40
	teeth
	Coefficient of	+	24.29 8.77 0.006 1.52
	variation of
	tooth height
	Relative	-	24.38 8.86 0.006 1.40
	ovipositor
	length bearing
	teeth
	Mean tooth	+	24.76 9.24 0.005 1.40
	height

= Non-pollinating-ovary galler (receptive flowers)

= Receptacle galler (pre-receptive flowers) 1 = Non-galler3 = Non-galler (galled flowers)

Acknowledgements

We thank Liam J. Revell (University of Massachusetts) for advice about the use of phytools functions and Atsushi Kawakita (Kyoto University) for discussion of the correlation between ovipositor morphology and life history traits. We are also indebted to Anne Le Ralec (Agrocampus Ouest, Rennes) for valuable discussion of ovipositor morphology, to Huan Gang, Yang Pei and Wenjuan Ma (XTBG) for help with field work in Xishuangbanna Tropical Botanical Garden, and to Rodrigo Silva (FFCLRP -Universidade de São Paulo) and Laurent Soldati (CBGP, Institut National de la Recherche Agronomique) for help with Scanning Electron Microscopy. We thank Elettra Greene for the English revision.

Appendix A. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.actao.2017.10.007.

Funding This work was supported by the Sao Paulo Research Foundation (FAPESP), grant numbers 2007/59059-3 and 2013/01918-1 (L.G. Elias); 2015/06430-2 (F.H.A. Farache); 2011/09477-9 (E.A.B. Almeida); 2015/25417-7 (R.A.S. Pereira), and by the National Council for Scientific and Technological Development (CNPq) grant number 306078/2014-7).